COLUMN MATERIALS AH - Sepharose-4B, Sephadex G-100...
Transcript of COLUMN MATERIALS AH - Sepharose-4B, Sephadex G-100...
MATERIALS AND METHODS
COLUMN MATERIALS
AH - Sepharose-4B, Sephadex G-100, Sephadex G-200,'
Blue dextran, Low molecular weight markers f~r gel
filtration, BSA (67,000), Ovalbumin (43,000), chymotryp
sinogen A (25,000), Ribonuclease A (13,700), were pro
ducts of Pharmacia Fine Chemicals,Sweden. All columns
used in the purific~tion were made of glass or Bio-Rad
columns.
Electrophoresis Materials
Acrylamide, N-N'-methylene bis-acrylamide and
brqrqophenol blue were purchased from Bio-Rad L~boratories,
USA. Commassie brilliant blue R-250, sodium dodecyl
sulfate, 2-mercaptoethanol, ammonium persulfate, TEMED,
were obtained from Sigma Chemical Co., St. Louis.
Molecular weight marker kit for SDS-PAGE, e. g'., Bovine
sert;tm albumin (67,000), ovalbumin (43,000), carbonic
anhydrase (30,000) ,Trypsin inhibitor (20,000), Lactal
bumin' (14,400) was purchased fromPharmacia Fine Chemi
cals, Sweden.
Radioactive Chemicals
r 125 (specific activity 100 mCi) were obtained
from the Radiochemical Centre, Amersham, Bucks, UK.
Ch~micals
Hyaluronic acid type-r (from human umbilical. cord) ,
gl utaraldehyde, glllcuronic acid, N-acetyl-gl ucosamine,
,D-glucuronolactone, N-acetyl-neuraminic acid, chondroitin-
4-sulphate, chondroi tin-6-sulphate, Protease type XIV
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(Pronase E) ,Tris, Conunassie brilliant blue R-25,O,
p-dimethylaminobenzaldehyde, EDTA, glycine, hyaluroni
dase; chondroitinase ABC, heparin, blue dextran, sialic
aCid,'thiobarbituric acid, cyanogen bromide, dye amido
black, cyclohexanone, Toluidine Blue, toluidine were
obtained from Sigma Chemical Co., St. Louis. Polyethy
lene glycol-20~000, was from Fluka, BuCha Switzerland,
Protein reagent was purchased from Bio-Rad Laboratorie~.
Iodogen was from Pierce Chemical Col, Rockford, USA.
Iodogenis a trademark for chemical 1,3,4,6-Tetrachloro-
30(.., 6 oc;..-Diphenyl-glycouril. Mono-sodium citrate,
methyl-cellosolve, Thiodiglycol, Brij-35, sodium-hydroxide,
sodium chloride, sodium acetate hydrazine-sulphate,
Ninhydrin were .from E. Merck., England .. Some of the other
chemicals were purchased from E.Merck and BDH (AR grade) .
All other solvents and acids used, were obtained from
conunercial sources in highest purity grade available.
Millipore immercible CX-10 single use - ultrafiltration
unit were purchased from Millipore, USA~
Processing of Dialysis Tubing
Dialysis tubing obtained from Medicell International
Ltd., Liverpool, London, (England) ,was boiled for fi~e
min in 10 mM EDTA solution and then washed with distilled
wa ter.
Maintenance of Rats
Wistar rats of different age groups were purchased • from Haryana Agricultural University, Hissar and All
In~ia Institute of Medical Sciences, New D~lh{. The
rats ~ere maintained with proper facility and bred f6r
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newborn (2-3 days), 3-4 week (young ones) of age rats in
our animal house. Animals were fed ad libitum -the diet
obtairied from Hindustan Lever Ltd.
