J. Biol. Chem. 1986 Shechter 66 70
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Transcript of J. Biol. Chem. 1986 Shechter 66 70
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8/9/2019 J. Biol. Chem. 1986 Shechter 66 70
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8/9/2019 J. Biol. Chem. 1986 Shechter 66 70
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Oxidation and Reduction of Methionine
67
activity of ACTH and insulin were determined by lipolysis (23) and
to Rodbell (25).
lipogenesis (24 ), respectively. Rat fat c ells were prepared according
RESULTS
Conversion of Methionine to Methionine Sulfoxide by Me2S0
under Different Experimental Conditions-Table I summa-
rizes varying conditions for the oxidation of methionine to
methionine sulfoxide by Me2S0. The reaction was found to
be strongly pH dependent. No oxygen transfer was observed
in an aqueous solution of 0.1 M sodium bicarbonate, pH 8.5,
and only slight reaction in 0.01
M
HC1 (Table
I .
The reaction
occurs readily, however, at 0.1-1
M
HC1. Acetic acid was a
poor substitute for hydrochloric acid. Some reactions, how-
ever, occurred at high concentrations (5 M ) of acetic acid
(Table I . The chloride anion, however, seems to be by far
more efficient in the acid-promoted S-0 bond breaking that
is required to oxidize methionine by Me2S0 in queous acidic
solutions.
Selectivity of Me2SO/HCl toward Methionine Residues-A
mixture of amino acids were treated with Me2SO/HC1 (Table
11 .
As shown in the Table, after exposure of the amino acids
to
1
M
Me2S0, 2
M
HC1 for 3 h at room temperature, methi-
onine was completely oxidized to methionine sulfoxide. Other
amino acids were recovered quantitatively (Table
11 .
There
wereno detectablequantities of cysteic acid, methionine
sulfone, or any otherinhydrine-positive derivatives of amino
acids. To examine whether tryptophan residues are modified
byMe2SO/HC1 the ultraviolet absorption spectrum of
N -
acetyltryptophan (0.35 mM in
2
M HCl) was monitored (Fig.
1).MezSO was then added to a final concentration of 1M and
the spectrum was monitored again after 2 hat room temper-
ature. No change in the spectrum (other han a small decrease
due to dilution) is detected, indicating that the ryptophanyl
moiety has remained unmodified. As an additional control,
about 20 eq of chloramine-T were added to the quartz cell
(Fig.
1).
This resulted in amarked decrease in the bsorbance
at 280 nm as xpected from the conversion of the tryptophanyl
moiety to 2-oxyindolealanine (26). Thus, aqueous Me2SO/
HC1 at room temperature is selective toward the oxidation of
methionine among the common amino acids side chains. This
selectivity is even preserved at elevated temperatures (i.e. at
50 and 100 C) as demonstrated in the study of Lipton and
Bodwell (19).
To examine whether free S H groups are oxidized by aqueous
Me2SO/HC1, reduced glutathionine (3.6 mM) was incubated
for several hours, either in H2 0, 10% NJV-dimethylformam-
ide, 1.2
M
HCl, or a t two different concentrations of Me2SO/
HC1. Aliquots were withdrawn a t intervals to determine SH
TABLE
Oxidation of methionine to methionine
sulfoxide
at different
experimental conditions
Th e reaction mixture contained 0.2 mM m ethionine and 0.2 M
Me2 S0 n themedium specified in the T able. The reaction was carried
out for
2
h at room temperature.
Medium
Methionine
sulfoxide
% of to ta l
Hz0
100 0
0.1 M NaH C03, pH 8.5 100 0
0.01
M
HCl 92
8
0.1 M HCl 30 70
1
M
HCl
0
100
1 M Acetic acid
100
0
5 M Acetic acid
80 20
a Determined by autom atic amino acid analyses.
TABLE1
Recov ery of am ino acids after incubation with Me2SO-HC1
The amino acid mixture (0.5 pmollml of each) was made 2
M
in
HC1 and 1 M n M e2S 0. The reaction was carriedout for 3 h at 25 C
before an aliquot was evaporated and its content analyzed.
