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BENSON, Ann Marie, 1936-THE AMINO ACID SEQUENCE OF LEUCAENAGLAUCA FERREDOXIN.
University of Hawaii, Ph.D., 1969Biochemistry
University Microfilms, Inc., Ann Arbor, Michigan
THE AMINO ACID SEQUENCE OF
LEUCAENA GLAUCA FERREDOXIN
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE
UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN BIOCHEMISTRY
JANUARY 1969
by
Ann Marie Benson
Dissertation Committee:
Kerry T. Yasunobu, ChairmanLawrence H. PietteMorton MandelJohn B. HallJohn A. Hunt
DEDICATION
To my mother
ACKNOWLEDGMENTS
To the National Institutes of Health
for support, 1967 to 1969.
To Mrs. Kerry T. Yasunobu and Chrissie and Steven Yasunobu
for gathering a great many pounds of Leucaena glauca
leaves.
To Dr. Hans Georg Kloepfer
for felling numerous Leucaena glauca trees.
TABLE OF CONTENTS
LIST OF TABLES •
LIST OF FIGURES
ABBREVIATIONS
ABSTRACT . . • • .
v
vi
.. viii
x
1. INTRODUCTION
A. Discovery of Ferredoxins .
· . . .· ......
1
1
B. Classification, Properties, and PhysiologicalFunctions of Ferredoxins . . . . . 3
C. Primary Structure of Ferredoxins • 15
D. Statement of the Problem .
II. MATERIALS AND METHODS
A. Materials . . . . · · . . · . · · . ./.
l. Materials and Reagents ObtainedCommercially . · · . . · . · ·
2. Materials Obtained as Gifts · ·3. Materials Obtained by Preparative Methods
B. Methods
1. Isolation of Ferredoxin .
25
33
33
33
34
34
35
35
2. Preparation of the CarboxymethylcysteineDerivative .• . . . . • . . . . . . . . 41
3. Methods of Hydrolysis by Endopeptidases . 42
c. Bacillus subtilis Neutral Protease.
· . . . . . . . . . . .
a. Trypsin
b. Chymotrypsin.
d. Thermolysin
· . . . . . . . . . .42
42
42
43
4. Methods of Peptide Purification. . . . . . .ii
43
a. Chromatographic Separation ofTryptic Peptides . • • • . . . . . . . 43
b. Chromatographic Separation ofChymotryptic Peptides . . . . . 44
c. Further Purification of Trypticand Chymotryptic Peptides • . . . . . . . 44
d. Chromatographic Separation ofB. subtilis Neutral ProteaseFragments of Tryptic Peptide T-7a 45
e. Partition Chromatographic Separationof B. subtilis Neutral ProteaseFragments of Chymotryptic Peptide C-lO 46
f. Chromatographic Separation of ThermolysinFragments of Chymotryptic Peptide C-lO •. 46
5. Determination of Amino Acid Composition • •• 47
6. Methods of Amino Acid Sequence Determination 48
a. Methods of NH2-terminal Analysis . . • •. 48
1. Edman Degradation • • . • • • 48
. . .
50
50
50
50
51
52
. . .. .
. . .2. Carboxypeptidase A
3. Carboxypeptidase B
2. Dinitrophenylation
3. Dansylation • . • .
b. Methods of COOH-terminal Analysis
1. Hydrazinolysis
7. Studies on Ferredoxin from Individual Trees. 52
. . . .A. Isolation of Ferredoxin
III. RESULTS • . . • . . . . . . . . . .. . . .
. . 54
54
B. Preparation of the CarboxymethylcysteineDerivative . . • . • . • • • • • • • •
C. Amino Acid Composition of Ferredoxin • •
. . . 54
54
111
D. Amino and Carboxyl Terminal Residues · · · · · · 62
E. Tryptic Peptides • · · · · · · • · · · 62
1- Initial Chromatography · · · · · · 62
2. Further Purification of Tryptic Peptides 68
3. Amino Acid Composition · · · · · · · 68
4. Amino Acid Sequence · · · · · 75
F. Chymotryptic Peptides · • · · · · · 83
1- Initial Chromatography · · · · · · · 83
2. Further Purification of ChymotrypticPeptides . . · · · · · · · · · · · · · · 83
3. Amino Acid Composition · · · · · · · · · 86
4. Amino Acid Sequence · · · · · · · · · · · · · 86
G. Complete Amino Acid Sequence of Leucaenaglauca Ferredoxin • · · · · · · · · · · · · • · 113
H. Distribution of Sequence Heterogeneityamong the ~. glauca Population · · · • · · · · · 113
IV. DISCUSSION AND CONCLUSIONS · · · • · · · · · · · · 120
A. Characteristics of the Amino Acid Sequenceof L. glauca Ferredoxin · · · · · · · · · • · · 120
B. Comparison of Plant and Algal Ferredoxinsfrom Four Species · · · · · · · · · · · · · · · 121
C. Acidic Residues in Plant and AlgalFerredoxins. . . · · · · • · · • · · · · · · · · 125
D. Genetic and Evolutionary Aspects ofVariations in Ferredoxin Sequences · · · · · 127
1- Plant and Algal Ferredoxins · · · · · · · 127
2. Heterogeneity in ~. glauca Ferredoxin · · 132
E. Techniques in Ferredoxin Purification · · · 140
F. Specificities of Proteolytic EnzymesEmployed in these Stud1.es · · · · · · · · • · · 143
iv
G. Determination of the Complete Structureof L. glauca Ferredoxin • • • • . • • • • • • .• 144
. . . . . . . . . . . . . . . . . . . .V.
VI.
SUMMARY
BIBLIOGRAPHY • • . . . . . ... . . . . . . . . . . .146
148
LIST OF TABLES
I. Amino Acid Compositions of Ferredoxins fromL. gl~uca and Other Plants • • • • • • • • •
II. Tryptophan Content of ~. glauca Ferredoxin • · . .
v
61
63
III. Amino Acid Composition and Properties ofTryptic Peptides . . . . . . . . . . . · · · . 71
IV. Amino Acid Composition and Properties ofChymotryptic Peptides . . . . . . . . · · · . 97
V. Distribution of Amino Acid Residues at ThreePoints of Heterogeneity in L. glauca Ferredoxin 119
VI. Acidic Residue Content of Proteins of VariousType s • • • • •• ••••.••••••• · . 126
VII. Mutation Distances and Values of MinimumBase Difference per Codon • . . . . • • .
VIII. Types of Mutations in L. glauca FerredoxinHeterogeneity•.•••••••••••••
· . . .
· . . .
130
141
vi
LIST OF FIGURES
1. Spectra of Type I ferredoxins •• . . . . . . . . . 6
2. Spectra of typical Type II and Type IIIferredoxins • • • • . • • • . • . . . . . . . . 8
3 a. Summary of the functions of ferredoxin ingreen plants •• • • • • • • • • . • • • . . . . . 11
3 b. Summary of the functions of ferredoxin inphotosynthetic bacteria • • • • . • • . • . 13
20
3 c. Summary of the metabolic reactions in whichType III ferredoxin is known to function • • . •. 17
4 a. Amino acid sequences of three Type IIIferredoxins • • • • . • • • • . . • • •
4 b. The structure of a possible precursor ofType III ferredoxins • • • • • • • • . • · . . 22
5. Comparison of the amino acid sequences offerredoxins from C. butyricum and Chromatium
6. Comparison of spinach and Clostridialferredoxins . • • . • • • • • • . • • • • • • · . .
24
27
60· . .
7. Amino acid sequences of ferredoxins from alfalfa,spinach, and Scenedesmus, a green alga • • • • •• 29
8. Photograph of a specimen of Leucaena glauca • • 32
9. Procedure for isolation of ~. glauca ferredoxin 38
10. Photomicrograph of ~. glauca ferredoxin crystals 56
11. Absorption spectrum of ~. glauca ferredoxin. • 58
12. Purification of CMFd by gel filtration througha column of Sephadex G-25 • • • • • . • • • •
13. Action of carboxypeptidase A on CMFd . . . . 65
14. The elution pattern of the tryptic digest ofCMFd from AG l-X2 • • . • • • • 67
15. Purification of tryptic peptide T-2 on AG 50-X2 70
16. Summary of evidence establishing the sequenceof tryptic peptide T-7a • • • • • • • • • • • • 81
· . .
17. Elution pattern of the chymotryptic digestof CMFd from a column of AG 50-X2 • • • • •
18 a. Purification of peptide c-4 by gel filtration .
18 b. Purification of peptide C-5 by gel filtration.
19. Resolution of peptides C-7a, C-7b, C-12, andc-14 on a column of AG l-X2 • . • • • • • • •
· .· .· .
vii
85
88
90
92
20 a. Pattern of elution of a mixture containingpeptide C-9 from Sephadex G-15 •• • . . • · . . . 94
. . . . .
20 b. Distribution of peptide C-9 and a contaminantin the effluent fractions from Sephadex G-15,as determined by paper chromatography . • • .
21. Summary of the evidence establishing thesequence of peptide C-lO • • . • • • . •
· . . 96
107
22. Elution pattern of peptides from thermolysindigest of chymotryptic peptide C-lO • • • •
23. Tryptic and chymotryptic peptides arrangedin order in L. glauca ferredoxin • • • • •
· . . .· . . .
110
115
. . . . . . .
24. Elution pattern of tryptic peptides of theferredoxin of Tree 1 from AG l-X2 • • • • • •
25. The amino acid sequences of ferredoxins fromL. glauca, alfalfa, spinach, and Scenedesmus
26. Phylogenetic tree of the ferredoxins
· . .· . .
118
123
134
27. Inheritance patterns of allelic and non-allelicgenes . . . . . . . . . . . . . . . . . . . . . 137
A
Ala
Arg
Asp
Asn
Asx
BSNP
C
CMCys
CMFd
CoA
Cys
DEAE
DNP
DNS
EDTA
G
Glu
GIn
Glx
Gly
His
lIe
Leu
Lys
LIST OF ABBREVIATIONS
Adenine
Alanine
Arginine
Aspartic acid
Asparagine
Aspartic acid or asparagine
Bacillus subtilis neutral protease
Cytosine
S-carboxymethylcysteine
Carboxymethylated ferredoxin
Coenzyme A
Cysteine or half-cystine
Diethylaminoethyl
2,4-Dinitrophenyl
5-dimethylamino-I-napthalene sulfonyl
Ethylenediaminetetraacetic acid
Guanine
Glutamic acid
Glutamine
Glutamic acid or glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
viii
Met
PCAW
Phe
Pi
PPNR
Pro
PTH
Rf
Ser
Th
Thr
TLCK
TPCK
TPN
Tris
Trp
Tyr
U
UV
Val
ix
Methionine
Pyridine-collidine-acetic acid buffer
Phenylalanine
Inorganic phosphate
Photosynthetic pyridine nucleotide reductase
Proline
Phenylthiohydantoin
Partition coefficient
Serine
Thermolysin
Threonine
L-(1-tosylamido-2-lysyl)ethyl chloromethyl ketone
L-(1-tosylamido-2-phenyl)ethyl chloromethyl ketone
Triphosphopyridine nucleotide
Trishydroxymethylamino methane
Tryptophan
Tyrosine
Uracil
Ultraviolet
Valine
x
ABSTRACT
Ferredoxin, a protein of unusually low oxidation
reduction potential, and also the first protein found to con
tain both iron and labile sulfide, plays a very fundamental
role in the photosynthetic process, that of acceptor of
electrons from light-activated chlorophyll.
Ferredoxin was isolated from the leaves of LeUcaena
glauca, a species of small leguminous tree. The carboxy
methylcysteine derivative of the combined ferredoxin from
many plants was used for the amino acid sequence studies.
The products of tryptic and chymotryptic hydrolyses were
purified by ion exchange chromatography, gel filtration, and
paper chromatography. Thermolysin and Baci'llus subtilis
neutral protease were uRed to further degrade large peptides.
Most of the sequence was determined by the subtractive
method of Edman degradation (J. BioI. Chern., 237, 2547, 1962).
The amino acid sequence, deduced primarily from the
structures of the tryptic and chymotryptic peptides, is as
follows: Ala-Phe-Lys-Val-Lys-Leua/Valb-Leu-Thr-Pro-Asp-Gly
Proa/Alab-Lys-Glu-Phe-Glu-Cys-Pro-Asp-Asp-Val-Tyr-Ile-Leu-Asp
Gln-Ala-Glu-Glu-Leu-Gly-Ile-Glux/Aspx-Leu-Pro-Tyr-Ser-Cys-Arg
Ala-Gly-Ser-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Leu-Val-Glu-GIy-Asp
Leu-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Glu-Gln-Ile-Glu
Glu-Gly-Trp-Val-Leu-Thr-Cys-Ala-Ala-Tyr-Pro-Arg-Ser-Asp-Val
Val-Ile-Glu-Thr-His-Lys-Glu-Glu-Glu-Leu-Thr-GlYx/Alax-COOH.
The sequence of this ferredoxin resembles those of
xi
spinach and alfalfa ferredoxins, showing a slightly greater
similarity to alfalfa, as would be expected from taxonomic
considerations. Over 70% of the sequence, including the five
cysteine residues, is invariant in these three species. Many
of the changes between species are conservative, but radical
changes involving charged residues and proline residues occur
also.
Heterogeneity was found in positions 6, 12, 33, and 96
within the L. gl"auca ferredoxin sequence, each of which was
occupied by two different amino acid residues, as shown.
However, the tryptic peptide containing residues 6 and 12
occurred in only two of the four possible forms, one of which
contained leucine and proline in positions 6 and 12, respec
tively, whereas the other contained valine and alanine. Thus
the presence of different genes is indicated, rather than
ambiguity in translation of the genetic code.
A study of the distribution of the different forms of
the protein among the ~. glauca population was undertaken.
In a normal distribution of allelic genes, 50% of the popula
tion would be expected to be homozygous. Ferredoxin was
isolated from ten different plants and three of the four
points of heterogeneity were investigated (residues 6, 12,
and 96). Heterogeneity was found in each of these positions
in each of the ten individual trees. Thus the probability
that the differing allelic nuclear genes are involved is less
than 0.001. The two most probable explanations for the
xii
observed sequence heterogeneity are (1) that differing non
allelic genes for ferredoxin are present, that is, that gene
duplication has occurred in the evolution of this species,
followed by point mutations in the individual genes, or (2)
that the ferredoxin genes are located not in the nucleus of
the call but rather in the chloroplasts, and their distribu
tion therefore follows a pattern quite different from that of
allelic nuclear genes.
I. INTRODUCTION
A. Discovery of Ferredoxins
Ferredoxins, a group of non-heme, non-flavin proteins
containing iron and labile sulfide and possessing unusually
low oxidation-reduction potentials, function as electron
carriers in cellular redox reactions driven by light energy
or by the hydrogenase system. Proteins of this type have been
found in anaerobic bacteria such asC16~t~idia (1), in photo
synthetic bacteria such as Chromatium (2), in algae such as
Nostoc (3) and in chloroplasts of spinach (4), alfalfa (5)
and other species of green plants.
In 1952 a soluble factor, then called "methaemoglobin
reducing factor," was isolated from parsley chloroplasts by
Davenport, Hill, and Whatley (6). This sUbstance, believed
to be a protein, catalyzed the reduction of methemoglobin in
the presence of chloroplast fragments and light. Four years
later, San Pietro and Lang (4) isolated a similar factor from
spinach chloroplasts, found it to be involved in the photo
reduction of NADP, and named it "photosynthetic pyridine
nucleotide reductase," or "PPNR." In 1957, Arnon, Whatley
and Allen (7) independently isolated the same factor from
spinach chloroplasts and named it "TPN-reducing factor."
When Davenport (8) reported in 1960 that the partially purified
methemoglobin reducing factor, which had since been found to
catalyze the photoreduction of other heme proteins as well (9)
and had been renamed "haem-reducing factor," was also active
2
in catalyzing the photoreduction of NADP by chloroplasts, it
became evident that the three separately isolated and named
factors, haem-reducing factor, PPNR, and TPN-reducing factor,
were identical.
In the following two years factors which were active in
the photoreduction of NADP, and which were similar in some
but not all respects to the plant factors previously found,
were isolated from bacteria. Losada, Whatley, and Arnon (2)
isolated a pyridine nucleotide reductase from Chromatium, a
photosynthetic bacterium, in 1961 and in the following year
Mortenson, Valentine, and Carnahan (10) reported isolation of
a non-heme iron protein, which they named "ferredoxin," from
Clostridium pasteurianum, a species of anaerobic non-photo
synthetic bacterium in which this protein served as an electron
carrier. Q. pasteurianum ferredoxin was obtained in crystalline
form by Tagawa and Arnon and was shown to be capable of cata
lyzing the photoreduction of NADP by illuminated spinach
chloroplasts (11). These investigators also found that the
chloroplast protein contained iron, as did the C. pasteurianum
ferredoxin (10), that it was reversibly oxidized and reduced,
with accompanying spectral changes, and that both of these
proteins had unusually low oxidation-reduction potentials,
E'o at pH 7.55 being -417 mv for C. pasteurianum ferredoxin
and -432 mv for the spinach protein (11). These values are
close to the potential of hydrogen gas and approximately 100
mv more negative than that of the pyridine nucleotides. The
3
interchangeability of the native spinach protein and C.
pasteurianum ferredoxin in the chloroplast reaction and the
fact that both of these proteins contain iron and have unusu
ally low oxidation-reduction potentials led to the proposal
by Tagawa and Arnon (11) that the name "ferredoxin" be extended
to include the chloroplast protein as well. Thus the class of
proteins known as "ferredoxins" has been defined as those non
heme, non-flavin, iron containing proteins which have an
oxidation-reduction potential close to that of hydrogen gas,
and which are capable of catalyzing the photoreduction of NADP
by washed chloroplasts.
B. Classification, Properties, and Physiological Functions of
Ferredoxins
Within the past few years ferredoxins have been isolated
from numerous other species, including plants, algae, and
photosynthetic and non-photosynthetic anaerobic bacteria.
Since the properties of these proteins was found to var~ with
their source, the ferredoxins were classified into three types
(12) according to their origins:
(I) Ferredoxin of green plants and algae.
(II) Ferredoxin of photosynthetic bacteria.
(III) Ferredoxin of non-photosynthetic anaerobic bacteria.
These three types of ferredoxin differ in molecular
weight, iron and labile sulfur content, and spectral character
istics.
Type 1 ferredoxins have the highest molecular weight,
4
approximately 11,500 (5,13,14) and contain two atoms of iron
and two atoms of labile sulfide (5) per molecule. The
characteristic absorption spectra of these plant and algal
ferredoxins have maxima at approximately 465, 420, 330, and
276 m~. The visible and UV spectra of ferredoxins from spinach
(11) and Scenedesmus (15), a green alga, are shown in Figure 1.