Activation of AH-Sepharose-4B with Glutaraldehyde
Cambiaso's(142) method was used for the activation
of AH-Sepharose-4B beads with ,glutaraldehyde. The glu
taraldehyde acts as a homobifunctional reactant. To
couple, the desired ligand, -Le., hyaluronic acid on one
hand and the commercially availableSepharose-4B beads
already substituted with 6-amino-hexyl side chains (AH
Sepharose-4B, from Pharmacia) on the other hand, made·
the task to conjugate HA to amino hexyl sepharose easier,
by means or two step reaction with glutaraldehyde and
later 'the conjugation ,with HA. The aminated gel, was
first washed with ten volumes of PBS (0. aIM Phosphate
buffer saline; pH 7.2,0.80% NaCl) on a sintered glass
filter. 7.0 ml PBS b~ffer containing one ml of 25%
glutaraldehyde was added to 3.0 ml of packed gel with
constant stirring so that the final concentration of
glutaraldehyde was 2.5% •. This represents a lOa-fold
molar excess of glutaraldehyde over the amino groups .of'
the aminated gels (8 p-mole NH 2/ml packed gel). A
yellow colour soon developed after the addition of gluta-
'raldehyde. The reaction was allowed to proceed for
twenty minutes at 40 C after which the tinreacted glutaral
dehyde' was eliminated by washing five times with 20 ml
PBS ..
Coupling of Hyaluronic Acid to the Glutarladehyde Acti
vated Amino-Hexyl Sepharose
As mentioned above, the aminated gel, which is now
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glutaraldehyde activated gel, we added 1 ml of hyaluronic
acid solution (Grade I) (10 mg/gm of gel) in PBS to 3 ml
of glutaraldehyde activated gel,under continuous mild
stirring. The reaction was allowed to proceed at 40
C ·for
20 min. After'the incubation period, the gel was washed
five times with 20 ml of PBS to allow the non-conjugated
hyaluronic acid to diffuse out of the gel beads. A
further washing with 0.2 M'glycine;buffer, pH 8.5 was
carried out to remove non-covalently bound hyaluronic
, acid~
The conjugation of hyaluronic acid to glutaral
dehyde activated gel was checked quantitatively. As the
quantity used to couple'HA to glutaraldehyde activated
gel,was known, and the total content of'HA in the wash
was determined according to Bitter and Muir(145) and
deducted from the absolute amqunt used for conjugation.
Thus, it was seen'that 2.2 mg of HA was bound perml of
packed Sepharose gel. The HA-coupled sepharose, AH
Sepharose-4B was scanned in Shimadzu UV240 from 200 to
300 nrnwavelength.Gel was suspended. in 42% sucrose
solution and scanned (Fig. 3). The gel was further in
cubated for sixteen hours at 40 C with 0.2 M glycine
buffer, pH 8.5, to block unreacted aldehyde groups, and
to completely prevent freshly activated gel from cova
lently binding hyaluronic acid.
prepara'tion of Chondroi tin-4-Sulphate-sepharose-4B Affinity
Gel
The method used to couple-chondroitin'"-4-sulphate
to AH-Sepharose-4B via glutaraldehyde was same as men
tioned in hyaluronate-Sepharose affinity gels. Accord.-
•
>-t---Vl Z UJ 0
---1 0.1 « LJ
t--0... 0
I I I I' I , , \ \ \ \ \ \ \ \ \ \ \ \ \
\ \ ,
\ \ \
'-..-.-....
'\ , " " " " , '" '"
\ .'
\
"
\ \ \ \ \
\
" " " ----'0.0 0 '---______ -----JL....-____ -_-_--=-:.==-~...oL
Fig. 3
200 300 WAVELENGTH nm
Absorbance spectrum of HA, HA-Sepharose, and AH-sepharose-4B. AH-Sepharose-4B and HA-sepharose Were suspended in 40% sucrose and their absorption spectra at 200-400 nm. __ ' ___ Hyaluronic acid -------- AH-Sepharose-4B -.-.-.-.-. HA-sepharose
400
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. , ing to Cambiaso et al. (142) to 3 ml of glutaralciehyde
activated gel, j ml of chondroitin-4~sulphate (Type-C)
(10 mg/gm of gel in PBS) was added, under constant mild
stirring. The reaction was allowed to proceed at 40
C
for twenty minutes after th'? incubation period.' The gel
was washed with 20 ml of buffer for five times to allow
the non-conjugated chondroitin-4-sulphate to diffuse out
of the gel beads. A further washing with 0.2 M glycine
buffer, pH 8.5, Vlas carried out to remove non-covalently (
bound chondroitin-4.;..sulphate.