Amino acid Recoverf
%
Aspartic acid 100
Threonine 99
Serine 100
Glutamic acid 104
Proline 98
Glycine 102
Alanine 100
Half-cystine 99
Valine 97
Methionine
0
Isoleucine 100
Leucine 103
Tyrosine 98
Phenylalanine 101
Lysine 103
Histidine 102
Arginine 99
Cysteic acidb 0
Methionine sulfoxide'00
Methionine sulfone' 0
Alanine was taken as 100% .
An o xidation product of cystine or cysteine.
Oxidation products of methionine.
Wavelenghhml
FIG. 1.
Spectrophotometric monitoring
of
N-acetyltrypto-
phan
treated with
Me2SO/HC1 or
chloramine-T.
The spectrum
of N-acetyltryptophan (final concentration 0.35 mM in 2
M
HCl) was
monitored
-).
MelSO was then added to a final concen tration of
1 M and the spectrum was remonitored after 2 h of incubation at
22 C
----).
Chloramine-T (20 m olar excess) was finally added and
the spectrum was remonitored
.
).
content.
As
shown in Fig.
2,
no detectable oxidation is ob-
served at the first2 h. Slow oxidation is obtained thereafter
(Fig. 2). As the oxidation of methionine residues by Me2SO/
HC1 are completed within shorter periods (next paragraph),
it implies tha t methionine can be modified selectively in the
presence of free SH groups.
Rate and Extent f the Reaction-When an excess of Me2S0
is applied over methionine (25-250
M
excess) in a fixed
concentration of HCl(1
M),
the rate of methionine oxidation
represents pseudo-first order rate (Fig. 3).Rateconstant
observed was calculated to be 0.23 f 0.015 M s at 22 C.
Methionine was quantitatively converted to methionine sulf-
oxide within 30 min (Fig. 3). Quantitative conversions were
still evident at 10 M excess of Me2S0 over methionine. Fifty
per cent conversion was obtained at about 4 M excess of
Me2S0 over methionine (not shown).
Oxidation
of
Methionyl Residues in Peptides
and
Proteins
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8/9/2019 J. Biol. Chem. 1986 Shechter 66 70
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68
Oxidation and Reduction of Methionine
I
I
2.6H
DHF 1
TABLE
Oxidation of methionyl
residues
in peptidesand proteins by aqueous
MezSO/HC1
0 L
vl
m
3.6M
HCI
1 2 3 L 5 6
Time (hours)
FIG.
. Oxidation of glutathione with time
by
MezSO/HCl.
Reduced glutathione (final concentration 3.6 mM) was incubated at
room temperature under the conditions specified in the figure. At
intervals, 25-p1 aliquots were withdrawn and added to solutions of 1
mM 5,5-dithiobis- 2-nitrobenzoic cid) in 0.3 M Tris-HC1 buffer, pH
7.4. The absorbance at 412 nm was then recorded.
L 8 12 16 20 2L
Time Iminutesl
FIG.
.
Rateof conversion of methionine to methionine sulf-
oxide by MeaSO/HCl. The reaction mixture contained 0 4 mM
methionine and the indicated molar concentrations of Me2S0 and
HCl.At intervals, 30-p1 aliquots were withdrawn, neutralized by
NH20H, lyophilized, and their Met and MetO content was deter-
mined.
by Me2SO/HC1-Table
I11
demonstrates the extent
of
methi-
onine oxidation of various peptides, polypeptides, and pro-
teins. Quantitative oxidations were obtained in methionyl-
containing di- and tripeptides. Also, the single methionyl
residues of ACTH, glucagon, and a-lactalbumin
of
bovine
milk were quantitatively oxidized. In bovine pancreatic ribo-
nuclease,
25%of
the methionyl residues were oxidized (Table
111).
Since in thisprotein only
1
out
of
the
4
methionyl
residues is exposed to modification by other oxidizing agents
2), it seems likely that aqueous Me2SO/HC1 discriminates
between buried and exposed methionines in large polypeptides
as well.