Type II ferredoxins, those of the photosynthetic bacteria,
have a molecular weight of approximately 10,000 (16), inter
mediate between Type I and Type III ferredoxins. They contain
five to six gram-atoms each of iron and labile sulfide per
mole (12). The typical Type II ferredoxin spectrum has maxima
at 390, 310, and 280 m~, as can be seen in Figure 2, which
shows the spectrum of Chromatium (16) ferredoxin. This figure
also shows the spectrum of ferredoxin from Clostridium
pasteurianum (1), a typical Type III ferredoxin, which has the
same visible maxima as ferredoxins of Type II. However, the
ferredoxins from non-photosynthetic anaerobic bacteria differ
from those of the photosynthetic bacteria in other properties,
having a lower molecular weight, approximately 6,000 (1), and
containing seven to eight atoms each of iron and labile sulfide
per molecule (12).
Type I ferredoxin plays a very fundamental role in the
process of photosynthesis, being the primary acceptor of light
activated electrons from chlorophyll, and thus the first stable
reductant formed as a result of the radiant energy trapped (11).
In 1962 Whatley, Tagawa, and Arnon (17) separated the light and
5
Figure 1. Spectra of Type I ferredoxins. Curve A is
the spectrum of spinach ferredoxin in 0.033 M Tris buffer of
pH 7.80. Curve B is the spectrum of Scenedesrnus ferredoxin
in 0.01 M Tris buffer of pH 8.0. These spectra, which
represent different concentrations of protein, both exhibit
maxima at 325, 420, and 463 m~. The ratios among the
absorbancies at these three maxima are identical in the
two spectra. In the region below 300 m~, the slight shift
in maxima and the differences in absorbancy ratios between
these maxima and the other maxima in the corresponding
spectra are due to differences in content of aromatic amino
acids, especially tryptophan. The spectra shown in the
figure are reproduced from pUblished spectra (11, 15).
I&JUZCma::o(I)IIIC
I&J>-t-C....I&J0:
250 350 450 550
-_......
WAVELENGTH (mfJ)
7
Figure 2. Spectra of typical Type II and Type III
ferredoxins. Curve A is the spectrum of ferredoxin from
Chromatium, a photosynthetic bacterium. The protein was
dissolved in 0.005 M phosphate bUffer, pH 7.6. Curve B is
the spectrum of Clostridium pasteurianum ferredoxin, in 0.07 M
Tris buffer of pH 7.3. These are reproductions of pUblished
spectra (16, 1). These spectra, which represent different
protein concentrations, are very similar. Each exhibits
maxima at 280 and 390 rn~ and a shoulder at 310 m~.
I&JUZea:aa:ofnmeIIJ>-l-e.J1&.1a:
300 350 400 450 500
WAVELENGTH (mfl)
9
dark reactions in electron transfer during photosynthesis,
showing that photoreduction of NADP occurs in two steps. The
first step is the reduction of ferredoxin by light-activated
chlorophyll. The reduced ferredoxin then reduces NADP in a
dark reaction which is mediated by a flavoprotein enzyme, NADP
reductase. Reduced ferredoxin was shown by these investigators
to be the physiological electron carrier in NADP reduction and
in cyclic and noncyclic photo-phosphorylation. Another func
tion of ferredoxin in plants involves nitrogen fixation.
Nitrate is converted to ammonia by illuminated spinach grana,
by a two-step reaction (18):
FMNN03- ------------------>
nitrate reductase
FerredoxinN02- ------------------> NH3nitrite reductase
In the second step of this reaction, reduced ferredoxin
supplies the electrons, via the reductase enzyme, for the
reduction of nitrate to ammonia. The functions of ferredoxin
in green plants are summarized in Figure 3 a.
Figure 3 b shows some of the functions of ferredoxin in
photosynthetic bacteria. Although these bacteria do not con
tain chloroplasts, the process of photosynthesis is similar
to that in plants. However, unlike plants, the photosynthetic
bacteria require a reductant other than water, the oxidation
of which supplies electrons to ferredoxin via photoactivated
chlorophyll. Since water cannot serve as the reductant,
oxygen is not evolved. In contrast to chloroplasts, which
have no hydrogenase, photosynthetic bacteria can photoproduce
Figure 3 a. Summary of the functions of ferredoxin in green plants.
I-'o
1'0:I:Z
f
.1'0oz
0..+
..J «
..J>-g: > ON
oa::g"""l::<_~O:I: ~U
a.. :I:o a..« _----:>....... 0Z «z
a..I«
0..+a..o«
t
II
Figure 3 b. Summary of the functions of ferredoxin in photosynthetic bacteria.
I-'I\)
H2-CO? :> PYRUVATE
CO2
Ir:==--~_~_N_A_DP--;;;~NADPH
ACETYL
CO2 . 01 -KETO-
SUCCINYL coA4 GLUTARATE
LIGHT > CHLOROPHYLL :> I FERREDOXIN
1REDUCTANT
14
hydrogen gas, which isa product of the reduction of hydrogen
ions by reduced ferredoxin.
In 1966 Evans, Buchanan, and Arnon (19) discovered a new
ferredoxin-dependent carbon reduction cycle in a green sulfur
photosynthetic bacterium, Chlorobium thiosulfatophilum. Each
turn of the cycle fixes four molecules of C02' yielding one
molecule of oxaloacetate. Compounds ranging in size from C2
to C6' including pyruvate and ot-ketoglutarate, are also
synthesized from C02 in this cycle. Although evidence for this
cycle has been obtained only with this species, these reactions
are believed to occur in other species of photosynthetic
bacteria also.
In the non-photosynthetic anaerobic bacteria ferredoxin
acts as an electron carrier in numerous oxidation-reduction
reactions. Ferredoxin can be reduced either by hydrogen in
the presence of bacterial hydrogenase (10), or by substrate,
as in the oxidation of pyruvate to CO 2 and acetyl phosphate
in C. acidi-urici, a species in which the hydrogenase enzyme
is not present (20). The reduced ferredoxin can then function
as an electron donor in the reduction of pyridine nucleotides
(11), the conversion of nitrite and hydroxylamine to ammonia
(21), the production of pyruvate from acetyl coenzyme A and
CO2 (22,23,24,25), the reduction of sulfite to sulfide (26),
and the reduction of urate to xanthine (27). Each of these
reactions is mediated by a specific enzyme (e.g. nitrite
15
reductase, xanthine oxidase). Oxidized ferredoxin has been
found to serve as the electron acceptor in the oxidation of
various sUbstrates, such as pyruvate, hypoxanthine (27), 0(
ketoglutarate (28), formate (29), and in the case of Methano
bacillus, acetaldehyde (30). Specific dehydrogenase enzymes
catalyze these reactions. The role of ferredoxin in
anaerobic non-photosynthetic bacteria is summarized in
Figure 3 c.
C. Primary Structure of Ferredoxins
Since the ferredoxins play such a fundamental role in the
processes of photosynthesis and nitrogen fixation, and since
they appeared to be such unique proteins with their extremely
low oxidation-reduction potentials, and, furthermore, were
the first proteins reported to contain both iron and labile
sulfide, there has been much interest in determining the
structure-function relationships eXisting in this class of
proteins. Also, since three types of ferredoxin have been
isolated, all of which are to some extent functionally inter
changeable, it was suspected that the primary structures of
these proteins might show evidence of evolutionary relation
ship among the three types. Thus, in 1962 the first steps
were taken toward elucidating the primary structure of a
ferredoxin, that of Clostridium pasteurianum. Since that
time, amino acid sequence determinations have been performed
on ferredoxins of various species, including representatives
of all three of the ferredoxin types previously described.
I-'0\
Figure 3 c. Summary of the metabolic reactions in which Type III ferredoxin is
known to function. The compounds at the left of the diagram are oXidized by ferredoxin,
which then acts as an electron donor in the reactions at the right.
ACETALDEHYDE
"'~>---'t::H2 -----------1FORMATE
HYPOXANTHINE
a-KETOGLUTARATE
S0'3 > S=
-~> NADH
N02
Y\NH 3
Y
ACETYLC~ PYRUVATE
CO2
,;
18
The sequence of C. pasteU:rianum (31) ferredoxin was completed
in 1964, followed by C. bu:tyrlcum (32) in 1966, and Micrococcus
aerogenes" (33) in 1968. These Type III ferredoxins, shown in
Figure 4 a, were each found to consist of a single polypeptide
chain, 54 to 55 residues in length. Each has eight residues
of cysteine, all of which are involved in iron binding, and
all of which occupy homologous positions in the sequence in
all three species. A striking similarity between the first
half and the second half of the ferredoxin molecule was noted
in the case of C. pasteuriartum (31), and was found in the
other two species as well. It was this similarity between the
two "half-molecules" which led to the postulation of a pre
cursor molecule (34), approximately half the size of these
ferredoxin molecules, which gave rise to the Type III
ferredoxins by gene duplication. Figures 4 a and 4 b show
possible precursor structures.
In 1968 the amino acid sequence of a Type II ferredoxin,
that of Chromatium (12), was completed. As shown in Figure 5,
a large amount of homology was found to exist between the
Type III ferredoxins and Chromatium ferredoxin, indicating a
close evolutionary relationship between these two groups of
proteins (12). Chromatium ferredoxin was found to contain an
insertion of nine amino acid residues and an extra 17 residues
at the carboxyl terminus, as compared with the' Clo'stridial
ferredoxins. Eight of the nine residues of half-cystine in
Chromatium ferredoxin were found to be homologous to the eight
19
Figure 4 a. Amino acid sequences of three Type III
ferredoxins. The six homologous half-molecules are aligned
for comparison. Lines (1), (2), and (3) show the amino acid
sequences of residues 1 through 28, and lines (4), (5), and
(6) show the sequences of residues 29 through 56, of the
ferredoxins of Clostridium pasteurianum, c. butyricum, and
Micrococcus aerogenes, respectively. The possible structures
of a precursor are shown on line (7) and include all the amino
acid residues found at each position in the six half-molecules.
-Ala-Asp-Ser-Cys-Val-Ser-Cys-Gly-Ala-
1
(1) Ala-Tyr-Lys-Ile-
(2) Phe Val
(3) Tyr Val
29
5
Asn
Asn
34
10
Val Ser
Ile Ala
39
20
Ile-Phe-Val-Ile-Asp-Ala-Asp-Thr-Cys-Ile-Asp-Cys-Gly-Asn-(4)
(5)
(6)
GIn Phe Val
Ile Tyr Ala
1 5
Thr
Ser
10
Asn
Ser
(7) Ala-Tyr-Lys-Ile-Asp-Ala-Asp-Ser-Cys-Val-Ser-Cys-Gly-Ala-Ile Phe Val Asn Thr Ile Ala AsnGIn Ala Asp Ser
Cys-Ala-Ser-Glu-Cys-Pro-Val-Asn-Ala-Ile-Ser-Gln-Gly-Asp-Ser
Cys-Ala-Asn-Val-Cys-Pro-Val-Gly-Ala-Pro-Val-Gln-Glu
(1)
(2)
(3)
(4)
(5)
(6)
14
43
14
Ala Gly
Lys Pro
Asn
Ser
20
49
20
Ser Ala
Asn
Thr
GIn
Asn GIn
Asn Pro
55
28
Asp Thr
Ser
Asp.
28
(7) Cys-Ala-Ser-Glu-Cys-Pro-Val-Asn-Ala-Ile-Ser-Gln-Gly-Asp-SerLys Gly Val Ser Pro Thr Pro Glu Thr
Pro Gly GInAsn Val
Asn
21
Figure 4 b. The structure of a possible precursor of
Type III ferredoxins. In this structure, each position is
occupied by the predominant residue in the corresponding
positions in the six half-molecules of Figure q a. Three
positions are each occupied by two amino acids since neither
dominates in the half-molecules. The placement of
phenylalanine in position 2 is based on the belief that
c. butyricum is the most conservative of the three species
compared.
1 7 14
22
Ala-Phe-Val-lle-Asp-Ala-Asp-Ser-Cys-lle-Asp-Cys-Gly-Ala-
15 21 28Glu lIe Gly
Ala-Asn-Val-Cys-Pro-Val-Gly-Ala-Pro-Asn-Gln-Glu-Asp-Ser
23
Figure 5. Comparison of the amino acid sequences of
ferredoxins from ~. butyricum and Chromatium. The numbers
below the two rows of sequences refer to the minimum number
of base changes for each codon which would be required to
convert one sequence to the other.
(A)
(B)
(A)
(B)
(A)
(B)
(A)
(B)
(A)
(B)
(B)
24
1£. butyricum A1a-Phe-Va1-I1e-Asn-Asp-Ser-Cys-Va1-Ser-Cys-
Chromatium A1a-Leu-Met-I1e-Thr-Asp-G1n-Cys-I1e-Asn-Cyso 1 1 0 1 0 2 0 110
G1y-A1a-Cys-A1a-G1y-G1u-Cys-Pro-Va1-Ser-A1a-I1e-Thr-G1n
Asn(G1x,Cys,Asx,Pro,G1x,Cys,Pro,Va1)G1y-A1a-I1e-Ser-G1n2 2 0 2 2 1 0 0 0 1 001 0
G1y-Asp-Thr-G1n-Phe-Va1-I1e-Asp-A1a-Asp-Thr-Cys-I1e-Asp
G1y-Asp-G1u-Thr-Tyr-Va1-I1e-G1u-Pro-Ser-Leu-Cys-Thr-G1uo 0 2 2 1 0 0 1 1 2 2 011
41 42Cys-G1y- -Asn-Cys-A1a-
41 51Cys-Va1-G1y-His-Tyr-G1u-Thr-Ser-G1n-Cys-Va1-Asp-Cys-Va1-o 1 101
Asn-Va1-Cys-Pro-Va1-G1y-A1a-Pro-Asn-G1n-G1u
G1u-Va1-Cys-Pro-I1e-Lys-Asp-Pro-Ser-His-G1u-G1u-Thr-G1u20001 210 110
81Asp-G1u-Leu-Arg-A1a-Lys-Tyr-G1u-Arg-I1e-Thr-G1y-G1u-G1y
25
half-cystine residues of the Type III ferredoxins.
Before amino acid sequence determinations had been per
formed on plant ferredoxins, it was thought that they might
have evolved by gene duplication from the Type III ferredoxins,
just as the latter appeared to have evolved from their postu
lated precursor. When the sequence of spinach ferredoxin (14)
was published, it could be seen that the evolution of this
protein had not followed such a simple course. However,
comparison of the spinach ferredoxin sequence with those of
Clostridial ferredoxins showed some regions of homology, as
shown in Figure 6, indicating possible evolution of these two
types of ferredoxin from a common precursor (12,14). The
amino acid sequence of alfalfa ferredoxin (35) has also been
determined and, as would be expected, is similar to that of
spinach ferredoxin. Recently, an algal ferredoxin, that of
Scenedesmus (36) was also sequenced and was found to be very
similar to the ferredoxins of the higher plants. Figure 7
shows the comparison of Scenedesmus ferredoxin with the two
plant ferredoxins.
D. Statement of the Problem
The amount of homology between spinach ferredoxin and
the clostridial ferredoxins is small enough to leave serious
doubts as to whether the plant and bacterial ferredoxins are
truly related in the evolutionary process. The possibility
of convergent evolution, in which similar structural patterns
evolve independently due to their advantage to the function
C\)
0\
Figure 6. Comparison of spinach and Clostridial ferredoxins. The amino acid
sequence of spinach ferredoxin, on line (2), is aligned with portions of the sequence of
Clostridium butyricum ferredoxin, on line (1), to show the regions of homology between
the structures of these two proteins. Gaps have been introduced into both sequences to
strengthen homology. The carboxyl terminal portion of spinach ferredoxin was not
homologous to any portion of the C. butyricum ferredoxin, and is therefore compared with
residues 27 through 34 of the spinach ferredoxin sequence. The numbers below the two
lines of sequences show the minimum base difference per codon. No value was assigned by
Matsubara et ale (12) in those positions where gaps are postulated in the sequences.--The average value obtained for the minimum base difference per codon for these alignments
was 1.07, which is interpreted (12) as an indication of evolutionary relationship between
spinach ferredoxin and C. butyricum ferredoxin.
1 20A1a-Phe-Va1-I1e-Asn-Asp-Ser-Cys-Va1-Ser-Cys-G1y-A1a-Cys-A1a-G1y-Glu-Cys-Pro-Va1
20-Leu-Val-Thr-Pro-Thr-Gly-Asn-Va1-Glu-Phe-G1n-Cys-Pro-Asp
1 2 212 0 2 212 1 0 0 1
1A1a-Ala-Tyr-Lys-Val-Thr
o 1 2 1 1
(1)
(2)
(1)
(2)
21 34 4 8Ser-A1a-Ile-Thr-Gln-Gly-Asp-Thr-Gln-Phe-Val-Ile-Asp-Ala-Ile-Asn-Asp-Ser-Cys-21 34 35 39 41Asp-Va1-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Glu-Gly-Ile-Asp-Ile-Pro-Tyr-Ser-Cys-Arg-A1a-212 1 1 1 1 1 1 3 1 2 2 1 1 210 0
(1)
(2)
9 29Va1-Ser-Cys-Gly-A1a-Cys-Ala-Gly-G1u-Cys-Pro-Va1-Ser-Ala-Ile-Thr-Gln-Gly-Asp-Thr-G1n-42 62Gly-Ser-Cys-Ser-Ser-Cys-A1a-G1y-Lys-L~u-Lys-Thr-Gly-Ser-Leu-Asn-Gln-Asp-Asp-Gln-Ser-
1 001 1 0 0 012 2 211 1 1 0 1 022
49-Asn-Cys-A1a-Asn-Val-Cys-Pro-Val
83-Tyr-Pro-Va1100
30Phe-Val-Ile-Asp-Ala-Asp-Thr-Cys-I1e-Asp-Cys-G1y63Phe-Leu-Asp-Asp-Asp-G1n-I1e-Asp-G1u-G1y-Trp-Va1-Leu-Thr-Cys-A1a-A1a-0120121 2 2 1 1 1 1 0 0 2
(1)
(2)
(1)
(2)
50 55G1y-Ala- -Pro-Asn-Gln-Glu84 89Ser-Asp-Val-Thr-Ile-Glu-Thr-
1 1 111 2
27 34(2) Ala-A1a-G1u-Glu-Glu-Gly-Ile-Asp
90 97His-Lys-Glu-G1u-G1u-Leu-Thr-Ala
2 2 0 0 0 2 1 1
N-.:I
28
Figure 7. Amino acid sequences of ferredoxins from
alfalfa, spinach, and Scenedesmus, a green alga. The resi
dues below the spinach ferredoxin sequence at positions 31
and 33 are believed to occupy these positions in a minor
form of the spinach protein. Amino acid residues which are
identical in all three species are underlined.