In order to check the coupling of chondroitin -4~
sulpha te to the gl utaraldehyde-acti va ted gel, the gel
was scanned in ShimadzuUV-240 . from 200 to 300 nm wave
length. The gel was suspended in 40% sucrose solution and
scanned (Fig. 4). The amount coupled was determined by
chondroitin-4.;..sulphate estimation of coupling solution
before and after coupling. Approximately 1.7mg of chondroitin-
4-sulphate was bound per ml ·of packed gel.
The gel was incubated for sixteen hours at 40 C with
0.2 M glycine buffer, pH 8.5, to block unreacted aldehyde
gr:o,ups, arid to completely prevent freshly activated gel
from covalently binding to chondroitin-4-sulphate. When
the gel was nOt in use, they were stored in 0.02% sodium
'azide in buffer to prevent microbial growth.
Activation of Sepharose .with Cyanogen Bromide:
Porath's (143) procedure was used for activation
of Sepharose 6B beads with cyanogen bromide. 10 gms of
Sepharose 6B was washed with 40 ml of 2M potassium phosphate
buffer pH 12.1 on a sintered glass funnel (G2 ). 'I'hen the
gel was suspended in 15 ml of 5M potassium phosphate
>-~ -C/)
z w 0
~
<i u -~ Q... 0
O. 2 .-r-~;----.---~--------~---4
0.1
I' I" I '\
l \ I I \ \ \ \ \ \ \
\ \ \
\ \
"
\
\ . \
" " ;\
" '- ....... \ .........
........ ....... \
" O.OO~--------------~--------~~~--~~
. 200
Fig. 4
300 WAVELENGTH nm
400
Absorption spectrum af chondraitin-4-sulphate chondroitin-4-sulphatesepharose, and Jl.H-sepharos-48. AH-sepharose-48 and chondroitin-5-sulphate-sepharose, were suspended in 40% sucrose and their absorption spectra at 200-400 nm.
-.-.-.-.-.
Chondroi tiri-4-sulphate AH-sepharose:.48 Chondra i t i n-4 -sulpha te -sepharose
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buffer pH 12.1. 6 ml of cyanogen bromide solution(lOOmg/
ml) was added in portions during a period of 2 min and
the reaction was allowed to continue for 8 more minutes
with gentle stirring. The temperature of S°";;lOoC was
maintained during the reaction time. The product was
washed with cold distilled water to neutral pH. Immediately;
after the washing the activated gel was tran~ferred ~o
heparin i.e., ligand solution, activation was done in a
well ven'tila ted place or under· furnehood. Precautions was
taken not to touch or inhale CNBr. vapours.
Activation of gel was checked qualitatively according
to the procedure described by Kohn and Wilchek (144). '1'0 a
few drops_of activated material one or two ml of qualitat
ive reagent was added. After 30 seconds a red purple
colour developed which,became maximum after 10 min. The
qualitative reagent was prepared by mixing 12 ml of pyridine ~
slowly with 2.5 ml of concentrated hydrochloric acid. 0.5 g
of barbituric acid was added ,to this and volume was made , .
upto 20 ml with distilled water. A clear. colourless solution
was obtained after stirring for 10 min.