In order to examine whether methionine can be modified
in thepresence of cysteinyl residues, the four disulfide bonds
of
a-lactalbumin were reduced and the random polypeptide
was subjected to Me2SO/HC1 for 2 h. The single methionyl
residue was quantitatively modified with no observable reduc-
tion in free SH groups within the first 2 h (Table 111).
Reduction of Methionine Sulfoxide by Me2S/HC1-Treat-
ment
of
methionine sulfoxide with dimethyl sulfide (Me2S)
and high concentrations of hydrochloric acid resulted in the
rapid conversion
of
methionine sulfoxide back to methionine
(Fig.
4).
The raection proceeds readily a t 10.7 M HC1 and the
rate decreases as theH 2 0proportion in the reaction medium
increases. Thus, at 4.4-10.7 M HC1, the reaction proceeds to
completion, while a t lower concentrations of HC1 (i.e. 1.0 M),
the extentof the reduction is retarded tremendously (Fig. 4).
Reduction of
1
Meto-a-Lactalbumin
by
Me2S/HC1-Table
IV
demonstrates that the reduction
of
a MetO residue in a
Substance oxidized
Methionine
Free SH
sulfoxide
Methionine
Methionyl-valine
Methionyl-aspartic acid
Methionyl-phenylalanine
Methionyl-phenylalanylglycine
Adrenocorticotropic hormone
Glucagon
Ribonuclease
A
a-Lactalbumin
Reduced a-lactalbumin
(ACTH)
%
100
100
100
100
100
100
100
25
100
lood loo
% initial
value
a
Extent of oxidation in the methionyl peptides and proteins was
determined by the cyanogen bromide method described under EX-
perimental Procedures.
One residue (out of 4) was modified.
The single methionyl residue of a-lactalbumin was quantitatively
modified.
Reduced a-lactalbumin (prepared as described under Experi-
mental Procedures was oxidized for 2 h at 22 C n aqueous Me2S0
(0.1 M), HCl 1M).
e Samples were withdrawn for SH determination after
1-
and 2-h
intervals.
72
W
.
X
?Li2lL
0
4
8
12 16
20
24
28
32
0.5MOM
6
M q S
CI
40
Time (minutes)
FIG. . Conversion of methionine sulfoxide to methionine
by MepS/HCl.
The reaction mixture contained 0.089 mM methionine
sulfoxide (MetO) and thendicated concentrations of dimethyl sulfide
(Me2S)and HCl. At intervals, 70-pl aliquots were withdrawn, diluted
10-fold with cold HzO, lyophilized, and their Met and MetO content
was analyzed.
TABLE
V
Reduction
of
1-Met0-a-lactalbum in under arious conditions
Conditions applied
Reduction
of
methionine
sulfoxide in a-l actalbumin
m o MetOlmol a lactalbumin
1M
Me2S in Hz0 0.97
10.7
M
HC1,.5
M
MezS 0.05
6 M HC1,.5
M
Me2S 0.03
4
M
HC1,.5
M
Me2S 0.03
2 M
HC1,.5
M
Me& 0.44
0.5 M HCl, 0.5 M MezS 0.69
The protein derivative (2 mg) was incubated for 2 h at 22 C in
0.25 ml of the medium specified in the Table. The tubes were then
evaporated
to
dryness, dissolved in 0.3 ml of 70% formic acid, and
cyanogen bromide (50 molar excess) was added. The reaction pro-
ceeded for 24 h at room temperature. The samples were then evapo-
rated and acid hydrolyzed. MetO is converted to methionine during
acid hydrolysis in 78% yield. Values were corrected according to this.
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8/9/2019 J. Biol. Chem. 1986 Shechter 66 70
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Oxidation and Reduction of Methionine 69
protein snotobtained by Me& in he absence of HCl.
Quantitative reductions, however, were obtained at 4-10.7
M
HCl and were incomplete at lower HCl concentrations (Table
IV).This is essentially the same pat tern of reduction as was
obtained with the free Met 0 residue (Fig.
4).