29
1 5 10 15:1) Alfalfa Ala-Ser-Tyr-Lys-Val-Lys-Leu-Val-Thr-Pro-Glu-Gly-Thr-Gln-Glu-Phe-Glu-C:
(2) Spinach Ala-Ala-Tyr-Lys-Val-Thr-Leu-Val-Thr-Pro-Thr-Gly-Asn-Val-Glu-Phe-Gln-C;
(3) Scenedesmus Ala-Thr-Tyr-Lys-Val-Thr-Leu-Lys-Thr-Pro-Ser-Gly-Asp-Gln-Thr-Ile-Glu-C:
23 30 35 40(1) Tyr-Ile-Leu-Asp-His-Ala-Glu-Glu-Glu-Gly-Ile-Val-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Se
(2) Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Glu-Gly-Ile-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-SeLys Met
(3) Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Al
48 55 60 65(1) Ala-Gly-Lys-Val-Ala-Ala-Gly-Glu-Val-Asp-Gln-Ser-Asp-Gly-Ser-Phe-Leu-Asp-Asp-Asp-GJ
(2) Ala-Gly-Lys-Leu-Lys-Thr-Gly-Ser-Leu-Asn-Gln-Asp-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Asp-G:
(3) Ala-Gly-Lys-Val-Glu-Ala-Gly-Thr-Val-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Ser-G:
73 80 85 go(1) Trp-Val-Leu-Thr-Cys-Val-Ala-Tyr-Ala-Gly-Ser-Asp-Val-Thr-Ile-Glu-Thr-His-Lys-Glu-G
(2) Trp-Val-Leu-Thr-Cys-Ala-Ala-Tyr-Pro-Val-Ser-Asp-Val-Thr-Ile-Glu-Thr-His-Lys-Glu-G
(3) Phe-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Ala-Thr-His-Lys-Glu-Q
29
5 10 15 20-Tyr-Lys-Va1-Lys-Leu-Va1-Thr-Pro-G1u-G1y-Thr-G1n-G1u-Phe-Glu-Cys-Pro-Asp-Asp-Val-
-Tyr-Lys-Va1-Thr-Leu-Va1-Thr-Pro-Thr-Gly-Asn-Val-G1u-Phe-Gln-Cys-Pro-Asp-Asp-Val-
-Tyr-Lys-Va1-Thr-Leu-Lys-Thr-Pro-Ser-Gly-Asp-GIn-Thr-Ile-Glu-~-Pro-Asp-Asp-Thr-
30 35 40 45Ala-G1u-Glu-Glu-Gly-I1e-Val-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ser-Cys-Ser-Ser-Cys-
·Ala-G1u-Glu-Glu-Gly-I1e-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-G1y-Ser-Cys-Ser-Ser-Cys-Lys Met
·A1a-G1u-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Ser-~-
55 60 65 10-Ala-G1y-G1u-Va1-Asp-Gln-Ser-Asp-Gly-Ser-Phe-Leu-Asp-Asp-Asp-Gln-I1e-Glu-G1u-G1y-
-Thr-G1y-Ser-Leu-Asn-G1n-Asp-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Asp-G1n-I1e-Asp-Glu-Gly-
-A1a-G1y-Thr-Va1-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Ser-Gln-Met-Asp-Gly-G1y-
80 85 90 91-Va1-Ala-Tyr-A1a-G1y-Ser-Asp-Val-Thr-I1e-G1u-Thr-His-Lys-Glu-Glu-Glu-Leu-Thr-Ala
-Ala-Ala-Tyr-Pro-Va1-Ser-Asp-Val-Thr-I1e-G1u-Thr-His-Lys-Glu-Glu-Glu-Leu-Thr-A1a
~Va1-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-A1a-Thr-His-Lys-Glu-G1u-Asp-Leu-Phe
I\I
!
f
30
of the molecules, must also be considered. In order to obtain
more information as to the relationships among the three types
of ferredoxins, it will be necessary to determine the amino
acid sequences of ferredoxins from additional species of
plants and bacteria. From such data it may be possible to
determine the evolutionary pattern and the types of mutations
which have occured between species.
Another important aspect of these comparative studies
involves structure-function relationships, since ferredoxin
from all of these sources can function in the photoreduction
of NADP. The functional interchangeability between bacterial
and plant ferredoxins suggests that, despite differences in
properties such as molecular weight, spectrum, and iron and
sulfur content, there is an active site common to all three
groups of ferredoxins. Thus, a comparison of the primary
structures of a greater number of bacterial and plant
ferredoxins may help in elucidation of the nature of this
active site.
Determination of the amino acid sequence of ferredoxin
from Leucaena glauca, a species of small leguminous tree
(Figure 8) abundant in Hawaii, was undertaken in hopes that
the results of this research would contribute some of the
information needed to resolve the as yet unanswered questions
involving structure-function and evolutionary relationships
among the ferredoxins.
31
Figure 8. Photograph of a specimen of Leucaena glauca.
This plant, like all legumes, has seeds in pods. The flower,
which can be seen at top center, appears similar to that of
alfalfa.
33
II. MATERIALS AND METHODS
A. MATERIALS
1. Materials and Reagents Obtained Conunercially:
a •..J. T. BakerChemic"al CO"ITipany; Ne·w ·Jersey
Hydrindantin
Sulfanilic acid
b. Biorad Laboratories, California
Cellex D
AG l-X2 (200-400 mesh)
AG 50-X2 (200-400 mesh)
c. Brown Company, New Hampshire
Solka-Floc
d. California Corporation for Biochemical Research,
California
5-Dimethylamino-l-naphthalene sulfonyl chloride
e. Cyclo Chemical Corporation, California
L-(1-tosylamido-2-phenyl)ethyl chloromethyl ketone
f. Eastman Organic Chemicals, New York
B-Dimethylaminobenzaldehyde
Phenylisothiocyanate
Trifluoroacetic acid
g. Mallinckrodt Chemical WOrks, Missouri
Ethylenediaminetetraacetic acid
h. Matheson, Coleman and Bel1 2 New Jersey
Hydrazine
Hydrazine sulfate
34
i. Nutritional Biochemicals 'Corporation, Ohio
Iodoacetic acid
Trishydroxymethylamino methane
j. Pharmacia Fine Chemicals, Incorporated, New York
Sephadex G-10, G-15 G-25F, G-75F
k. Pierce Chemical Company, Illinois
2,4-Dinitrofluorobenzene
Ninhydrin
Trichloroacetic acid
1. Carl Schleicher and SchuellCompany, New Hampshire
Diethylaminoethyl cellulose
m. Sigma Chemical Company, Missouri
Cellulose
B-Mercaptoethanol
n. Warner-Chilcott Laboratories, California
Silica gel G
o. Worthington Biochemical Corporation, New Jersey
Carboxypeptidase A
Carboxypeptidase B
Chymotrypsin, pancreatic
Trypsin, pancreatic
2. Materials Obtained as Gifts:
a. Thermolysin was a gift from Dr. H. Matsubara,
Space Sciences Laboratory, University of California,
Berkeley.
3. Materials Obtained by 'Prepa.rative Methods:
35
a. Bacillus subtilis neutral protease was isolated
by Dr. James McConn, by the method of McConn,
Tsuru and Yasunobu (37).
b. TLCK-Chymotrypsin was prepared from commercially
obtained chymotrypsin by Dr. James McConn, accord
ing to the method of Mares-Guia and Shaw (38).
c. TPCK-trypsin was prepared from commercially
obtained trypsin according to the method of
Wang and Carpenter (39).
B. METHODS
1. Isolation of Ferredoxin:
The isolation procedure included homogenization
and acetone fractionation by a method adapted from
that of San Pietro and Lang (40). Modifications in
cluded the addition of acetone without prior dilution,
filtration, or centrifugation of the homogenate, and
removal of particulat material from the 35% acetone
treated homogenate by filtration through Solka-Floc
rather than by centrifugation. Chromatography on
columns of DEAE-cellulose and ammonium sulfate
fractionation, carried out by procedures similar to
those of Tagawa and Arnon (41), were followed by
adsorption of the ferredoxin onto DEAE-cellulose in
ammonium sulfate solution (0.5 gram per ml) and
elution with Tris buffer, and gel filtration through
Sephadex G-75 (42). The isolation procedure is
36
summarized in Figure 9.
Six kg of leaves, which had been picked at
various locations in the vicinity of the University
of Hawaii campus and stored in the freezer, were
divided into batches of 500 grams. Each batch was
homogenized in a Waring blender at 4° for 2-10
minutes at high speed with 750 ml 12.5 roM Tris-HCl
buffer of pH 7.2 which was 44 mM in NaCl. Twelve
batches of leaves yielded 15 liters of homogenate.
To each 2 1/2 liters of homogenate were added slowly
and with vigorous stirring 810 ml of acetone of -10°.
Each batch was immediately filtered through a layer
of Solka-Floc in a Buchner funnel at 4°. All partic
ulate matter was retained by the filter and
approximately 2.3 liters of brown filtrate were
obtained per batch of homogenate. To each 2 liters
of filtrate were added, slowly and with stirring,
3.2 liters of acetone at -10°. The flask was packed
in dry ice and the precipitate was permitted to
settle for a few minutes. Most of the supernatant
was then siphoned off and the remaining suspension
was centrifuged at -10° for 5 minutes at 5000 rpm.
All steps after this point were carried out at 4°.
When 12 batches of leaves had been processed to
this point, the precipitates thus obtained were
combined and extracted with 1.2 liters of 0.01 M
Tris buffer of pH 7.3. After removal of the residual
31
Figure 9. Procedure for isolation of Leucaena glauca
ferredoxin.
.~
12 KG. LEAVESt Warino Blender
HOMOGENATE
~ +Acetone (to35%) i FilterFILT RAT Et +Acetone(to 75%)\ DecantiCentrifuge
PRECIPI TATEExtract; DialyzeDEAE-cellulose column (5 x50cm)
Wash: 0.1 ~IO.2M Tris, pH 7.3Elute l 0.5 M Tris,pH-r.3
RED BAND (1650ml)Dilute 2, -foldDEAE 2 (3 x20cm)DEAE 3 (3 x 20 cm)DEAE 4 (3 x 6 cm)
RED BI\ND (85 mt)t + (NH4)2S04 (,O.5gm/ml),Centrifuge
SUPERNATANTDEAE 5 (I x 15 em)
Wash:O.l M Tria, pH7.3, containing0.5 gm/ml(NH4)2S04
Elute: 1M Tris, pH 7.3RED EFFLUENT (35ml)t Sephadex G-75 (4x70cm)PURE FERREDOXIN (240 mg)
t +(N~~S04(O.50m/ml)CRYSTALLINE FERREDOXIN
39
precipitate by centrifugation at 8500 rpm for 10
minutes at 0°, the supernatant was dialyzed over
night against 4 liters of 0.01 M Tris (pH 7.3) at
4°, centrifuged again to remove precipitate which
-f-ormed during dialysis, and then applj.ed to a
column (5 x 50 cm) of DEAE-cellulose which had been
equilibrated with 0.1 M Tris. All Tris buffers
were adjusted to pH 7.3 with HC1. One liter of the
same buffer was then passed through the column to
remove any residual acetone. A dark brown band
could be observed at the top of the column, with a
red band immediately below it. The material was
left at this stage for 24 hours while another 6 kg
of leaves were processed to this point. The material
thus obtained was applied to the same DEAE-cellulose
column. The column was then washed with 2 liters of
0.1 M Tris buffer followed by 1 liter of 0.2 M Tris
buffer. The effluent, at first orange and finally
pale yellow, was discarded. Ferredoxin was eluted
from the column in 1650 ml of 0.5 M Tris buffer.
This solution was diluted 2 1/2 fold, to yield a
Tris concentration of 0.2 M, and applied to a 3 x 20
cm column of DEAE-cellulose. Development of this
and sUbsequent columns followed the procedure used
for the initial column. After chromatography on
another 3 x 20 cm column followed by a 3 x 6 cm
40
column, the ferredoxin was contained in 85 ml of
buffer. Ammonium sulfate (0.5 gram per ml) was
then added. Although the procedure of Tagawa and
Arnon (41) for isolation of spinach ferredoxin
specifies 0.6 gram ammonium sulfate per ml, it was
found that this higher concentration of ammonium
sulfate caused partial precipitation of Leucaena
glauca ferredoxin, whereas when 0.5 gram per m1 was
used no ferredoxin could be detected in the precip
itate. After removal of the precipitate by
centrifugation, the supernatant solution was applied,
directly and without dilution, to a 1 x 15 cm
column of DEAE-ce11u1ose. Passage of 200 ml of 0.1
M Tris buffer containing 0.5 grams ammonium sulfate
per mI. through the column yielded a yellow effluent
which was discarded. Ferredoxin was eluted in 35 ml
of 1 M Tris and applied to a 4 x 70 cm column of
Sephad~x G-75 which was equilibrated and developed
with 0.1 M Tris buffer. Fractions of 16 ml were
collected. The UV and visible spectra of the
effluent fractions were determined by use of the
Beckman DK-2A ratio recording spectrophotometer.
Eight fractions, containing the majority of the
ferredoxin, were combined. Ammonium sulfate (0.5
gram per ml) was added and ferredoxin crystallized
readily. The crystals and supernatant solution were
41
applied to a 1 x 8 cm DEAE-cellulose column and the
ferredoxin was eluted in 15 ml of 1.44 M Tris
buffer of pH 8.6.
2. Preparation of the Carboxymethylcysteine Derivative:
The method of Crestfield, Moore and Stein (43)
was used, with modification of the volume of the
reaction mixture and the time of reduction. To 189
mg of native ferredoxin, dissolved in 8 ml of 1.44
M Tris buffer of pH 8.6, were added 6.4 grams of
twice recrystallized urea and 0.8 ml of a solution
containing 50 mg of EDTA per mI. The resulting
solution was flushed with nitrogen for 10 minutes,
after which 0.27 ml of mercaptoethanol was added.
After an additional 20 minutes of flushing with
nitrogen, the reaction vessel was sealed. The
reduction was permitted to proceed at room tempera
ture for 24 hours, during which the color of the
solution changed from dark red to pale yellow.
Carboxymethylation of the cysteine residues was
accomplished by addition of 947 mg of thrice
recrystallized iodoacetic acid. The reaction
proceeded under nitrogen in the dark for 15 minutes
after which the reaction mixture was applied to a
1.5 x 83 cm column of Sephadex G-25 in 0.02 M
NH4HC03' Fractions of 3.4 ml were collected. The
carboxymethylated protein was located by its
42
absorbance at 277 mlJ. The appropriate fractions
were combined and dialyzed against 3 liters of
deionized water.
3. Methods of Hydrolysis by Endopeptidases:
a. Trypsin
TPCK-trypsin, in which chymotryptic activity
is inhibited, was used. A solution of 83 mg of
CMFd in 3 m1 of deionized water was adjusted to
pH 8.0 by addition of 1 M NaOH. TPCK-trypsin,
equivalent to 1% of the weight of sUbstrate, was
added initially and after 1 hour. The pH was
maintained between 7.9 and 8.2 by periodic addition
of 1 M NaOH. The hydrolysis proceeded at 40 0 for
4 hours and was stopped by application of the
digest to a column of AG 1-X2.
b. Chymotrypsin
A solution of 79 mg of CMFd in 3 to 4 m1 of
deionized water was adjusted to pH 8.0 by addition
of 1 M NaOH. Initially and after 3 hours, 1% (by
weight) portions of TLCK-chymotrypsin (in which
tryptic activity is inhibited) were added. After
9 hours of hydrolysis at pH 8.0 to 8.2 and 400 ,glacial acetic acid was added to lower the pH to
3.0.
c. Bacillus subti1is Neutral Protease
An aliquot of an acetone suspension of the
43
enzyme was centrifuged at 10,000 rpm for 15
minutes at -100 C. The precipitate was dissolved
in 1 ml of buffer of pH 7.1 which was 2 roM in
cacodylate and 2 roM in CaC12, and dialyzed for 4
hours against 2 liters of the same bUffer. The
enzyme concentration was calculated from the
absorbance at 280 m~. Approximately 1 ~mole of
peptide, dissolved in 0.02 M ammonium acetate
buffer of pH 7.1, was hydrolyzed by 2% (by weight)
B. subtilis neutral protease at 300 for times
ranging from 45 minutes to 5 hours. Reaction was
stopped by addition of glacial acetic acid to
lower the pH to 4.5, thus inactivating the enzyme.
d. Thermolysin
This enzyme was used to hydrolyze one of the
large chymotryptic peptides so as to produce
fragments of sizes suitable for sequence determi
nation. The peptide (0.7 ~mole) was dissolved in
1 ml of 0.2 M ammonium acetate buffer of pH 8.2.
Initially and after two hours, portions of
thermolysin equal to 2% of the weight of peptide
were added. The hydrolysis proceeded at 400 for
7 hours after which the digest was dried in a
stream of nitrogen.
4. Methods of Peptide Purification:
a. Chromatographic Separation of Tryptic Peptides
44
The tryptic digest was applied to a column
(2.0 x 107 cm) of AG l-X2 which had been equili
brated with starting buffer. The column was
developed with 360 ml pyridine-collidine-acetic
acid buffer of pH 7.8 (44), a linear gradient
(1800 ml) from the same buffer to 0.4 M acetic
acid, 500 ml of 0.4 M acetic acid, and finally
300 ml of 50% acetic acid. Fractions of
approximately 6 ml were collected and 0.1 ml was
taken from each fraction for ninhydrin assay (45).
b. Chromatographic Separation of Chymotryptic Peptides
The chymotryptic digest, adjusted to pH 3.0,
was applied to a 1.6 x 105 cm column of AG 50-X2,
equilibrated with starting buffer. Elution with
1360 ml of 0.2 M pyridine acetate of pH 3.0 was
followed by a linear gradient (2 liters) from the
same buffer to 2.0 M pyridine acetate of pH 5.0.
The fraction size ranged from 3.6 to 8.2 mI. A
suitable aliquot was taken from each tube for
ninhydrin assay.
c. Further Purification of Tryptic and Chymotryptic
Peptides
The purity of the tryptic and chymotryptic
peptides was determined by amino acid analysis,
paper chromatography in the butanol-acetic acid
water (4:1:5) solvent system (46), hereafter
45
referred to as Solvent I, and thin layer
electrophoresis on cellulose layers in pyridine
acetate buffers of pH 6.5. Peptides which re
quired desalting were passed through a 0.9 x 100
cm column of Sephadex G-15 in 0.02 M ammonium
bicarbonate, deionized water, or 0.2 M acetic
acid. Peptide mixtures remaining after the
initial chromatography were resolved by gel
filtration through Sephadex G-15, by chromatography
on columns of AG 50-X2 in pyridine acetate buffers
or AG l-X2 in pyridine collidine acetate and
acetic acid, or by paper chromatography in the
upper phase of Solvent I or, in case of two large
tryptic peptides, in pyridine-isoamyl alcohol-
0.1 M ammonia (6:3:5) (47), hereafter referred to
as Solvent II.
d. Chromatographic Separation of B. subtilis Neutral
Protease Fragments of Tryptic Peptide T-7a
The digest was lyophilized, dissolved in a
small volume of starting bUffer, and applied to
a column-(0.8 x 57 cm) of AG 50-X2. The column
was developed with 100 ml of 0.2 M pyridine
acetate, pH 3.0, followed by a 300 ml linear
gradient from the same buffer to 2.0 M pyridine
acetate, pH 5.0. Fractions of approximately 2 ml
were collected. From each tUbe, 0.05 ml was taken
46
for ninhydrin assay. Fractions contained in each
ninhydrin positive peak were pooled.
e. Partition Chromatographic Separation of B.
subtilis Neutral Protease Fragments of
Chymotryptic Peptide C-10
The digest was dissolved in a few drops of
the upper phase of Solvent I and applied to a
0.6 x 65 cm column of Sephadex G-25F equilibrated
with the lower phase of the same solvent. The
upper phase was used to develop the column.