Preparation of Heparin Sepharose:
20g of activated Sepharose 6B was suspended in 100 ml.
of heparin (1 mg/ml) solution made in 0.1 !vI N,aHC0 3 contain
ing O.SM Kcl. The suspension was kept at 300 C 'for overnight
with gentle stirring. The gel was filtered on a sintered
glass funnel (containing O.lM NaHC03 and O.SM Kcl) and O.SM
Kcl solution. Heparin concentration in coupling solution
was determined before and after coupling reaction by using
to~uidene blue. To 20 pl of sample, 0.5 mI. of O.lSM Nacl
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solution was added. To thisO.Sml buffer containing O.lM
Boric acid, O.ISM NaCl, pH 8.5 and 0.2Sml. of Toluid:tne
Blue (0.015%) were added absorption at SDQ nm was
recorded (Fig. S);
All these column chroma~ographic analysis were
conducted in Pharmacia Frac-100 Fraction Collector with
uV-I monitor, peristaltic pump and Recorder I with the
efficiency of monitoring as low as S fl9 of protein.
'Assay of Hyaluronic acid:
I, Hyaluronic acid was estimated according' to the
method of Bitter and Muir (14S) S ml. of sulphuric
acid reagent (0.02SM sodium tetraborate, 10H2 0 in
sulphuric acid sp. gr. 1.84) was cooled at 4o C, 1 mI.
of the sample is lay~redon to the acid. The tubes were
closed with glass stoppers. The tubes were shaken at
first gently and then vigorously with conStant cooling;
Th,e tubes were then heated for 10 min. in a boiling
water bath and cooled at room temperature. 0~2 ml of
carbazole reagent (0.12S% carbazole in absolute ethanol)
was then added, the tubes were shaken again and heated
in a boiling water bath for further lS mi~. It was then
cooled to room temperature. The optical density was read
at S30"rnu. Glucuronolactone standards 'of 20-120 umoles/ml
were used as shown in Fig 6 by dilution with deionized
water saturated wi,ttl benzoic acid from a stock standard
in water saturated with benzoic acid.
'Fig. 5
2
.-. I \. .
w I \ u . z I \ <i "'-, (I) 1 I
I , /. , \ a:: \
0 i: .' \ Vl , .,
m II , " \. <i
., .~'
,. ~- ---...........
.........
OL..------....I.------....J 190 240 .290
WAVELENGTH (nm)
Absorbance spectrum of heparin, CNBr-Sepharose and sepharose CN3r-sepharose and heparin-sepharose were in 40% sucrose and their absorption spectrum were taken.
- ..... -.-.-.
heparin CNBr-sepharose Heparin-sepharose
heparinsuspended
1.2
·1.0
E c 0.8
(:)
rn LI"I
.t-:- 0.6 4:
>-t-:- . -l/) 0.4 ·z UJ Cl
. ....J 4: 0.2 u -t-o... 0
0.02 0.04 0.06 0.08 0.10 0.12 ..
( 0 NeE NT RAT ION 0 F G L U CUR 0 NO LAC TON E (f mol e·s )
~6.. Standard Curve for hyaluronic acid.
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Assay of Hyaluronidase
Hyaluronidase activity was measured by the method of
polansky and Toole (88). Hyaluronidase activity was
determined by the measurement of the·terminal N-accetyl
glucosarnine in oligosaccharides released during incubation
of hyaluronate with hyaluronidase.
Hyaluronidase'activity was measured simultaneously
for standard hyaluronidase and for purified hyaluronic
acid binding protein.
For the standard hyaluronidase, the incubation·
mixture consisted of 0.8 ml of HA (0.8 mg/ml, in 0.1 M \
sodium formate buffer,· pH 3.7, containing 0.15 M Nacl)
and O. '4 mI. of standard hyaluronidase (Sigma, from umbi
lical cord) (200ug ml of 0.1 M sodium formate buffer,
pH 3.7) whereas, the incubation mixture for purified
hyaluronic aciq binding protein consisted of 0.8 ml of
HA, (0.8 mg/ml, in 0.1 M'sodium formate buffer, pH 3.7),
containing 0;15 M Nacl) and 0.4 ml of purified HB~
(200 ug in PBS). The standard hyaluronidase as well
purified, HBPwith the 'above incubation mixture was
, incubated for six hours in a water bath at 37o C. pre
c~ution,was taken to. check microbial growth during the
i'ncuba tion by adding 15 rnM Saccharolactone to the
incubation mix~ures. The blank corisisted 0.4 ml of
purified HBP and'0.8 ml of 0.1 M sodium formate buffer,
PH 3.7 containing 0.15 M NaCl. Enzymatic reaction was
terminated by boiling the tube at 100~C for 3 minutes:.