Specifici ty of the Redu ction by Me,S/HCl Inactivation and
Reactivation of ACTH-As shown in Table
V,
under the
conditions used for achieving quantitative conversion of me-
thionine sulfoxide to methionine i e . at 10.7 M HC1,0.5 M
Me2S for
2
h a t room temperature), none of the standard
mixture of amino acids is modified. All the 17 amino acids
were recovered in quantitative yield. The same reaction con-
ditions, when applied to insulin did not cause any irreversible
denaturation of the hormone (not shown). As shown previ-
ously, treatment of ACTH with aqueous Me2S 0.1M)/HC~
1 M )
for 3 h at 22 C leads to oxidation of methionine to
methionine sulfoxide (Table 111). The modified derivative
retained about4% of the native hormonal activity (Table VI).
Treatment of the oxidized protein with 10.3 M HC1,0.3 M
Me2S for 15 min a t 37 C resulted in nearly full reactivation
of the oxidized derivative (Table VI).
It,
therefore, seems that
Me2S/HC1 can be used to reduce and reactivate proteins of
TABLE
Recovery
of
amino acids after incubation with
Me2S/HC1
The amino acid mixture (0.217 pmol/ml of each amino acid) was
incubated for 2 h at room temperature in 10.4M HCl, 1M Me2S. The
sample was then evaporated and an aliquot of its content analyzed.
Alanine was taken as 100%.
~ ~ ~~~~
Aminocid
Recovery
%
Aspartic acid 100
Threonine 104
Serine 97
Glutamic acid 100
Proline 98
Glycine
107
Alanine 103
Half-cystine 97
Valine 96
Methionine 100
Isoleucine 97
Leucine 100
Tyrosine 103
Phenylalanine 101
Lysine 96
Histidine 96
Arginine 108
TABLE
I
Biological activity of oxidized and reduced ACTH
Derivative EDrn'
Relative
bioactivitg
ng ml-
ACTH 2.7 100
Oxidized ACTH' 68
4
Oxidized and reduced 4.9 55
ACTHd
The ACTH or modified ACTH concentration that produces half-
maximal effect in lipolysis.
etermined by lipolysis in rat adipocytes according to Ref. 23.
22 C.
'Oxidation was performed at 1 M HCl, 0.1 M Me2S0 for 2 h at
An aliquot of the oxidized ACTH was evaporated and reduced in
10.3 M HC1, 0.3 M Me2S for 15 min a t 37 C. The sample was then
loaded on a Sephadex G-10 olumn (20 X 1cm), equilibrated and run
with
M
HCl, 0.1 M NaCl. The protein ractions were ooled.
ACTH concentration was determined by hydrolyzing an aliquot and
determined its amino acid content.
biological interest after oxidation of their methionyl residues.
DISCUSSION
The present study shows that Me2S0, Me2& and HCl can
be efficiently used for the selective oxidation and reduction
of methionyl residues in peptides and proteins. The reactions
involved and conditions applicable to peptides and proteins
are summarized in Scheme
1.
Oxygen exchange between
Me2S0and methionine is acid-dependent (Table I). The
chloride anion is effective in promoting the exchange. High
concentrations of HCl, however, are not required and the
reaction proceeds readily to completion at 0.5-1.0 M HCl
(Table I, Fig. 3). Similarly, the reaction is completed at low
concentrations of Me2S0 i x . at 0.01 M Me2S0, Fig. 3),
therefore eliminating the possibility of protein denaturation
due to high concentrations of an organic solvent. The oxygen
exchange between M e 8 0 and methionine can be considered
specific to this mino acid residue, since other side chains are
not modified (Fig. 1, Table 11),with the exception of cysteinyl
groups (Fig.
2).
The latter , however, are oxidized very slowly
and the reaction is initiated only after a lag period of
2
h at
room temperature (Fig.
2).
This issue also agrees with the
work of Snow et al.(27) who demonstrated that the xidation
of the SH roup of penicillamine by Me2S0 at7 C is grossly
retarded a t low pH values i.e.at pH 1-2, Ref. 27).Methionine,
methionyl containing di- and tripeptides, as well as exposed
methionyl residues of proteins are eadily oxidized by Me2SO/
HCl (Table 111). Methionines, which are buried within the
three-dimensional core of the protein molecule (such as the3
methionyl residues of ribonuclease A), do not seem to be
modified either by Me2SO/HC1 (Table 111)or by chloramine-
T
(Ref. 2). The methionyl residue of reduced a-lactalbumin
was quantitatively modified within
2
h with no concomitant
oxidation of the cysteinyl residue of the polypeptide chain
(Table 111).