Fractions of 1 ml were collected. From each
fraction, one large drop was spotted on filter
paper, dried, and sprayed first with ninhydrin
solution and then with Ehrlich reagent for
tryptophan (48) to determine the pooling of
fractions.
f. Chromatographic Separation of Thermolysin
Fragments of Chymotryptic Peptide C-10
The digest was dissolved in starting buffer
and applied to a 0.6 x 90 cm column of AG l-X2.
Elution with a 120 ml linear gradient from
pyridine-collidine acetate buffer of pH 7.8 to
0.4 M acetic acid was followed by 90 ml of 0.4 M
acetic acid, an 80 ml linear gradient from 0.4 M
to 1.0 M acetic acid, 50 ml 1 M acetic acid, and
finally, 50 ml of 50% acetic acid. An aliquot
41
was taken from each tube for ninhydrin assay and
fractions were pooled accordingly.
5. Determination of Amino Acid Composition:
Samples for amino acid analysis were hydrolyzed
in sealed evacuated tubes in glass distilled constant
boiling HCl containing a small amount of phenol to
prevent oxidation of tyrosine (49). Hydrolysis was
carried out at 1050 for 18 to 96 hours, after which
the HCl was evaporated in a stream of nitrogen. The
residue was dissolved in 0.2 M sodium citrate, pH 2.2.
The Beckman/Spinco Model 120 amino acid analyzer (50)
was used for quantitative determination of all amino
acids except tryptophan, which was determined by the
method of Opienska-Blauth et ale (51). In this pro
cedure, three samples (0.063, 0.090, and 0.135
~moles) of CMFd, were each dissolved in 1 ml deionized
water, and to each were added 2 ml of glacial acetic
acid containing 56 mg ferric iron per ml, followed by
2 ml of concentrated H2S04 (specific gravity 1.84).
The reaction mixtures were shaken, allowed to stand
for 1 hour during which the color developed, and their
absorbances at 545 m~ were read against a blank in the
Coleman Jr. spectrophotometer. The same procedure was
applied to a series of tryptophan samples of varying
concentrations. Thus a standard curve was obtained
from which the amount of tryptophan in the protein
lt8
samples could be determined.
6. Methods of Amino Acid Sequence Determination:
a. Methods of NH2-terminal Analysis
1. Edman Degradation
The phenylisothiocyanate procedure of
Edman (52), as modified by Konigsberg and
Hill (53), was used in a sequential manner
to determine most of the amino acid sequence
of the peptides. The coupling reaction was
carried out at 40° for 2 hours in
N-ethylmorpholine acetate buffer of pH 8.8.
The reaction mixture was dried in a stream
of nitrogen at 40°. After extraction of the
excess phenylisothiocyanate into benzene,
the residue was again dried thoroughly.
Anhydrous trifluoroacetic acid (0.1 to 1 ml)
was added and the cyclization reaction was
allowed to proceed at room temperature for
1 hour. After evaporation of the
trifluoroacetic acid in a stream of nitrogen
at room temperature, the residue was dissolved
in 1 ml O.lt M acetic acid. In some cases this
solution was then maintained at 40° C for 10
minutes (54). The PTH-amino acid was extracted
into two 1 ml portions of benzene and in some
cases was identified by thin layer
chromatography on layers of silica gel G in
the solvent systems of Randerath (55). In a
few instances the PTH-amino acid was hydrolyzed
in vacuo in constant boiling HCl at 1500 C
for 16 hours (56) and the regenerated amino
acid was then identified on the amino acid
analyzer. However, the method used to the
greatest extent was the subtractive procedure
of Konigsberg and Hill (53), whereby the NH2
terminal residue is identified by its disap
pearance from the amino acid analysis of the
peptide after Edman degradation. After
extraction of the PTH-amino acid, an aliquot
is taken from the aqueous phase which contains
the residual peptide. This aliquot is
hydrolyzed in HC1 and subjected to amino acid
analysis. In the case of acidic peptides which
were not very soluble in acetic acid, the
aqueous phase was dried at 40 0 C in a stream
of nitrogen and the residue was dissolved in
the N-ethylmorpho1ine buffer for the next
stage coupling reaction before an aliquot was
taken for analysis. This procedure was also
useful when PTH-arginine was present.
PTH-arginine is incompletely extracted from
the aqueous phase (54) and is hydrolyzed by
50
HCl to yield arginine, which thus appears in
the analysis of the residual peptide. How
ever, when the sample is first dissolved in
the basic N-ethylmorpholine buffer, the
PTH-arginine decomposes and is eliminated as
an artifact in the analysis.
2. Dinitrophenylation
The procedure used was that of Sanger
(57) as modified by Fraenkel-Conrat (58).
The DNP-amino acids were identified by thin
layer chromatography on layers of silica gel
G as described by Randerath (59).
3. Dansylation
The reaction of dansyl chloride (5
dimethylamino-l-naphthalene sulfonyl chloride)
with amino groups to yield a fluorescent
derivative and the hydrolysis in constant
boiling HCl, which releases the DNS-amino
acid from the NH2-terminal position in a
peptide or protein, were carried out as
described by Gray (60). The DNS-am1no acids
were identified by thin layer chromatography
on layers of silica gel G in the solvent
systems of Deyl and Rosmus (61).
b. Methods of COOH-terminal Analysis
1. Hydrazinolysis
51
The method of Bradbury (62) was used,
with minor modifications. To 0.02-0.03 pmole
peptide were added 26 mg hydrazine sulfate
and 0.2 ml 96% hydrazine. The reaction was
carried out in a sealed, evacuated tube for
16 hours at 60°. After the sample had been
dried in a stream of nitrogen, 1 ml of 0.2 M
sodium citrate buffer of pH 2.2 was added.
The pH was adjusted to 2.2 by dropwise addi
tion of 6 M HCl, and the sample was then
analyzed directly on the amino acid analyzer.
2. Carboxypeptidase A
The method used for carboxypeptidase A
digestion was a modification of that of
Harris (63). To 1 ml of cold water were
added 10 pI of 1 M NaOH and 1 drop of an
aqueous suspension of the enzyme. The high
pH caused immediate solublization of the
enzyme. Eight pI of 1 M HCl were added to
lower the pH, followed by 0.6 ml of 0.02 M
NH4HC03 (pH 8-9). The concentration of the
enzyme was determined spectrophotometrically
(E~18 mp = 19.4). To the peptide or protein,
dissolved in 0.02 M NH4HC03
, was added an
aliquot of enzyme solution such that the
weight ratio of substrate to enzyme was 20:1.
52
The reaction mixture was incubated at 40°.
At various time intervals, aliquots were
taken and pipetted into 0.2 M sodium citrate
buffer of pH 2.2, thus inactivating the
enzyme. The amino acid analyzer was used to
quantitatively determine the amino acids
released.
3. Carboxypeptidase B
This enzyme was used to release lysine
and arginine from the carboxyl terminal
positions of tryptic peptides. The method
used was similar to that for carboxypeptidase
A, except that solublization is not required.
The weight ratio of enzyme to substrate varied
from 2 to 3%. Digests were carried out in
0.1 M sodium phosphate buffer of pH 8.0, at
40° for 2 to 8 hours.
7. Studies on Ferredoxin from Individual Trees:
Leaves were gathered from ten individual L. glauca
trees at various locations on the island of Oahu - in
Manoa Valley, on Round Top, and on Mount Tantalus.
Ferredoxin was isolated separately from each tree and
carboxymethylated as previously described. After
passage through a column of Sephadex G-25, the CMFd
was divided into three portions, for amino acid
analysis, hydrolysis by carboxypeptidase A, and
hydrolysis by trypsin. Acid hydrolysis and amino
53
acid analysis were performed as previously described.
Carboxypeptidase digests were carried out for five to
ten hours at 40° with an enzyme:substrate ratio of
1:20. Tryptic digests were performed in 0.02 M
NH4HC03
at 40° for one to seven hours with 2% (by
weight) TPCK-trypsin. After drying in a stream of
nitrogen, each tryptic digest was dissolved in a
minimal amount of starting buffer and applied to a
0.6 x 36 cm column of AG l-X2. The column was
developed with 6 ml of pyridine-collidine-acetic acid
buffer of pH 7.8, followed by a 60 ml linear gradient
from the same buffer to 0.4 M acetic aCid, then 6 ml
of 0.4 M acetic aCid, and finally 50% acetic acid.
Fractions of approximately 2 ml were collected, and
an aliquot was taken from each fraction for ninhydrin
assay. Fractions within each ninhydrin-positive peak
were pooled and aliquots were taken for amino acid
analysis.
54III. RESULTS
A. Isolation of Ferr~doxin
The yield of ferredoxin from the purification pro
cedure described was 20 mg per kg of leaves. A
photomicrograph of ferredoxin crystals is shown in
Figure 10. Figure 11 shows the spectrum of the native
protein. The ratios of absorbances at 463, 420, and
325 m~ to that at 277 m~ are 0.43, 0.49, and 0.65,
respectively, as compared with 0.43, 0.48, and 0.65 for
the corresponding maxima in the spectrum of alfalfa
ferredoxin.
B. Preparation of the Carboxymethylcysteine Derivative
The pattern of elution of the carboxymethylation
reaction mixture from a 1.5 x 84 cm column of Sephadex
G-25F is shown in Figure 12. The yield of carboxymethylated
ferredoxin (CMFd) was 15.6 ~moles, or 100%. The molar
absorptivity index, E, at 277 In~ was 1.59 x 103 cm-l , with
the protein concentration based on amino acid analysis.
C. Amino Acid Composition of Ferredoxin
The results are summarized in Table I. Each value
is the average of those obtained from analyses of duplicate
hydrolysates of CMFd. Half-cystine was determined as
carboxymethylcysteine. The amino acid compositions of
ferredoxins from spinach (13), alfalfa (35), and
Scenedesmus (36) are included in the table for comparison.
Tryptophan determinations carried out according to the
55
Figure 10. Photomicrograph of L. glauca ferredoxin
crystals. The photograph was taken at l500X magnification.
iI
Figure 11. Absorption spectrum of ~. glauca ferredoxin. Measurements were made on
a Beckman DK-2A ratio recording spectrophotometer. The solvent was 0.1 M Tris-Hel buffer
of pH 1.3.
V1~
U)
d10 q- rt> "!d d d 0
3::>N\f8~OS8\f 31\I.l\f13~
.o
o(\Jq-
oen(\J
--
59
Figure 12. Purification of CMFd by gel filtration
through a column of Sephadex G-25. The eu1tion pattern shows
the separation of CMFd, which was detected by its absorbance
at 276 m~, from the carboxymethy1ation reagents after
passage through a 1.5 x 84 cm column of Sephadex G-25F in
0.02 M NH4HC03.
17070 100 135
......... -- ~-- ..... -.- ~ ----- -- -----
-
f-
~
-
l-
• CMFd\J
.~4 • • •
1.4
0.2
0.0
35
1.2::L
Ew \.0l"-N
r-c::x: 0.8wuz
0.6c::x:CD0::0(f)
0.4CD<t
EFFLUENT ml.
61TABLE I
Amino Acid Compositions of Ferredoxins fromL. glauca and Other Plants
AminoL. glauca
from from Alfalfa Spinach ScenedesmusAcid Analyses Sequence
residuesLysine 5.10 5 5 4 4
Histidine 0.91 1 2 1 1
Arginine 1.91 2 1 1 1
Aspartic acid 10.55 10+ 8 11 12
Asparagine 0 1 2 0
Threonine 3.94 4 6 8 10
Serine 6.72 7 8 7 8
Glutamic acid 16.5 12+ 13 9 6
Glutamine 4 3 4 4
Proline 4.50 4+ 3' 4 4
Glycine 6.87 6+ 7 6 7
Alanine 6.91 7 9 9 10
Half-cystine 4.98 5 5 5 6
Valine 6.61 6+ 9 7 5
Methionine 0.0 0 0 0 1
Isoleucine 3.87 4 4 4 3
Leucine 8.98 9+ 6 8 7
Tyrosine 2.73 3 4 4 4
Phenylalanine 3.11 3 2 2 3
Tryptophan 0.92 1 1 1 0
Total 96 97 97 96
62
method of Opienska-B1auth et a1. (51) gave the data in
Table II, which includes values obtained with tryptophan
samples of known concentration and the results from three
samples of CMFd. The value obtained for CMFd, 0.92
residue of tryptophan per molecule, was an average of the
results of the three determinations.
D. Amino and Carboxyl Terminal Residues
Dansy1ation (60) yielded only DNS-a1anine, which was
identified by thin layer chromatography on silica gel G,
in the solvent system, benzene, 16: pyridine, 4: glacial
acetic acid, 1, of Dey1 and Rosmus (61). Hydrolysis of
CMFd by 5% (by weight) carboxypeptidase A at 40° was
carried out for at total of five hours, with a1iquots
taken periodically. The results are shown in Figure 13.
E. Tryptic Peptides
1. Initial Chromatography
The elution pattern of the tryptic digest from
AG 1-X2 is shown in Figure 14. Peptide numbers were
assigned according to the order of the peptides in
the sequence (rather than according to elution
position); thus T-1 is the amino terminal tryptic
peptide. The fractions represented by each
ninhydrin-positive peak were pooled as indicated.
The results of paper chromatography, paper electro
phoresis, and amino acid analyses indicated that
peptides T-1, T-3a, T-3b, T-5, T-7a, and T-7b did not
TABLE II
Tryptophan Content of L. glauca Ferredoxin
Sample llgrams pmolesAbsorbance at llgrams
545 mu vs. blank tryptophan(calculated)
Molestryptophanper mole
of protein
Tryptophan 0.00 -----
" 4.08 0.008
" 8.16 0.018
" 16.3 0.040
" 24.5 0.060
" 40.8 0.096
CMFd 0.0632 0.028 11.8 1.00
" 0.0900 0.035 14.8 0.88
" 0.135 0.052 22.0 0.88
0\W
Figure 13. Action of carboxypeptidase A on CMFd. The amount of each amino acid
released by the exopeptidase is plotted against time. Leucine (--0- ---- ~ ---),
threonine ( --0 0), glycine (- .. - - -), and alanine
~ -A- - - 04- - -) were released from the protein by carboxypeptidase A.
'".l=
.<1o a
\ ~ 1I 'w
\~ ~I 'z,-u
lz
w \~~
1«,.-J -<!>\
en,« Q)-~ ~~
:::s
~ ~ 0 c
~, r() E
\ \-
~\w
\ \ :iE
\ \t-
, 1,.~ LO
~\ \~~ \"
"~ "-,.., ~,
\ "4.-_---0°0 0
0 LO 0• .
00
N131.0tkJ .::10 310~ ~3d S310~
Figure 14. The elution pattern of the tr~ptic digest of CMFd from AG l-X2. CMFd~
was hydrolyzed by trypsin and the resulting peptide mixture was chromatographed on a
2.0 x 107 cm column of AG l-X2 with pyridine-collidine-acetic acid buffers. The peptides
were detected in the eluate by ninhydrin analysis of an aliquot from each fraction. The
absorbance at 570 mp represents the color obtained by ninhydrin reaction. The bars on
the abscissa indicate the fractions pooled.
0"10"1
o -fI"---~I-
.a,.------I-
EIt)
l- t!- t-Z 0 ZW W- ~C ..J« I.L0: I.LC)
LLI
~---===============-:-u +~
~ ~:I:
-oe~
o
CX)r--::I:0. _
~ t!-« '}I~====-----o
C1- f's;:::============::;:===:;::=--_1-.s.:::......LIt)---~o::-----~It):------:(\J.----~It)~
~ a ~ d d(rfwOL9) 30N'fe~OS8'f 3/\ I.L'f13 ~
68
require further purification.
-2~ Further P~rification of Tryptic Peptides
Peptide T-2 was first desalted and then
chromatographed on a 1.0 x 58 cm column of AG 50-X2
(200-400 mesh). Elution with a 300 ml linear gradient
from 0.2 M pyridine acetate of pH 3.0 to 2.0 M
pyridine acetate of pH 5.0 yielded two ninhydrin
positive peaks, as shown in Figure 15. The major
component was peptide T-2, whereas the minor com
ponent was found to be identical in composition to
T-l.
The material eluted from the original AG l-X2
column with 50% acetic acid, when concentrated to
dryness in vacuo and subjected to partition
chromatography on Whatman 3 MM paper in Solvent II,
gave four ninhydrin positive bands. The Rf values
were 0.15, 0.30, 0.37, and 0.45, and the visually
estimated relative intensities of ninhydrin color
were +5, +4, +5, and +3, respectively. After elution
of the peptides from the paper in 0.02 M NH4HC03
,
aliquots were taken for amino acid analysis. The
results showed that the two slower moving bands were
peptide T-4, whereas the two bands of higher Rf
corresponded to peptide T-6.
3. Amino Acid Composition
Table III shows the amino acid compositions,
69
Figure 15. Purification of tryptic peptide T-2 on
AG 50-X2. This peptide was separated from a lesser amount
of T-l by ion exchange chromatography on a 1.0 x 58 cm
column of AG 50-X2. Elution was performed with a 300 ml
linear gradient from 0.2 M pyridine acetate of pH 3.0 to
2.0 M pyridine acetate of pH 5.0. Ninhydrin assay was used
to detect the peptides.
E 0.4o~lO
~wuZ<t~ 0.3oenCD«w>-....<t-ILaJa::
180
T-2
200 220 240 260
EFFLUENT VOLUME (mU
71
TABLE III
Amino Acid Composition and Properties of Tryptic Peptides
Amino Acid
Lysine
Histidine
Arginine
CM-cysteine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Tryptophanb
Total residues
Yield (JlIDoles)
Purificationd
Rfe
fElectrophoresis
T-l
0.95(1)
0.22
0.20
1.00(1)
3
3.15
0.28
-0.61
T-2
residuesa0.92(1)
1.08(1)
2
3.88
AG 50
0.16
-0.73
T-3a
0.90(1)
0.97(1)
0.91(1)
2.24(2)
1.04(1)
1.94(2)
8
1.61
0.34
0.0
72TABLE III, continued
Amino Acid T-3b T-4 T-5
residuesaLysine 0.94(1) 1.10(1)
Histidine
Arginine 0.9(1)
eM-cysteine 1.5(2)c 1.97(2)
Aspartic acid 1. 08(1) 3.9(4)
Threonine 1.01(1) 0.2
Serine 1.0(1) 2.71(3)
Glutamic acid 5.3(5)
Proline 1.21(1) 2.0(2)
Glycine 1.10(1) 1.3(1) 2.17(2)
Alanine 0.77(1) 1.1(1) 2.06(2)
Valine 0.95(1) 1.1(1)
Isoleucine 2.0(2)c
Leucine 1.20(1) 3.1(3)
Tyrosine 1.9(2)
Phenylalanine 1.0(1)
Tryptophanb
Total residues 8 26 10
Yield (flmo1es) 1.54 2.41 3.75
Purificationd PC
R e 0.21 (0.30) 0.02f
E1ectrophoresis f 0.0 ----- +0.29
73
TABLE III, continued
Amino Acid T-6 T-7a T-7b
residuesaLysine 1.00(1) 0.91(1)
Histidine 0.93(1) 0.85(1)
Arginine 0.8(1)
CM-cysteine 1.1(1)
Aspartic acid 5.2(5) 1.01(1) 1.11(1)
Threonine 0.8(1) 1.77(2) 1. 79(2)
Serine 1.5(2) 0.98(1) 1.11 (1)
Glutamic acid 6.9(7) 3.76(4) 4.10(4)
Proline 1.2(1)
Glycine 1.8(2) 0.88(1)
Alanine 1.8(2) 0.98(1)
Valine 2.1(2) 1.58(2)c 1.83(2)
Isoleucine 1.3(1) 1.09(1) 0.91(1)
Leucine 3.7(4) 1.18(1) 1.02(1)
Tyrosine 1.3(1)
Phenylalanine 1.2(1)
Tryptophanb (1)
Total residues 32 15 15
Yield (J-lmo1es) 2.75 2.95 2.01
Purificationd PC
R e (0.37) 0.15 0.07fE1ectrophoresis f ----- +0.47 +0.14
TABLE III, continued
aResidue values calculated from amino acid analysis data
are given, followed by the assumed number of residues in
parentheses.
bDetermined by Ehrlich reaction.
cValues obtained after 96 hours of hydrolysis.
dpC refers to paper chromatography in Solvent II.
AG 50 refers to ion exchange chromatography on columns of
AG 50-X2.
eparentheses indicate Solvent II. All other values refer
to Solvent I.
fMovement at pH 6.5 relative to alanine (=0), lysine (=-1),
and glutamic acid (=+1).
75
yields, Rf's, electrophoretic characteristics, and
purification procedures for the tryptic peptides.
Tryptophan was determined qualitatively by Ehrlich
reaction (48) since only one residue of this amino
acid is present in the protein. All composition data
were from 22 hour hydrolysates, except for those
indicated in the table to be values obtained after 96
hours of hydrolysis. Amino acids present in analyses
in amounts less than 0.15 residue are not included in
the table.
4. Amino Acid Sequence
The data are expressed in table form, rather than
the usual text form, wherever the ease of understand
ing the data and the interpretation thereof could be
enhanced by this form of presentation. The amino
acid sequence of each peptide is given, with the
residues of which the sequence has been determined
separated by hyphens, whereas unsequenced sections
are enclosed in parentheses with the residues therein
separated by commas.
The methods which were used are indicated in the
sequence given for each peptide. Arrows to the left
and right represent the use of carboxypeptidase A or
B and Edman degradation, respectively. A broken
arrow indicates dinitrophenylation. Dansylation and
hydrazinolysis are represented by single and double
underlinings, respectively. Aligned immediately
76
below each peptide sequence, the amino acid composition
is given for comparison with the sequence data. For
those amino acids which appear more than once in the
same peptide, the residue value is given to correspond
only with the first appearance of that amino acid.
Step 1, Step 2, etc. refer to the results of Edman
degradation. A dash in this data indicates that the
corresponding amino acid was not determined in that
step. The amino acid disappearing at each step is
indicated by underlining of the residue value. Values
less than 0.1 are not included except as required to
show the disappearance of a residue. Carboxypeptidase
and hydrazinolysis data are expressed as residues of
amino acid liberated, based on the amount of peptide
used.
T-l: ~-Phe-kYs
Composition: 1.1 1.0 1.0
Step 1: 0.3 1.1 1.0
Carboxypeptidase B (2 hours): Lys 0.56.
T-2: Val-Lys-'7
Composition: 1".1 0.9
Step 1: 0.0 1.0
T-3a: Leu-Leu-Thr-Pro-As~-Gl~Pro-Lys--,.~ ~ ---'7
Composition: 1.9 0.9 2.2 1.0 1.0 0.9
Step 1:
Step 2:
1.0
0.2
1.0 1.9 1.1 1.1
1.0 1.9 1.0 1.1
--
77
Step 3: 0.1 0.2 2.0 1.0 1.0
Step 4 : 1.0 0.9 1.1
Step 5: 1.0 0.3 1.0
Step 6: 1.0 0.4 0.6
Direct identification in 5th step: PTH-Asp.
Carboxypeptidase B (20 hours): no residues liberated.
Lysine was placed at the COOH-termina1 according
to the specificity of trypsin.
T-3b: Val-Leu-Thr-Pro-As~-G1y-A1a-kYs~ -., --""'7~ -
Composition: 1.0 1.2 1.0 1.2 1.1 1.1 0.8 0.9
Step 1: 0.0 1.0 0.9 1.1 1.0 1.2 0.8
Step 2: 0.2 0.9 1.1 1.0 1.1 0.9
Step 3: 0.2 1.0 1.0 1.2 0.8
Step 4: 0.2 0.3 1.0 1.2 0.8
Step 5: 0.4 1.1 0.9
Direct identification in 5th step: PTH-Asp.
Carboxypeptidase B (8 hours): Lys 0.54.
--Hydrazino1ysis of residual peptide: Ala 0.97.
T-4: ~-Phe-G1x-(CMCYS2,ASx4_,Ser,G1x3+,Pr02,G1Y,
A1a,Va1,I1e2,Leu3,Tyr2)-~rg
Edman degradation - direct
identification in 1st step: PTH-G1u
Dansy1ation: DNS-G1u
Dansy1ation after step 1: DNS-Phe
Dansy1ation after step 2: DNS-G1u
Carboxypeptidase B (2 hours): Arg 0.71
78
T-5: A1a-G1~-Ser-CMCys-Ser-Ser-CMCYS-A1a-G1y-kYs--'? --'7 7 -,~ 7 ----'7
Composition: 2.1 2.2 2.7 2.0 1.1
Step 1: 1.1 2.2 2.8 1.8
Step 2: 1.1 1.3 2.1 2.0
Step 3: 1.0 1.1 1.9 1.8
Step 4 : 1.0 1.1 1.1 1.2
Step 5 : 1.0 1.0 1.2 1.1
Step 6: 0.9 1.1 0.4 0.8
Step 1 : 0.9 1.1 0.2 0.2
Step 8: 0.4 1.0 0.3 0.3
Carboxypeptidase B (1 hours): Lys 0.85.
Hydrazino1ysis of residual peptide: G1y 0.72.
T-6: ~-~-G1X-(CMCys,AsP5,Thr,Ser2,G1U6,Pro,
G1Y2,A1a2,Va1,I1e,Leu3,Tyr,Phe,Trp)-Arg
Step 1: Leu) Identified as free amino acids)
Step 2: Val) after acid hydrolysis of
pheny1thiohydantoin.
Dansy1ation after step 1: DNS-Va1
Dansy1ation after step 2: DNS-G1u
Arginine was placed at the COOH-termina1 according
to the specificity of trypsin.
T-1a: Ser-As~-Va1-Va1-I1e-G1u-Thr-His-LYS-G1u--, ---'7 --;J'~
Composition: 1.0 1.0 1.6 1.1 3.8 1.8 0.9 1.0
Step 1: 0.2 1.0 1.8 1.2 4.1 1.8 0.9 0.6
Step 2: 0.3 1.9 0.9 4.0 1.8
Step 3: 1.1 1.0 4.0 1.9
79Step 1.1 : 0.1 1.0 1.1.1 1.8
Step 5: 0.4 3.9 1.8
T-7a (cont.) Glu-Glu-Leu-Thr-Gly
Composition (cont.) 1.2 0.9
Step 1: 1.0 1.1
Step 2 : 1.0 1.1
Step 3: 1.0 1.1
Step 4 : 1.0 1.1
Step 5 : 1.0 1.3
Values for valine and isoleucine in the first 3
steps are those obtained after 96 hours of hydrolysis.
Direct identification in 2nd step: PTH-Asp
Hydrazinolysis: Gly 0.1.13
The data used to establish the sequence of T-7a are
summarized in Fig. 16.
Hydrolysis of T-7a with Bacillus subtilis neutral
protease (2% by weight) for 1 hour yielded 3 peptides
which were then separated on AG 50W-X2.
Step 5 (direct analysis): 0.0
1.1 1.0
0.9 1.0
0-.9 1.0
1.0 1.0
0.0 1.0
0.2 1.1
ser-As~-Val-Val-Ile-G1U~ ---:;:>' ---::0" -.,..
0.9 1.0 1.9 1.0 1.1
0.1 1.0 1.9
0.4 1.8
T-7a-BSNP-l (59%):
Composition:
Step 1:
Step 2:
Step 3:
Step 4:
80
Figure 16. Summary of evidence establishing the se
quence of tryptic peptide T-7a. The fragments obtained by
hydrolysis with B. subtilis neutral protease are BSNP-l,
BSNP-2, and BSNP-3. The arrows to the right above the se
quence represent Edman degradation steps performed on the
fragments, while those below the sequence refer to degrada
tion of T-7a. The amino terminal sequence of T-7a is the
same as that of BSNP-l; therefore BSNP-l is the amino
terminal fragment of T-7a. BSNP-3 must be the carboxyl
terminal fragment of T-7a, since hydrazinolysis released
only glycine from the latter. The placement of BSNP-2 thus
becomes unequivocal.
-d.z83
2zenm
~zenm
'1111:fa1~",__1
.a(!)
1
1<31~
1
1'§'I
1:f1.!.~
82
The values for Val and Ile in the composition and
in the first 3 steps of Edman degradation were
obtained after 96 hours of hydrolysis.
T-7a-BSNP-2 (76%): Thr-His-Ly~-Glu-Glu-Glu~ --:?' --:7' ~
Composition: 0.9 1.0 0.9 3.3
Step 1: 0.2 0.9 0.8 3.3
Step 2: 0.3 0.6 3.2
Step 3: 0.2 0.2 3.0
Direct identification in 4th step: PTH-Glu
Direct identification in 5th step: PTH-Glu
Direct analysis of residue after 5th step: Glu only
T-7a-BSNP-3 (79%):
Composition:
Step 1:
Step 2:
Leu-Thr-Gly~~
1.0 1.0 1.1
0.2 0.9 1.1
0.1 1.0
T-7b had an amino acid composition identical to that
of T-7a, except for the presence of an alanine residue
in place of the sole glycine residue of T-7a.
T-7b: Ser-(Asp-Val-Val-Ile-Glu-Thr-His-Lys-Glu-----;7
Composition: 1.1 1.1 1.8 0.9 4.1 1.8 0.9 0.9
Step 1: 0.4 1.1 2.0 0.9 4.0 2.0 0.9 0.9
T-7b (cont.): Glu-Glu)-Leu-Thr-Ala~
Composition (cont.):
Step 1:
1.0
1.1
1.0
1.0
83
The values for valine are those obtained after 96
hours of hydrolysis.
Carboxypeptidase A (4 hours, 40°): Ala 0.8S.
T-7b was hydrolyzed by B. subtilis neutral protease
under the same conditions used for T-7a and the
sequence of the alanine containing COOH-terminal
fragment was determined.
T-7b-BSNP-3 (8S%): Leu-Thr-Ala--;i" --;i"
Composition: 1.0 1.0 1.0
Step 1: 0.1 1.0 1.0
Step 2: 0.1 1.0
The sequence of the residues in parentheses in T-7b
is based on analogy with the structure of T-7a.
F. Chymotryptic Peptides
1. Initial Chromatography
The elution pattern of the chymotryptic digest
from AG SO-X2 is shown in Figure 17. Peptide contain
ing fractions which were pooled are indicated.
Peptides C-2, C-3, C-6, and C-10 did not require
further purification, as indicated by amino acid
analyses.
2. Further Purification of Chymotryptic Peptides
Chromatography of C-l on a column of AG l-X2
yielded a single ninhydrin positive peak which proved
to be the pure peptide. Gel filtration through a
column of Sephadex G-1S in 0.2 M acetic acid separated
peptide C-4 from contaminating material which was
Figure 17. Elution pattern of the chymotryptic digest of CMFd from a column of AG
50-X2. CMFd was hydrolyzed by TLCK-chymotrypsin and the resulting peptides were
fractionated by chromatography on a 1.6 x 105 cm column of AG 50-X2 in pyridine acetate
buffers. The bars on the abscissa indicate the fractions pooled under each ninhydrin
positive peak.
00J::"
C-sC-IO0, I
~ 0t:'v " ·':1t:= ·'1' ~ •
~ 250 350 450 500 850 950 1450 1550 1650U')
l-
I C-4«f'_Q 1\
~ 0.2
~~ 0.1
V \. C-13 C-2 C-3en I C-8 Im« 0 • , • '2350 2450 II 30501950 2150 3150 3250
EFFLUENT VOLUME (m I )
86
present in minor amount. The elution pattern is
shown in Figure 18 a. Peptide C-5 was also obtained
in pure form after gel filtration. The elution pattern
is shown in Figure 18 b. C-7, C-12, and c-14 were
resolved by chromatography on AG l-X2, as shown in
Figure 19. C-7 was separated into two peaks, C-7a and
C-7b. c-8 was obtained in pure form after chromatography
on AG l-X2. C-9 yielded a single asymmetric ninhydrin
positive peak on gel filtration through Sephadex G-15,
as shown in Figure 20 a. However, paper chromatography
of aliquots taken from fractions in the region of
asymmetry showed two sets of ninhydrin positive spots
of different Rf , the relative intensities of which are
plotted in Figure 20 b. Peptide C-9 was then obtained
in pure form by pooling fractions as shown. Peptide
C-ll was separated from minor contaminants by gradient
elution from a column of AG l-X2.
3. Amino Acid Composition
Table IV shows the amino acid compositions, yields,
Rr's, electrophoretic characteristics, and purification
procedures for the chymotryptic peptides. Samples for
amino acid analysis were hydrolyzed for 22 hours, except
where 96 hours of hydrolysis is indicated in the table.
The only Ehrlich positive peptide, C-IO, contains the
sole tryptophan residue of the protein.
4. Amino Acid Sequence
87
Figure 18 a. Purification of peptide c-4 by gel
filtration. This peptide was passed through a 1 x 100 em
column of Sephadex G-15 in 0.2 M acetic acid and effluent
fractions of 1.5 ml were collected. Two ~l from each frac
tion were spotted onto filter paper and sprayed with a 0.2%
solution of ninhydrin in acetone. The intensity of the blue
color which developed on heating gave the pattern shown when
plotted on a a to +5 scale. The UV spectrum of each fraction
was also measured. The phenylalanine containing c-4 was in
fractions 19 through 24 (indicated as pooled) and tyrosine
containing contaminants in the relative amount of 14% were in
the earlier ninhydrin positive fractions.
+5
a::0--l0 +4u
z-a::o·>- +3:cz-zw> +2- C-4~<t--lW0:
+1
10 15 20 25
FRACTION NUMBER
"~.
89
Figure 18 b. Purification of peptide C-5 by gel
filtration. This peptide was obtained in pure form in the
fractions indicated after passage through a 1 x 218 cm
column of Sephadex G-15 in deionized water. The tyrosine
containing C-5 was readily detected by its absorbance at
280 mu.
0.0 ""--&.-.. .....L-..L-__..L..-L --J
30 40 50
EFFLUENT VOLUME (mil
\DI-'
Figure 19. Resolution of peptides C-7a, C-7b, C-12, and C-14 on a column of AG 1-X2.
The elution pattern was obtained by ninhydrin assay of a1iquots from the effluent frac
tions from a 1.0 x 55 cm column of AG 1-X2. Elution by a 200 m1 linear gradient from,
pyridine-collidine-acetic acid buffer of pH 7.8 to 0.4 M acetic acid was followed by
elution with acetic acid as indicated. Peaks which are not numbered did not contain
significant amounts of peptide material, as determined by amino acid analysis.
C-12
C-7a
C-7b
C-14
OJ2
IIlIf GRADIENT >1< 0.4 M ACETIC ACID
0.0a
0.04
0.00 I J ! ,-,,,, .£ ,....,C"""'!-, , L - '=' ," I60 100 150 200 260 320 380
E~lO
~I.LJ()Z«CD0::oCJ)
CD«I.LJ>-l-e:(...JIJJ0::
EFFLUENT VOLUME (mU
93
Figure 20 a. Pattern of elution of a mixture containing
peptide C-9 from Sephadex G-15. The effluent fractions from
a 1 x 100 cm column of Sephadex G-15 in 0.2 M acetic acid
were assayed by spotting on filter paper, spraying with
ninhydrin solution, and heating to develop the color. The
intensity of the blue color was then rated visually on a a
to +9 scale and plotted against fraction number. Incomplete
resolution of the components of the peptide mixture is
indicated by the asymmetry of the ascending portion of the
peak.
3020O-----------.l"-----~
10
+1
+9
a:: +80.-J0u +7z-a:: +6c>-::I:
+5z-Z
IJJ +4>-J- +3«.-Jwa:: +2
FRACTION NUMBER
95
Figure 20 b. Distribution of peptide C-9 and a con
taminant in the effluent fractions from Sephadex G-15, as
determined by paper chromatography. A1iquots from fractions
in the region of asymmetry in the peak shown in Figure 20 a
were chromatographed on Whatman #1 paper in butano1-pyridine
water (1:1:1). Ninhydrin color was developed as previously
described. Two rows of ninhydrin positive spots of different
Rf values were observed. Their intensities were plotted
separately on a 0 to +5 scale as shown. Fractions 22 through
26 were pooled, as indicated by the bar on the abscissa, to
yield peptide C-9 in pure form.
a::o<3 +5uziE +4o>-:r:6 +3z
w2:: +2I-<{.-JWa:: +1
I \I \
I \
o LL-L...L..::'~=:::J:::::L---J
15 20 25 30
FRACTION NUMBER
97TABLE IV
Amino Acid Composition and Properties of Chymotryptic Peptides
Amino Acid
Lysine
Histidine
CM-cysteine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
PhenylalaninebTryptophan
C-l
1.04(1)
0.96(1)
C-2
aresidues1.89(2)
0.2!4
0.23
0.12
1.12(1)
0.29
C-3
1.92(2)
1.24(1)
c-!4
0.84(1)
0.98(1)
....-·0.92(1)
0.24
1.10(1)
1.!44(1.!4)
1.08(1)
0.58(0.6)
1.06(1)
Total residues 2
Yield (pmoles) 1.45
Purificationd AG 1
Rf in Solvent I 0.62
Electrophoresise 0.0
3
0.53
0.15
-0.92
5 8
0.40 3.90
G-15
0.22 0.26
-0.86 +0.22
98TABLE IV, continued
Amino Acid C-5 c-6 C-7a C-7b
residuesaLysine
Histidine
Arginine
CM-cysteine 0.92(1)
Aspartic acid 2.02(2) 1.07(1) 0.99(1)
Threonine
Serine 0.19 0.20 0.12
Glutamic acid 1.19(1) 2.92(3) 1. 04 (1)
Proline 1.12(1) 0.93(1) 1.05(1)
Glycine 0.23 0.24 0.96(1) 0.99(1)
Alanine 1.02(1)
Valine 0.90(1)
Isoleucine 0.16 0.81(1) 1.03(1) 0.99(1)
Leucine 0.24 1.86(2) 1.12(1 ) 1.02(1)
Tyrosine 0.85(1) 0.93(1) 0.96(1)
Phenylalanine
Tryptophanb
Total residues 7 8 6 6
Yield (pmo1es) 1.45 2.44 0.59 1.45
Purificationd G-15 AG 1 AG 1
Rf in Solvent I 0.37 0.64 0.64 0.66
E1ectrophoresise +1.11 +0.76 +0.26 +0.39
-- .