Using Me2S and HCl, the opposite reaction, namely the
reduction of methionine sulfoxide to methionine,
also
pro-
ceeds to completion (Fig.
4,
Table IV).This reaction, however,
will be completed only a t high concentrations of HC1 i . e .4-
10.7
M ),
since an increased proportion of
H 2 0
in the medium
is unfavorable for the reaction to proceed in the right direc-
tion. A concentration range of 0.3-0.5 M Me2S was found to
be sufficient to obtain a complete conversion
of
methionine
sulfoxide to methionine. This was found to be valid for both
the free amino acid (Fig.
4)
or for methionine residues of a
protein molecule (Tables IV and VI).
The present study has several potential applications in
chemical and biological sciences. Examples are a ) to study
-NH-Ui-CO-
NH- CO-
I
Fh 0s
F
CH,
cn
S
0.5MHCI,O.IMMe$30 s.0
cn
22
OC,30-180min
a 3
-NH-CH-CO- -NH-CH-CO-
I I
t
ScY 7
CH,
S=O 4.0-K17MHCl.a3 ~~S S
I
I
cn
22
OC,30-180min
SCHEME
.
Oxidation and reduction
of
methionyl residues
cy
by MezSO and MezS.Optimal conditions for the reactions.
-
8/9/2019 J. Biol. Chem. 1986 Shechter 66 70
5/5
70
Oxidation and Reduction of Methionine
the oxygen exchange between sulfoxides and sulfides of mac-
10. Burstein,
Y.,
and Patchornik, A. (1972) Biochemistry 1 1 ,4 6 4 1 -
romolecules under hydrous or semianhydrous conditions and
4650
b ) to
determine the biological significance of certain methi-
11.
Hachimori,
Y.,
Horinishi, H., Kurihara, K., and Shibata, K.
onines in macromolecules of interest and the relation
O f
the
12. Toennies, G., and Callan, T. p. (1939) J . B ~ Lh m . 1 2 9 , 4 8 1 -
three-dimensional structure of the macromolecule o the ate
490
of methionine oxidation by Me2SO/HC1. The reduction pro-
13. Morihara, K. 1964) Bull. Chem. SOC. pn 37,1781-1784
cedure may also be useful for radioactively iodinated her-
14.
Tashjian,
A.
H., Jr., Ontjes, D. A., and Munson, p.
L. (1964)
mones and proteins, since oxidation of methionines to methi-
15.
Bodwell,
F.
G.,
a;ld
Pitt,
B.
M.
(1955)
J
Am
Chem.
sot,
,7,
onine sulfoxide is an undesirable side reaction which occurs
572-577
readily during iodination due to the presence of chloramine-
16.
Searles,
S.,
and Hays, H. R.
(1958) J.
Org.
Chem. 23,2028-2029
T
2)
or hydrogen peroxide
28-30).
17.
Hull,
C.
M., and Bargar,
T.
W.
(1975)
J Org.
Chem. 40 , 3152-
18.
Ruffato,
V.,
and Miotti,
V. (1978)
Gazz.
Ch im ica ZtaZ. 1 0 8 , 9 1-
and th e careful reading of the manu script bY Dr. Cynthia Webb are
19.
Lipton, S. H., an d Bodwell, C. E.
(1976)
J
Agric.
ood
C h m .
gratefully acknowledged.
2 4 ,2 6 -3 1
20.
Bovio,
A,.
and Miotti. V.
(1978)
J .
Chem. Soc.
Perkin Trans. I .
(1964) Biochim. Bwphys. Acta 9 3 .3 4 6 -3 6 0
Biochemistry 3 1175-1182
3154
Acknowledgments-The skillful technic al assistance of B. Zarmi
96
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