99
TABLE IV, continued
Amino Acid c-8 C-9 C-10 C-11
residuesaLysine 0.93(1)
Histidine
Arginine 0.94(1) 0.11
CM-cysteine 1.63(2) 1.09(1)
Aspartic acid 4.94(5)
Threonine
Serine 2.71(3) 1.00(1) 1.70(2)
Glutamic acid 0.27 0.25 6.92(7)
Proline
Glycine 1.29(1) 1.06(1) 2.04(2)
Alanine 1.13(1) 0.98(1) 0.14
Valine 1.33(1) 0.90(1)
Isoleucine 0.88(1)
Leucine 0.23 0.95(1) 2.20(2) 1.10(1)
Tyrosine
Phenylalanine 0.11 1.00(1)
Tryptophanb (1)
Total residues 8 6 22 2
Yield Cj.1mo1es) 1. 32 2.64 2.51 1.78
Purificationd AG 1 G-15 PC
Rf in Solvent I 0.07 0.25 0.13 0.62
E1ectrophoresise +0.10 0.0 +1.00 0.0
100
TABLE IV, continued
Amino Acid C-12 C-13 C-11.1
residuesaLysine 1.06(1)
Histidine 0.85(1)
Arginine 0.88(1)
CM-cysteine 0.81.1(1)
Aspartic acid 0.91(1)
Threonine 0.98(1) 1.90(2) 0.96(1)
Serine 0.20 1.16(1) 0.23
Glutamic acid 4.17(1.1)
Proline 0.98(1)
Glycine 0.15 a.al}(1)
Alanine 2.14(2) 0.51 1.04(1)
Valine 2.01(2)C
Isoleucine 0.92(1)c
Leucine 1.07(1)
Tyrosine 1.01.1(1)
Phenylalanine
Tryptophanb
Total residues 5 16 2
Yield (Jlmo1es) 0.1.10 1.45 0.66
Purificationd AG 1 G-15 AG 1
Rf in Solvent I 0.35 0.05 0.30
E1ectrophoresise +0.43 +0.21 0.0
101
TABLE IV, continued
aResidue values calculated from amino acid analysis data
are given, followed by the assumed number of residues in
parentheses.
bDetermined by Ehrlich reaction.
cValues obtained after 96 hours of hydrolysis.
dpC refers to paper chromatography in Solvent I.
G-15 refers to gel filtration through columns of
Sephadex G-15.
AG 1 refers to ion exchange chromatography on columns
of AG l-X2.
eMovement at pH 6.5 relative to alanine (=0), lysine (=-1),
and glutamic acid (=+1).
0.9 1.8 1.2
0.8 1.0 1.1
0.3 1.0 1.0
0.0 1.0-
102
The sequence data are presented in the same form
as were the data for the tryptic peptides.
C-l: Ala-Phe---;;?
Composition: 1.0 1.0
Step 1: 0.0 1.0
C-2: LY2--Val-~Ys
Composition: 1.9 1.1
Step 1: 1.0 1.0
Carboxypeptidase B (5 hours): Lys 1.04.
C-3: LY~-~LY~-~-Leu
Composition: 1.9 1.9 1.2
Step 1:
Step 2:
Step 3:
Step 4:
c-4: Thr-(Pro,Asp,Gly,Pro/Ala,Lys)-Glu-Phe~ =-==~
Composition: 0.9 1.4 1.0 1.1 0.6 0.8 1.1 1.1
Step 1: 0.1 1.4 1.0 1.1 0.6 0.7 1.1 0.9
Carboxypeptidase A (18 hours): Phe 0.62.
Hydrazinolysis of residual peptide: Glu 1.07.
Electrophoresis, pH 6.5: Acidic; therefore
Asp & Glu.
This portion of the sequence overlaps six residues
of the tryptic peptides, T-3a and T-3b, which differ
in the residue in position 12. This position is
occupied by proline in T-3a and by alanine in T-3b.
This sequence heterogeneity accounts for the non
integral values of proline and alanine in C~4.
103
Direct identification in 1st step:
Direct identification in 4th step:
Direct identification in 5th step:
Carboxypeptidase A (4 hours):
Hydrazino1ysis of residual peptide:
~-CMCY~-~-ASg.-Ast;.-~-'tyr
1.2 0.9 1.1 2.0 0.9 0.9
C-5:
Composition:
Step 1:
Step 2 :
Step 3 :
Step 4 :
Step 5:
0.3 1.0 0.8 2.3
0.2 0.0 0.8 2.2
0.0 2.1
1.2
0.6
1.0 0.6
1.1 0.9
0.8 1.1
0.9 0.9
1.0 1.0
PTH-G1u
PTH-Asp
PTH-Asp
Tyr 1.0
Val 0.79
C-6: I1e-LeU-As~-G1n-A1a-G1u-G1u-LeU--;7~ -,~~-,
Composition: 0.8 1.9 1.1 2.9 1.0
Step 1: 0.1 1.9 1.1 3.0 1.1
Step 2: 1.1 1.0 2.9 1.0
Step 3 : 1.0 0.4 2.9 1.0
Step 4 : 1.0 0.2 2.2 1.0
Step 5 : 0.9 2.1 0.1
Step 6: 0.9 1.1
Step 7: 1.0 0.0
Direct identification in 3rd step: PTH-Asp
Direct identification in 4th step: PTH-G1n
Direct identification in 6th step: PTH-G1u
104
Direct identification in 7th step: PTH-Glu
C-7a:
Composition:
Step 1:
Step 2:
Step 3:
Step 4:
Step 5:
Gl~-Ile-Glu-Leu-Pro-Tyr-, -r ---r~1.0 1.0 1.0 1.1 0.9 0.9
0.1 1.0 1.0 1.1 1.0 0.9
0.1 1.1 1.0 1.0 0.9
0.3 1.1 1.0 0.9
0.1 0.2 1.0 1.0
0.2 1.0
Direct identification in 3rd step: PTH-Glu
C-7b:
Composition:
Step 1:
Step 2:
Step 3:
Step 4:
Gl~-~-As~-~-Pro-Tyr
1.0 1.0 1.0 1.0 1.1 1.0
0.2 1.0 0.9 1.0 1.0 1.0
0.1 0.1 1.0 1.0 1.0 1.0
0.2 1.0 0.9 1.1
0.2 1.0 1.0
Direct identification in 3rd step: PTH-Asp
Hydrazinolysis: Tyr 0.81
C-8: ser-CMCYS-Ar~-(Ala)Gly,Ser,CMCys,Ser)~ 7
Composition: 2.7 1.6 0.9 1.1 1.3
Step 1 : 1.8 2.0 0.8 1.1 1.1
Step 2: 1.9 0.9 0.7 1.0 1.2
Step 3: 2.0 0.9 0.2 0.8 1.2
Since the remaining five residues are contained in
tryptic peptide T-5, of which the sequence has been
determined, no further studies were performed on c-8.
C-9: Ser-(CMCys,Ala,Gly,Lys)-Leu~ .........
105
Composition: 1.0 1.1 1.0 1.1 0.9 1.0
Step 1: 0.3 1.0 1.0 1.1 0.7 0.9
Hydrazinolysis: Leu 0.24
Since the first five residues of this peptide are
contained in peptide T-5 and have been sequenced in
that peptide, no further studies were performed on
C-9.
C-lO: Val-Glu-Gly-Asp-Leu-Asp-Gln-Ser-Asp-Gln-Ser-Phe---;:;-
Comp: 1.3 6.9 2.0 4.9 2.2 1.7 1.0
Step 1: 0.4 7.1 2.2 4.8 2.1 1.8 1.0
C-lO (cont): Leu-Asp-Asp-Glu-Gln-Ile-Glu-Glu-~-!fp
Composition (cont.) 0.9 (1)
Step 1: 1.1 (1)
Carboxypeptidase A (16 hours): Trp 0.54
Hydrazinolysis of residual peptide: Gly 0.84
Thus C-lO has valine as its amino-terminal residue
and -Gly-Trp as its carboxyl-terminal sequence. The
data used to establish the sequence of C-lO are sum
marized in Fig. 21. The internal amino acid sequence
was determined by studies on fragments obtained by
enzymatic hydrolyses of C-lO. C-lO was digested with
~. subtilis neutral protease for 5 hours and the re
sulting fragments were resolved by partition chromato
graphy on a 0.6 x 65 cm column of Sephadex G-25
equilibrated with the lower phase of Solvent I.
Figure 21. Summary of the evidence establishing the sequence of peptide C-IO.
Peptides Th-l and BSNP-l both have valine as the amino terminal residue, as does C-IO,
and are therefore placed at the amino terminal of C-IO. BSNP-l provides an overlap,
showing that Th-l is followed by Th-2 and Th-3. Since the tryptophan containing peptide,
BSNP-2, must represent the carboxyl terminal portion of C-IO, the placement of Th-4 is
established. Edman degradation, hydrazinolysis, and the use of carboxypeptidase A,
indicated by arrows to the right, double underlining, and arrows to the left, respec
tively, established the remainder of the sequence.
I-'o0'\
Q)~
a.~
Q)
Cf)
~re-l
I..
rt')~r (\J &11
I (\JI
II
.r:. I ~ra..
:::J-I- ,s::::.
ZC)
l-I
en I
~rCD ~r
I
I
- ~r~r
Ia.. I
z ~r
c:-en
C)I
CDI
~II~rI
I V
-~ ir
I- I
~
C),s::::.
l-I l-
I
~r irI-
I
~r ~r
108
Elution was performed with the upper phase of the
same solvent system. Two peptides, accounting for
15 of the 22 residues, were obtained in pure form.
C-lO-BSNP-l (45%):
Val-(Glu,Gly,Asp,Leu,Asp,Gln,Ser,Asp,Gln)~
Composition: 0.8 3.2 1.1 3.0 1.1 1.0
Step 1: 0.1 3.3 1.2 2.7 0.6 1.1
Thus C-lO-BSNP-l has valine at its amino-terminus
and is therefore the amino-terminal fragment of C-lO.
C-lO-BSNP-2 (13%): Ile-Glu-(Glu,Gly,Trp)~-r
Composition: 0.9 2.1 1.0 (1)
Step 1: 0.2 2.1 1.0 (1)
Direct identification in 2nd step: PTH-Glu
The presence of tryptophan.in this peptide was
shown by a positive Ehrlich reaction.
The residual peptide after step 2 was sUbjected to
thin layer electrophoresis at pH 6.5 and found to be
acidic. Thus the remaining Glx residue in this peptide
is glutamic acid and not glutamine. Since -Gly-Trp has
been shown to be the carboxyl-terminal sequence of C-lO,
the sequence of C-lO-BSNP-2 is Ile-Glu-Glu-Gly-Trp.
The remainder of the sequence of C-lO was obtained
from studies on peptides formed by the action of
thermolysin in a 7 hour digest. These peptides were
resolved by chromatography on a 0.6 x 90 cm column of
AG l-X2. The elution pattern is shown in Fig. 22.
I-'o\0
Figure 22. Elution pattern of peptides from the thermolysin digest of chymotryptic
peptide C-lO. The pattern was obtained by ninhydrin assay of portions of the effluent
from a 0.6 x 90 cm col~n of AG l-X2. Elution was· performed as follows:, 120 ml linear
gradient from pyridine collidine acetate of pH 7.8 to 0.4 M acetic acid; 140 ml of 0.4 M
acetic acid; 80 ml linear gradient from 0.4 M to 1.0 M acetic acid; 50 ml of 1.0 M acetic
acid; and 40 ml of 50% acetic acid. Th-l and Th-2 were eluted in 0.4 M acetic acid, Th-3
and Th-4 were eluted in 1.0 M acetic acid, and the residual C-lO was eluted in 50% acetic
acid. The bars on the abscissa indicate the fractions pooled under each ninhydrin positive
peak.
E-
....ZIJJ::>...JIJIJW
o~
-?--------~
I-
oT.--_-------0.--.. 0
~
o~
I _---------
~ -----_---....1 ~
-o•o
It)
od
(rfWOL9) 3~N\i8~OS8" 31\1J."13~
III
0.0 2.0 2.1 2.1 0.9
1.2 2.0 1.8 0.9
1.0 1.3 1.7 0.8
0.9 1.2 1.1 0.9
0.4 1.1 1.0 0.8
0.2 0.3 1.0 1.0
Leu-(Asp,G1n,Ser,Asp,G1n,Ser)---,1.0 1.8 2.3 1.8
0.2 2.0 2.3 1.7
LeU-AS~-G1n~Ser-As~-G1n-(Ser,Phe)---, ---, ---, -r1.0 1.9 2.1 2.0 0.9
Hydrazino1ysis:
C-10-Th-2 (37%):
Composition:
Step 1:
C-10-Th-3 (34%) :
Composition:
Step 1:
Step 2:
Step 3:
Step 4 :
Step 5:
Step 6:
C-10-Th-1 (71%): Va1-G1u-G1y-Asp-r --'7
Composition: 1.0 1.0 1.1 1.0
Step 1: 0.1 1.0 1.1 1.0
Step 2: 0.3 1.0 1.0
Direct identification in 2nd step: PTH-G1u
Asp 0.49
Direct identification in 2nd step: PTH-Asp
Direct identification in 3rd step: PTH-G1n
Direct identification in 5th step: PTH-Asp
Direct identification in 6th step: PTH-G1n
C-10-Th-4 (29%): Leu-As~-As~-G1u-G1n~ ----::r'
Composition: 0.9 2.0 2.1
Step 1: 0.1 2.0 2.1
Step 2: 1.2 2.0
Step 3: 0.3 2.0
112
Step 4 : Direct analysis of the residue yielded GIn,
0.80 residue.
Direct identification in step 2: PTH-Asp
Direct identification in step 3: PTH-Asp
Direct identification in step 4 : PTH-Glu
C-ll: Val-Leu---,Composition: 0.9 1.1
Dinitrophenylation: DNP-Val
C-12:
Composition:
Step 1:
Step 2:
Step 3:
Step 4:
Thr-CMCys-Ala-Ala-Tyr---'7 7 ---"7 ---,
1.0 0.8 2.1 1.0
0.0 1.0 2.0 1.0
0.2 1.9 1.1
Ll 0.9
0.0 1.0
C-13: ~-Ar~-(Ser,Asp,val,val,lle,GlU,Thr,His,
Composition: 1.0 0.9 1.2 0.9 2.0 0.9 4.2 1.9 0.9
Step 1:
Step 2:
0.3 0.8 1.1 1.0 1.9
0.3 1.0 1.0 1.7
LO 4.3 1.8 0.9
1.0 4.3 1.9 0.8
C-13 (cont.): Lys,Glu,Glu,Glu,Leu,Thr,Gly/Ala)
Composition (cont.):
Step 1 (cont.):
Step 2 (cont.):
1.1
0.9
0.8
1.1
1.2
1.2
0.8 0.5
0.8 0.5
0.8 0.5
The values for Val and lIe are those obtained after
96 hours of hydrolysis.
Position 96 is one of the points of sequence
heterogeneity, as shown in the studies on tryptic
113
peptides T-7a and T-7b. This accounts for the
fractional residue values for glycine and alanine in
C-13. The remaining portion of the sequence was
determined in studies on T-7a and T-7b.
C-14: Thr-Ala~
Composition: 1.0 1.0
Step 1: 0.0 1.0
G. Complete Amino Acid Sequence of Leucaena glauca Ferredoxin
The complete sequence was deduced primarily from the
structures of the tryptic and chymotryptic peptides and
the overlaps among them. Analogy with the sequences of
spinach (14) and alfalfa (35) ferredoxins was used to
establish the position of c-6 with respect to C-7a and
C-7b, and of C-11 with respect to C-12. Figure 23 shows
the complete sequence and the positions of the tryptic and
chymotryptic peptides. The four positions of heterogeneity,
positions 6,12,33, and 96, are enclosed in rectangles. The
correlation between residues at positions 6 and 12 is known,
since peptide T-3a contains leucine and proline, whereas
peptide T-3b contains valine and alanine, in these two
positions, respectively.
H. Distribution of Sequence Heterogeneity among the L. glauca
population
The individual trees yielded from 1 to 5.6 kg of
leaves. The recovery of ferredoxin ranged from 30 to 100%
of the previous value of 20 mg per kg of leaves. Trees
with a large proportion of new leaf growth yielded higher
Figure 23. Tryptic and chymotryptic peptides arranged in order in L. glauca
ferredoxin. The numbers of the tryptic peptides are given above, and those of the
. chymotryptic peptides, below, the amino acid sequence. The residues at the points of
heterogeneity are enclosed in rectangles.
I-'I-'~
:r-I~T.2-t T-3g
Ia-Phe-Lys-Va1-LY~LeUl-Thr- Pro-Asp-Glywrol-l..¥sLGlu-Phe-Glu-Cys-Pro-Asp-Asp-Va1-TYfrIle-Leu-1 ~ C- 2 ~1YgJJ 10 ~ 1 20
C-I-t-C-3 C-4 C-5----- ~--
T- 4 t T-5 1Asp-G1n-Alo-GIU-GIU-~~1GIY-Ilet~LeU-Pro-TYlSer- Cys-Arg-:~-G,y.ser.Cys-SelSer-Cys- AIa-GIy- Lys-
c-s t c- 7g t c-a t-- c-g
t T- 6
;~ ivaI-Glu-GIY-Asp-Leu-ASP-Gln-Ser-Asp-~~-ser-Phe-LeU-Asp-ASP-GIU-Gln-I le-GIu-~t'IY-Trp,Val- L.eUT
-4 c -10 +-C-II-1
r T-7 g •IThr.cYS-AIO-Ala-TYrt-~~- Arg-Ser-Asp-VoI-VaI-Ile-Glu-Thr-His-':~GIU-GIU-G Iu-LeUIThr-~i~I
~C-14C-12 C-13 JI
116
recoveries of ferredoxin. Carboxypeptidase A released
alanine, glycine, threonine, and leucine from the
carboxymethylated protein in each case. The chromatography
of the tryptic digests on AG l-X2 yielded five ninhydrin
positive peaks. A typical elution pattern is shown in
Figure 24. The results of amino acid analysis showed
that the first peak contained peptides T-l and T-2; the
second peak was a mixture of T-3a and T-3b; the third peak
contained only peptide T-5; the two carboxyl terminal
peptides, T-7a and T-7b, were eluted together as the fourth
peak; and finally, peptides T-4 and T-6 were eluted in 50%
acetic acid. No evidence for heterogeneity other than
that found in the sequence studies was evident in the
amino acid composition data obtained from the tryptic
peptides of the ferredoxin of the individual trees. The
amino acid composition of peak T-3 (peptides T-3a and T-3b)
showed fractional residue values for leucine, valine,
alanine, and proline in each of the ten trees studied.
Thus, heterogeneity exists in positions 6 and 12 in each
case. Also, T-7 (peptides T-7a and T-7b) invariably
contained both glycine and alanine, in fractional residue
amounts, confirming that heterogeneity exists in position
96 in the ferredoxin of each of the ten individual trees,
as was indicated by the data obtained in the
carboxypeptidase studies. The results of the amino acid
analyses of T-3 and T-7 from the individual trees are
summarized in Table V.
Figure 24. Elution pattern of tryptic peptides of the ferredoxin of Tree 1 from
AG l-X2. The 0.6 x 36 cm column was developed with 6 ml of pyridine collidine acetate
of pH 7.8, a 60 ml linear gradient from this buffer to 0.4 M acetic acid, 6 ml of 0.4 M
acetic acid, and finally 20 ml of 50% acetic acid. The elution pattern was obtained by
ninhydrin assay of an aliquot from each fraction. The pooling of fractions is indicated
by the bars on the abscissa.
......
......~
•-E...Z
011I
·3~
•°0
CD
N•o.C • •
..-J~<D0%
~~il ~
010
2"-~ 00 ~
•I-
~C. ~
U ~C0%:
:i¢ .0 •-
t It)III: .....~ ~c
(J0-..r-
.~z ~ rt)L\J- c:: t!-oc""')a::
(!)
4~ --N
~- t tLt-L
TABLE V
Distribution of Amino Acid Residues at Three Points
of Heterogeneity in L. glauca Ferredoxin
119
T - 3 T - 7Tree Residue 6 Residue 12 Residue 96
Leucine Valine Proline Alanine Glycine Alanine
1 0.45 0.40 0.69 0.44 0.62 0.38
2 0.57 0.53 0.41 0.51 0.66 0.37
3 0.50 0.53 0.50 0.53 0.69 0.34
4 0.52 0.39 0.43 0.36 0.62 0.45
5 0.47 0.44 0.50 0.37 0.67 0.42
6 0.60 0.50 0.47 0.53 0.68 0.36
7 0.50 0.39 0.38 0.56 0.63 0.45
8 0.45 0.38 0.40 0.38 0.6-3 0.34
9 0.26 0.57 0.31 0.60 0.66 0.37
10 0.52 0.49 0.51 0.46 0.64 0.35
Mean 0.48 0.46 0.46 0.47 0.65
Standarddeviation 0.03 0.02 0.03 0.03 0.01 0.01
120
IV. DISCUSSION AND CONCLUSIONS
A. Characteristics of the Amino Acid Sequence of L. glauca
Ferredoxin:
In this study, L. glauca ferredoxin was found to
contain 96 amino acids in a single chain. Methionine
and asparagine are absent from this protein, and
tryptophan and histidine are present only in the amount
of one residue each. L. glauca ferredoxin contains two
residues of arginine, in contrast to other plant and-"...,
algal ferredoxins in which only one residue of this amino
acid has been found. The number of cysteine residues is
of special interest because cysteine has been implicated
in the chelate structure through which the two atoms of
iron are bound to the plant ferredoxin molecule (5).
L. glauca was found to contain five residues of cysteine,
as do spinach (13) and alfalfa (35) ferredoxins.
Heterogeneity was found to exist at four positions in the
~. glauca ferredoxin sequence, at residues 6, 12, 33, and
96. The finding of both glycine and alanine in position:
96, the carboxyl terminal position, permitted interpreta
tion of the data previously obtained in the carboxypeptidase
A studies on the carboxymethy1ated protein. In each of
three of the four positions of heterogeneity, two very
similar amino acid residues are present. These include
position 6 (leucine and valine), position 33 (aspartic
acid and glutamic acid), and position 96 (glycine and
121
alanine). However, in position 12, alanine is present
in one form of the protein, whereas proline, an amino
acid which can have a drastic effect on the secondary
structure of protein molecules (64), is present in the
other form.
B. Comparison of Plant and Algal Ferredoxinsfrom Four Species:
In Figure 25, the amino acid sequence of L. glauca
ferredoxin is given in full, with the residues which
differ in alfalfa, spinach, and Scenedesmus ferredoxins
on the lines below the L. glauca sequence. The residues
are numbered according to the system used in the published
sequence of spinach ferredoxin (14). Thus, in ~. glauca
ferredoxin, which has one less residue at the amino
terminus when the homologous residues of the four
sequences are aligned, the amino terminal residue is
numbered "2". This difference at the amino terminal
accounts for the fact that only 96 amino acid residues
are found in L. glauca ferredoxin, whereas spinach and
alfalfa ferredoxins each have 97. Scenedesmus ferredoxin,
which also has only 96 residues, is lacking one residue
at the carboxyl terminal, as compared with the ferredoxins
of the higher plants~
Each of the four above-mentioned species has a
cysteine in each of positions 18, 39, 44, 47, and 77.
Scenedesmus ferredoxin has one additional cysteine
resi~ue, at position 85. It~has been found that all of
Figure 25. The amino acid sequences of ferredoxins from ~. glauca, alfalfa, spinach,
and Scenedesmus. The L. glauca ferredoxin sequence is given in fUll, with the additional
residues present at points of sequence heterogeneity shown on the line above. Residues
in alfalfa, spinach, and Scenedesmus ferredoxins which differ from the corresponding
residues in L. glauca are given on the lines below the L. glauca ferredoxin sequence, as
indicated. A minor form of spinach ferredoxin, having lysine in position 31 and methionine
in position 33, is not shown.
~NN
Val 10 Ala 20La. -Ala-Phe-Lys-Val-Lys-Leu-Leu-Thr-Pro-Asp-Gly-Pro-Lys-Glu-Phe-Glu-Cys-Pro-Asp-Alf. ~Ser Tyr Leu Val Glu Thr GinSp. Ala Tyr Thr Leu Val Thr Asn Val GinSc. AlaThr Tyr Thr Leu Lys Ser Asp Gin ThrIle
21 30 Asp 40L.g. Asp-Val- Tyr-lle-Leu-Asp-GIn-Ala-Glu~Glu-Leu-Gly-Ile-Glu-Leu-Pro-1Yr- Ser-Cys-Arg-
. Alt. His Glu ValSp. Ala Glu AspSc. Thr Ala Ala Leu Asp
41 50 60L.g. Aia-Gly-Ser-Cys-Ser-Ser-Cys-Aia-Gly-Lys-Leu-Val-Glu-Gly-Asp-Leu-Asp-Gln-Ser-Asp-Alt. Vol Ala Ala Glu ValSp. Lys Thr SIr AspSc. Ala Val Glu Ala Thr Val
61 70 80L.g. Gln-Ser-Phe-Leu-Asp-Asp-Glu-Gln-I le-Glu-Glu -GIy-Trp-Val-Leu-Thr -Cys-Ala-Ala-Tyr-Alf. Gly Asp Val~ ~p A~Sc. Ser Met Asp Gly Phe VaI
81 90 AlaL.g. Pro-Arg-Ser-Asp-Val-Val-Ile-Glu-Thr-His-Lys-Glu-Glu-Glu-Leu-Thr-GlyAlt. Ala Lys Thr AlaSp. Val Thr AlaSc. Thr Cys Thr Ala Asp Phe -
124
the cysteine residues in spinach ferredoxin are titrable
by heavy metals (5) and Matsubara et ale (14) have
suggested a model in which the folding of the polypeptide
chain brings together the five cysteine residues, which
are proposed to be chelated to the iron atoms. The three
residues of cysteine at positions 39, 44, and 47 are
located in a portion of the molecule in which the sequence
varies very little in these four species. This section,
from residue 35 through residue 50, includes only one
site, residue 43, at which variation is observed.
The similarities in the amino acid sequences of
these ferredoxins are more striking than are the differences
among them. Fifty-eight per cent of the sequence is
identical in these four species. Residues which do not
vary are: 4, 5, 9, 10, 12, 18-21, 23-26, 28-30, 32, 35-42,
44-50, 54, 58, 60, 62-66, 68,72, 74-77, 79, 80, 83, 84,
87, 89-93, and 95. If the ferredoxins of only the three
species of higher plants are compared the similarity is
even greater, with over 70% of the sequence invariant.
Many of the changes between species involve amino acid
residues which are structurally similar. Among these are
Tyr ---> Phe, Val ---> Leu, Ala ---> Gly, and Glu ---> Asp.
Such changes are considered conservative, since they would
not be expected to exert any significant effect on activity.
However, radical changes, such as those involving residues
which differ in charge and those involving proline
125
residues, occur also. It can be concluded that those
residues which vary with species are not essential to the
function of the protein. Thus, tryptophan and methionine,
and the histidine residue in position 27, which are found
in some but not all species, must not be directly involved
in catalysis or in the binding of iron. Many other
residues may also be considered non-essential on these
grounds. However, it may be noted that the requirements
for activity, even when not specifying that a certain
amino acid must occupy a given position in the molecule,
might nevertheless control the type (acidic, basic,
hydrophobic) of residue which may be present.
c. Acidic Residues in Plant and Algal Ferredoxins:
The amino acid sequences of the four plant and algal
ferredoxins shown in Figure 25 reveal an unusually high
content of acidic residues (aspartic and glutamic acids).
Table VI lists a number of proteins of various types and
the per cent of acidic residues in each, as established
in each case by amino acid sequence determinations. Most
of these proteins, including the bacterial ferredoxins,
have a content of acidic residues less than 15%. In
plant and algal ferredoxins these values range from 19%
in Scenedesmus to 24% in L. glauca. Of the proteins
listed, only rubredoxins, another class of non-heme iron
proteins, equal plant ferredoxins in their content of
acidic amino acids.
126TABLE VI
Acidic Residue Content of Proteins of Various Types
ProteinAcidic Total %Acidic Ref.Residues Residues Residues
Bacillus subtilisBPN' subtilisin 11 275 4 (65)
Bovine trypsinogen 14 229 6 (66)
Bovine ribonuclease 10 124 8 (67)
Rattlesnakecytochrome c 9 104 9 (68 )
Human hemoglobin 27 287 9 (69)
Human cytochrome c 11 104 11 (70)
E. coli tryptophan28 267 (71)synthetase Alpha 11
Clostridium butyricumferredoxin 7 55 13 (32)
Clostridium pasteurianumferredoxin 7 55 13 (31)
Human growth hormone 35 188 19 (72 )
Scenedesmus ferredoxin 18 96 19 (36)
Spinach ferredoxin 20 97 21 (14)
Alfalfa ferredoxin 21 97 22 (35)
....... Peptostreptococcuselsdenii rubredoxin 12 52 23 (73)
Leucaena glaucaferredoxin 23 96 24
Micrococcus aerogenesrubredoxin 13 53 25 (74 )
127
Of the 23 acidic residues in ~. glauca, 12 occur in
clusters in the sequence (residues 20-21, 29-30, 65-67,
70-71, and 92-94, as numbered in Figure 24). Such group
ings of acidic residues have also been noted (14) in the
sequence of spinach ferredoxin and can be seen in the
alfalfa and Scenedesmus ferredoxin sequences as well. In
comparing the four sequences shown in Figure 25, only
four clusters, including nine acidic residues (residues
20, 21, 29, 30, 65, 66, 92, 93, and 94), are found to be
common to all four species. It is not known whether any
of these groupings are required for the function of the
protein. It may be noted that no clustering of acidic
residues is found in the amino acid sequences of the
clostridial ferredoxins, which are also capable of
catalyzing the photoreduction of NADP.
D. Genetic and Evolutionary Aspects of Variations in
Ferredoxin Sequences:
1. Plant and Algal Ferredoxins
The differences among the ferredoxins of the
four species shown in Figure 25 clearly indicate three
types of mutations which may be responsible: addi
tion, deletion, and point mutations. The differences
in chain length, which involve the presence of one
less residue at the amino terminus of L. glauca
ferredoxin and at the carboxyl terminus of
Scenedesmus ferredoxin, are probably examples of
128
addition to or deletion from the corresponding genes
of one codon in each case. In the case of ~. glauca,
deletion rather than addition seems to be more
probable, since the other three species, including
the phylogenetically more primitive Scenedesmus, all
have an amino acid residue in this position. The
"missing residue" at the carboxyl terminal of
Scenedesmus, however, cannot be assumed to imply that
addition of a codon has taken place in the evolution
of the higher species, since a deletion in the
ferredoxin gene of Scenedesmus or of one of its
evolutionary precursors after divergence from the
precursor of the higher plants would be an equally
probable explanation. It may also be noted that it
is possible in the case of terminal residues that a
point mutation in the corresponding codon could
result in loss of one residue without affecting the
rest of the gene, thus giving the appearance of a
deletion, if the point mutation resulted in a chain
termination codon. However, in the two cases under
consideration, the amino acid present in the three
other species is alanine, which would require a
minimum of two point mutations within the codon for
such a change to occur. The codons considered are
those listed by Brimacombe et al. (75).
All of the variations within the interiors of
129
these sequences appear to have been caused by point
mutations in the ferredoxin genes. No addition or
deletion of interior residues is evident in the four
sequences, and although variation is found to exist
in as many as five consecutive positions in the
sequences, the codons corresponding to the amino
acids involved are such as to provide no evidence for
the occurrence of a frame shift. The differences
among the ferredoxin sequences in all four species
could be accounted for by a total of 45 single and
14 double mutations, with transversions occurring at
more than twice the frequency of transitions.
A method for relating phylogenetic differences
to the mutation distances between species has been
described by Fitch and Margoliash (76), who define
mutation distance as the minimal number of nucleotides
to be altered in order for the gene for one protein
to code for the other. A low value for mutation
distance between homologous proteins from different
species indicates close phylogenetic relationship
between the species. The mutation distances between
the ferredoxins of the four species are given in
Table VII. Also included in the table are the values
of the minimum base difference per codon which is
equal to the mutation distance divided by the number
of codon pairs compared (77).
130TABLE VII
Mutation Distances and Values of Minimum
*Base Difference per Codon
Ferredoxin LeucaenaAlfalfa Spinach ScenedesmusSource glauca
Leucaena glauca 24 29 44
Alfalfa (0.25) 27 40
Spinach (0.30) (0.28) 39
Scenedesmus (0.46) (0.42) (0.41)
*Mutation distances are given in the upper right half of thetable. Values of minimum base difference per codon are inthe lower left half of the table and are enclosed inparentheses.
131
The amino acid sequence of L. glauca ferredoxin
differs in 18, 21, and 25 positions from those of
spinach, alfalfa, and Scenedesmus, respectively, and
the corresponding mutation distances are 29, 24, and
44. The mutation distances indicate that L. glauca
is closest to alfalfa in the evolutionary scheme.
This is qualitatively in agreement with phylogenetic
considerations, since L. glauca and alfalfa are both
of the family Legl~inosae of the order Rosales of the
group Calyciflorae (cup flowers) whereas spinach is
of the group Thalamiflorae (receptacle flowers) and
Scenedesmus is a green alga (Chlorophyta). However,
from the closeness of the phylogenetic relationship
between alfalfa and L. glauca, it is surprising that
the calculated mutation distance is not much smaller.
Assuming that Scenedesmus is more primitive than
the higher plants, it can be seen that spinach and
alfalfa ferredoxins, with mutation distances from
Scenedesmus ferredoxin of 39 and 40, respectively,
have evolved equally since their divergence from a
common evolutionary precursor. L. glauca ferredoxin
must have diverged from the alfalfa line of evolution
within a relatively short time after the divergence
of the spinach and alfalfa precursors. These
evolutionary relationships, which are based on the
assumption of uniform rates of change in all groups,
132
are shown in Figure 26, in the form of a phylogenetic
tree.
2. Heterogeneity in ~. glauca ferredoxin
Heterogeneity has been found in the amino acid
sequences of ferredoxins from spinach and alfalfa.
In each of these species one major form of the protein
was found to be present in far greater amount than the
minor components. In~. glauca ferredoxin, however,
each of the four positions of heterogeneity is
occupied by two amino acid residues in ratios ranging
from 1:1 to 2.5:1, as determined from peptide yields.
Thus it is apparent that the heterogeneity in L.
glauca ferredoxin is not due to recent mutations,
since in that case the mutant forms would be present
in much lower amount than the original forms.
The tryptic peptide including residues 6 and 12
occurred in only two of the four possible forms, one
of which contained leucine and proline in positions
6 and 12, respectively, whereas the other contained
valine and alanine. Therefore the heterogeneity in
these two positions must be due to the presence of
different genes rather than to ambiguity in transla
tion of the genetic code, as may be the case in
positions 33 and 96. Since the four points of
heterogeneity thus represent a maximum of three
variables, there may be as many as eight forms of the
133
Figure 26. Phylogenetic tree of the ferredoxins. The
evolutionary relationships indicated by the mutation distances
among amino acid sequences of the ferredoxins are shown diagram
matically. All of these ferredoxins have regions of homology
among their primary structures, indicating possible common
evolutionary origin. The ferredoxins of the anaerobic
nonphotosynthetic bacteria have the most primitive structure,
having evolved little since their apparent origin by the
doubling of a precursor gene. The ferredoxin of Chromatium is
quite similar in structur~ and is therefore placed on the same
branch of the tree. The ferredoxins of the higher plants and
that of Scenedesmus have evolved much further. The branching
off of Scenedesmus ferredoxin and the inter-relationships among
the ferredoxins of the higher plants as shown here are based on
the degree of homology existing among the amino acid sequences
and are discussed in the text.
LEUCAENAGLAUCA ALFALFA SPINACH
ANAEROBICNONPHOTOSYNTHETIC
BACTERIA
135
protein in this species.
The population distribution of the different
amino acids at three points of heterogeneity was
studied in order to obtain more information as to
the genetic causes for the presence of multiple forms
of ferredoxin in ~. glauca. If the heterogeneity at
positions 6 and 12 were due to differing allelic
nuclear genes of equal frequencies, the distribution
of the two forms of ferredoxin among the L. glauca
population would be expected to follow a 1:2:1 ratio,
as shown in Figure 27. Thus 50% of the trees would
be heterozygous and contain both forms of the protein
(with respect to residues 6 and 12). However, of the
other 50% of the trees, half would be expected to be
homozygous for each form of ferredoxin. Thus, in
selecting trees at ~a~dom from the L. g!~ popula
tion, the probability that any given tree would be
homozygous and contain only one of the two forms of
the protein is 50%, or 1/2.
On the other hand, if the differing genes were
non-allelic, as would be the case if duplication of
the ferredoxin gene had· occurred in the evolution of
this species, each individual tree would be expected
to contain both forms of the protein, probably (but
not necessarily) in approximately equal amounts.
Such a pattern of distribution of the different forms
136
Figure 27. Inheritance patterns of allelic and non
allelic genes. The distribution among progeny of allelic
genes of equal frequencies, in this case genes for two
forms of ferredoxin as shown, according to classical
genetic principles, would follow a 1:2:1 pattern, with
equal numbers of homozygotes and heterozygotes resulting.
The distribution of non-allelic genes, however, would yield
only progeny which are homozygous for both forms of the
protein, as shown at the right.
•
..C\JX--t---...-....
(.)-.-.JW-.J-.J«
(.)
:Jw.-.J.-.J«I
zoz
• •• •
I · I
I II II II II £
« 0~ a::L~__-;;:;-I « c..
It] I I-"iii""""rtJ- : 1~ ::::>~ ~I II II III II
LLt.
138
of ferredoxin might also be observed if the ferredoxin
genes were located in the chloroplast DNA rather than
in the nuclear chromosomes, since an individual plant,
while inheriting a total of only one diploid set of
nuclear genes, may receive numerous proplastids which
develop into chloroplasts.
The present studies show that, of the ten trees
examined, all contained both forms of ferredoxin with
respect to residues 6 and 12. The probability that
allelic nuclear genes are responsible is thus (1/2)10,
or less than 0.001. However, it is not possible to
distinguish between the other two possibilities, non
allelic nuclear genes and allelic genes of the
chloroplasts, from the data obtained in these studies.
The fact that the ratio of glycine to alanine in
T-7 is 1.7:1, which is quite different from the 1:1
ratio found to exist between the residues present in
positions 6 and 12, indicates that the genetic causes
of the heterogeneity in this protein are more complex
than the presence of two non-allelic genes, which
would yield the same ratio between amino acids at all
points of heterogeneity. The fact that both glycine
and alanine are found at position 96 in the ferredoxin
of each of the ten trees studied shows that allelic
genes cannot be responsible for this heterogeneity.
If multiple non-allelic genes are responsible, there
139
must be more than two such genes to account for the
differences in ratios of amino acids present at the
different points of heterogeneity. Three non-allelic
genes could not account for these ratios unless they
had different activities. Four non-allelic genes of
the same activity could at best give a 3:1 ratio of
glycine to alanine, in contrast to the 1.7:1 ratio
actually found. If the ferredoxin genes were contained
in the chloroplast DNA rather than in the nucleus of
the cell it would be possible for three o~ more differ
ing ferredoxin genes, present in different amounts in
the cell, to account for the observed ratios. However,
it seems probable that more variation in ratios of
amino acids at each point of heterogeneity would be
found in different trees if this were the case.
Another, more probable explanation for the
different ratios of residues present at the points of
heterogeneity would be ambiguity in translation of the
terminal codon. Ambiguous translation of a special
codon has been suspected to occur in the genes for the
c(-chain of horse hemoglobin (78), with either lysine
or glutamine being inserted in position 60. Another
example of possible ambiguity in coding, involving
serine and threonine, has been found recently in
studies on mouse hemoglobin (79). The possibility
that the finding of more glycine than alanine in
140
position 96 might be an artifact of the methods
used for tryptic hydrolysis or peptide separation is
counteracted by the results of hydrolysis of the
carboxymethylated protein by carboxypeptidase, which
indicate the presence of more glycine than alanine at
the carboxyl terminus. The most probable explanations
for heterogeneity in L. glauca ferredoxin therefore
appear to be:
1. Either two non-allelic nuclear genes or
differing chloroplast genes, in the case of
residues 6 and 12.
2. Ambiguity of translation at position 96.
The cause of heterogeneity at position 33 has not been
investigated.
The amino acid differences and types of mutations
involved in the observed heterogeneity are summarized,
in Table VIII. In each case a single point mutation
could be responsible. All appear to be transversions,
and all involve the guanine-cytosine base pair, with
the possible exception of the aspartic acid-glutamic
acid change in which the nucleotides involved can not
be ascertained.
E. Techniques in Ferredoxin Purification:
The procedure used for purification of ferredoxin
from ~. glauca leaves included three modifications which
may be of use in the isolation of this protein from other
141
TABLE VIII
Types of Mutations in L. glauca Ferredoxin Heterogeneity
ResidueNumber Amino Acid Changes Codon Changes
RequiredMutation
Type
6 Leu <-----> Val UuA or CUX <--> GUX TransversionG
12 Pro <-----> Ala CCX <---------> GCX Transversion
33 Asp <-----> G1u GAU <---------> GAA TransversionC G
96 G1y <-----> Ala GGX <---------> GCX Transversion
142
species as well. The first of these was the addition of
acetone directly to the unfiltered homogenate. This was
necessary in the case of L. glauca due to the fact that
the leaf homogenate, in contrast to that from some other
species, was very viscous and could not have been
filtered without the prior addition of a large amount of
buffer, thus greatly increasing the volume to be processed
in sUbsequent steps. The second modification, the use of
a bed of Solka-floc for filtration of the acetone-treated
homogenate, had the advantage of being much faster than
centrifugation, and also succeeded in removing all
particulate homogenate material from the extract in one
step, along with the material precipitated by 35% acetone.
The third modification was introduced at a later stage
after the ferredoxin had been treated with 0.5 gm per ml
ammonium sulfate to precipitate much of the remaining
contaminating protein material. The ferredoxin, in
solution with this high concentration of ammonium SUlfate,
was ~pplied directly and without prior dilution to a
column of DEAE-cellulose, which was then washed with 0.1 M
Tris buffer of pH 7.3 to which had been added 0.5 gm
amm0nium sulfate per mI. The ferredoxin was absorbed as
a dark red band at the top of the column, while a large
amount of yellow material passed through. This property
of fe~redoxin, that of being strongly bound by DEAE
cellulose in very high salt concentration is highly
unusual and therefore results in a very large amount of
purification with little or no loss of ferredoxin. The
reason for this phenomenon is not known, but logically
would appear to involve a decreased solubility of the
protein in the ammonium sulfate solution in the presence
of DEAE-cellulose, rather than simple ionic interaction
between the ferredoxin and the DEAE-cellulose, such as
occurs in solutions of low salt concentration.
F. Specificities of Proteolytic Enzymes Employed in these
Studies:
Some observations may be made regarding the
specificities of the proteolytic enzymes used in the
sequence studies. TPCK-trypsin hydrolyzed both of the
Arg-X bonds but only four of the five Lys-X bonds in the
carboxymethylated protein. The unhydrolyzed bond was
Lys-Glu, in positions 90-91. However, another Lys-Glu
bond, in position 13-14, was hydrolyzed in the tryptic
digest. Thus it may be assumed that the presence of an
acidic residue following lysine is not in itself
inhibitory to the splitting of the bond, but that other
factors, involving additional portions of the sequence,
are involved. In the chymotryptic digest, one Lys-X
bond was partially cleaved, where X is either leucine or
valine, in position 5-6, even though the enzyme had been
pretreated with TLCK to inhibit tryptic activity. Four
of the nine Leu-X bonds were not cleaved; the four X
144
groups were three residues of aspartic acid and one of
proline. The Phe-Leu bond (positions 62-63) was not
hydrolyzed by the TLCK-chymotrypsin. In studies on
spinach ferredoxin (14) this bond was found to be
hydrolyzed to only a minor extent in the chymotryptic
digest. One unusual cleavage by the TLCK-chymotrypsin
was the bond between two serine residues, at positions
44 and 45. Apparently the action of the enzyme was
responsible, since this bond is stable in tryptic
peptide T-5.
In the hydrolysis of peptides T-7a and C-IO by
B. subtilis neutral protease, two of the bonds that were
cleaved, GIn-Leu (residues 67-68), and Glu-Leu (residues
93-94), were consistent with the known specificity of
the enzyme for linkages in which a hydrophobic residue
contributes the amino group (80, 81, 82). However, two
other bonds, Gln-Ser (residues 60-61) and Glu-Thr (residues
87-88) were also hydrolyzed. Thermolysin, which has a
specificity (83) like that of the B. subtilis neutral
protease, hydrolyzed only those bonds in peptide C-IO
which were in keeping with its known specificity, Asp-Leu
(residues 54-55), Ser-Phe (residues 61-62), Phe-Leu
(residues 62-63), and GIn-lIe (residues 67-68).
G. Determination of the Complete Structure of L. glauca
Ferredoxin:
The present studies have elucidated only the primary
145
structure of this protein. The secondary, tertiary, and
chelate structures remain to be determined. Possibly the
most definitive method of determining the complete
structure of L. glauca ferredoxin will be by X-ray
diffraction studies of the crystalline protein. To this
end samples of the protein have been sent to Dr. Lyle
Jensen at the University of Washington and, hopefully,
the determination of the complete structure will be
possible.
146
v. SUMMARY
Ferredoxin, a non-heme iron protein involved in
photosynthesis and nitrogen fixation in plants, was isolated
from the leaves of Leucaena glauca, a species of small
leguminous tree. The protein was carboxymethylated and
hydrolyzed by trypsin and chymotrypsin to yield two sets of
peptides which were then isolated by ion exchange and
partition chromatography. Chemical and enzymatic methods
were used to determine the amino acid sequences of the
peptides, from which the sequence of the protein was
reconstructed.
L. glauca ferredoxin was found to contain 96 amino acid
residues in a single chain. Comparison of its sequence with
those of spinach (14), alfalfa (35), and Scenedesmus (36)
ferredo~ins showed that 58% of the residues are identical in
these four species, including five cysteine residues which
are believed to be involved in the binding of the two iron
atoms to the polypeptide chain. The types of mutations which
appear to be responsible for the differences among the
ferredoxins of these four species are addition, deletion, and
point mutations. The mutation distances among these
ferredoxins indicate that L. glauca is more closely related
in evolution to alfalfa than to the other species, and that
it has evolved further from Scenedesmus than have spinach and
alfalfa.
Heterogeneity was found in the amino acid sequence of
147
~. glauca ferredoxin. A study of the distribution of the
observed heterogeneity within the ~. glauca population showed
that the genes responsible for the different forms of the
protein are not allelic nuclear genes, but may be either
chloroplast genes or non-allelic genes of the nucleus.
148VI. BIBLIOGRAPHY
1. Lovenberg, W., Buchanan, B. B., and Rabinowitz, J. C.,
J. BioI. Chern., 238, 3899 (1963).
2. Losada, M., Whatley, F. R., and Arnon, D. I., Nature,
,190, 606 (1961).
3. Arnon, D. I., Science, 149, 1460 (1965).
4. San Pietro, A., and Lang, H. M., Science, 124, 118 (1956).
5. Keresztes-Nagy, S., and Margoliash, E., J. BioI. Chern.,
241, 5955 (1966).
6. Davenport, H. E., Hill, R., and Whatley, F. R., Proc.
Roy. Soc., ~., 139, 346 (1952).
7. Arnon, D. I., Whatley, F. R., and Allen, M. B., Nature,
180, 182 (1957).
8. Davenport, H. E., Biochern. ~., 77, 471 (1960).
9. Davenport, H. E., and Hill, R., Biochern. J., ~, 493 (1960).
10. Mortenson, L. E., Valentine, R. C., and Carnahan, J. E.,
Biochern. Biophys. Res. Commun., 1, 448 (1962).
11. ~agawa, K., and Arnon, D. I., Nature, 195, 537 (1962).
12. Matsubara, H., Jukes, T. H., and Cantor, C. R., Brookhaven
Symposia in Biology, Structure, Function, and Evolution
in Proteins, No. 21 (1968), in press.
13. Matsubara, H., Sasaki, R. M., and Chain, R. K., J. BioI.
Chern., 243, 1725 (1968).
14. Matsubara, H., and Sasaki, R. M., J. BioI. Chern., 243,
1732 (1968).
15. Matsubara, H., J. BioI. Chern., 243, 370 (1968).
149
16. Sasaki, R. M., and Matsubara, H., Biochern. Biophys. Res.
Commun., 28, 467 (1967).
17. Whatley, F. R., Tagawa, K., and Arnon, D. I., Proc. Nat1.
Acad. Sci. (U. S.), 7, 266 (1963).---- -- -18. Losada, M., Paneque, A., Ramirez, J. M., and del Campo,
F. F., in A. San Pietro (Editor), Non-heme iron proteins:
role in energy conversion, Antioch Press, Yellow Springs,
Ohio, 1965, p. 211.
19. Evans, M. C. W., Buchanan, B. B., and Arnon, D. I., Proc.
Nat1. Acad. Sci. (U. S.), 53, 1420 (1965).
20. Korkes, S., J. Bio1. Chern., 216, 737 (1955).
21. Valentine, R. C., Mortenson, L. E., Mower, H. F.,
Jackson, R. L., and Wolfe, R. S., J. Bio1. Chern., 238,
856 (1963).
22. Raeburn, S., and Rabinowitz, J. C., in A. San Pietro
(Editor), Non-heme iron proteins: role in energy
conversion, Antioch Press, Yellow Springs, Ohio, 1965,
p. 189.
23. Stern, J. R., in A. San Pietro (Editor), Non-heme iron
proteins: role in energy conversion, Antioch Press,
Yellow Springs, Ohio, 1965, p. 199.
24. Bachofen, R., Buchanan, B. B., and Arnon, D. I., Proc.
Nat1. Acad. Sci. (U. ~.), 51, 690 (1964).
25. Andrews, I. G., and Morris, J. G., Biochem. Biophys.
Acta, 97, 176 (1965).
- .....
150
26. Akagi, J. 00., Biochem. Biophys.oRes. COrnniun., 21,72
(1965 ).
27. Valentine, R. C., Jackson, R. L., and Wolfe, R. S.,
Biochem. Biophys. Res. Commun., 7, 453 (1962).
28. Valentine, R. C., and Wolfe, R. S., J. Bacterio1., 85,
1114 (1963).
29. Bradshaw, W. H., and Reeder, D. J., Bacterio1. Proc.,
110 (1964).
30. Brill, W. J., and Wolfe, R. S., Fed. Proceed., 24, 233
(1965).
31. Tanaka, M. T., Nakashima, T., Benson, A., Mower, H. F.,
and Yasunobu, K. T., Biochem. Biophys. Res. Commun. ~ 16,
422 (1964).
32. Benson, A.M., Mower, H. F., and Yasunobu, K. T., Proc.
Nat1. Acad. Sci., (U. S.), 55, 1532 (1966).
33. Tsunoda, J., Whiteley, H. R., and Yasunobu, K. T., J.
BioI. Chern., in press.
34. Eck, R. v., and Dayhoff, M. 0., Science, 152, 363 (1966).
35. Keresztes-Nagy, S., Perini, F., and Margo1iash, E., to
be pUblished, (1968).
36. Sugeno, K., and MatSUbara, H., Biochem. Biophys. Res.
Commun., 32, 951 (1968).
37. McConn, J. D., Tsuru, D., and Yasunobu, K. T., J. BioI.
Chern., 239, 3706 (1964).
38. Mares-Guia, 00., and Shaw, E., Fed. Proc., 22, 528 (1963).
151
39. Wang, S. S., and Carpenter, F. H., J. Bio1. Chern., 240,
1619 (1965).
40. San Pietro, A., and Lang, H. M., J. Bio1. Chern., 231,
211 (1958).
41. Losada, M., and Arnon, D. I., in Modern rnethods~f plant
analysis, Vol. VII, H. F. Linskens, B. D. Sanwa11, and
M. V. Tracey, (Editors), Springen, Vienna, 1964, p. 594.
42. Bendall, D. S., Gregory, R. P. F., and Hill, R., Biochern.
~., 88, 29P (1963).
43. Crestfie1d, A. M., Moore, S., and Stein, W. H., J. Bio1.
Chern., 238, 622 (1963).
44. Schroeder, W. A., Jones, R. T., Cormick, J., and McCalla,
K., Anal. Chem., 34, 1570 (1962).
45. Moore, S., and Stein, W. H., J. Bio1. Chern., f76, 367
(1948).
46. Katz, A. M., Dreyer, W. J., and Anfinsen, C. B., J.
Bio1. Chern., 234, 2897 (1959).
47. Harris, J. I., and Hindley, J., J. Mol. Bio1., 13, 894
(1965).
48. Smith, I., Nature, 171, 43 (1953).
49. Sanger, F., and Thompson, E. O. P., Biochern. Biophys.
Acta, 71, 468 (1963).
50. Spackman, D. H., Stein, W. H., and Moore, S., Anal. Chern.,
30, 1190 (1958).
51 •. Opienska-B1auth, J., Charezinski, M., and Berbec, H.,
Anal. Biochem.,~, 69 (1963).
152
52. Edman, P., and Sjoquist, J., Acta Chern. Scand., 10,
1507 (1957).
53. Konigsberg, W., and Hill, R. J., J. BioI. Chern., 237,
2547 (1962).
54. Konigsberg, W., in C. H. W. Hirs (Editor), Methods in
enzymology, Vol. XI, Academic Press, New York, 1967,
p. 464.
55. Randerath, K., Thin Layer Chromatography, Academic
Press, New York, 1966, p. 122.
56. Fraenkel-Conrat, H., Harris, J. I., and Levy, A. L.,
Methods Biochem. Anal., £, 391 (1955).
57. Sanger, F., Biochem. J., 39, 507 (1945).
58. Fraenkel-Conrat, H., Harris, J. I., and Levy, A. L.,
Methods Biochem. Anal., £, 388 (1955).
59. Randerath, K., Thin Layer Chromatography, Academic Press,
New York, 1966, p. 116.
60. Gray, W. R., in C. H. W. Hirs (Editor), Methods in enzy-
mology, Vol. XI, Academic Press, New York, 1967, p. 139.
61. Deyl, Z., and Rosmus, J., J. Chromatog., 20, 514 (1965).
62. Bradbury, J. H., Biochem. ~., 68, 475 (1958).
63. Fraenkel-Conrat, H., Harris, J. I., and Levy, A. L.,
Methods Biochem. Anal., £, 397 (1955).
64. Szent-Gyorgyi, A. G., and Cohen, C., Science, 126, 697
(1957).
65. Smith, E. L., Markland, F. S., Kasper, C. B., Delange,
R. J., Landon, M., and Evans, W. H., J. BioI. Chern., 241,
153
5974 (1966).
66. Walsh, K., and Neurath, H., Proc. Natl. Acad. Sci. U. ~.,
52, 884 (1964).
69. Braunitzer, G., Gehring-Muller, R., Hilschmann, N.,
Hilse, K., Hobom, G., Rudloff, V., and Wittrnann-Liebold,
B., ~. Physiol. Chernie, 325, 283 (1961).
70. Matsubara, H., and Smith, E. L., J. BioI. Chern., 237,
3575 (1962).
71. Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton,
B. C., Proc. Natl. Acad. Sci. U. S., 57, 296 (1967).
72. Li, C. H., Liu, W-K., and Dixon, J. S., J. Am. Chern.
Soc., 88, 2050 (1966).
73. Bachrnayer, H., Yasunobu, K. T., Peel, J. L., and Mayhew,
S., J. BioI. Chern., 243, 1022 (1968).
74. Bachmayer, H., Yasunobu, K. T., and Whiteley, H. R.,
Biochern. Biophys. Res. Commun., 26, 435 (1967).
75. Brirnacornbe, R., Trupin, J., Nirenberg, M., Leder, P.,
Bernfield, M., and Jaouni, T., Proc. Natl. Acad. Sci.
U. ~., 54, 954 (1965).
76. Fitch, W. M., and Margoliash, E., Science, 155, 279
(1967).
77. Cantor, C. R., and Jukes, T. H., Proe. Natl. Acad. Sci.
154
Q. s., 56, 177 (1966).
78. Kilmartin, J. V., and Clegg, J. B., Nature, 213, 269
(1967).
79. Rifkin, D. B., Hirsh, D. I., Rifkin, M. R., and
Konigsberg, W. H., Cold Spring Harbor Symp. Quant.
BioI., 31, 715 (1966).
80. Feder, J., Biochemistry ~, 2088 (1967).
81. Morihara, K., Biochem. Biophys. Res. Commun., 26, 656
(1967).
82. Benson, A. M., and Yasunobu, K. T., Arch. Biochem.
Biophys., 126, 653 (1968).
83. Matsubara, H., and Sasaki, R. M., Fed. Proc., 26,723
(1967).