The enzym'atic activity of blank was' terminated at zero
minut,e. Release of terminal reducing N-Acetyl hexosamine
as a result of hyaluronidase action was measured accord-,
, ing to Reissig ~t al (146).
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N- Acetylglucosarnine was estimated according to
Reissig et al (1461 .. To 0.5 ml of samples was added O.lml
of sodium tetraborate, pH 9.2. The tubes were kept in a
boiling water~bath for exactly 3 minutes and cooled in
tap water. 3 m'l. of DMAB reagent (10 .,9 of p.-DIV'lAB·was
dissolved in 100 ml of, glaci,al acetic acid which
contained 12.5% wjV ION Hcl. Before using it was diluted
with 9 volumes of glacial acetic acid) was then added
and immediately after mixing the tubes were then placed
in a water bath at 36-380 C. After precisely 20 minutes the
tubes are cooled in tap water ,and the optical density was
read at 578 mu. Standard curve for N-acetylglucosamine
is presented in Fig. 7.
Assay of Sialic Abid:
. The Sialic Acid content was measured according to
Warren etal (147). To a sample of 0.2 ml (approximately . -o .o? ~ole of N -acetylneuramn.ic acid) 0.1 ml,of periodate
in 9 M phosphoric acid} was added. The tubes were shaken
and a~lowed to stand at room temperature for 20 minutes.
Iml of arsenite solutism (10% sodium arsenite arid O.OSM
sodium sulfate in 0.1 M sulphuric acid) was added. The
tubes were shaken until a' yellow brown colour disappears.
Thiobarbituric acid solution (0.6% in O. 5M sodium, sulfate)
,was added, tubes were shaken capped with glass stopper
and heated in a boiling water bath for 15 min. The tubes
are then placed in cold water for five miriutes. This
,~ixture4.3 ml is tran;ferred to a tube containing 4.3 mI.
of cyclbhexanone. The tubes are shaken twice qnd then
centrifuged for 3' minutes in a centrifuge. The clear
upper cyclohexanone phase was red and the colour is more
0.6
-. E 0.5
-00 " 0.4 L()
t-e:(
>- 0.3 t- '
Ul Z w 0.2 c ...J
~ 0.1
t-o... o
.02 .04 .06 .08 ·10 '·1 ,1
CONCENTRATION OF N-Ac-Gm (fJ mol ) ,
~I Standard curve for N .... AcetyJ-gJucosamine.
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intense than in wat~r. Optical density of the organic
phase are -determined ,at 549 nm. Blank contained 0.2 mI.
of distilled water. N-acetylneuraminic acid was used
for the standard curve and presented in Fig. 8.
Protein Determination:
The protein content of the column effluent was
monitored at 280 nm using an ISCO UA-5 absorption
monitor with type 6 optical unit.
Protein is assayed by the method of Bradford
(148) using Biorad Bradford reagent and BSA as standard.
This method is based on the observation that cOmniassie
brilliant blue G-250 exists in two different colour
forms~ red and blue, the red form is converted to the
blue upon binding of the dye to the protein. The protein
dye has a high extinction coefficient thus leading to
great sensitivity in measuring protein. Th~ binding of
the dye to protein is a very rapid process (approx. 2 min)
and the protein-dye complex remains dispersed in solution
for relatively longtime (approx. 1 hr), thus making the
procedure very rapid and yet not requiring critical
timing for assay.,
preparation ot Bradford Reagent:
Commassie brilliant blue G-250 (100 mg) was
dissolved in 50 ml. of 95% ethanol. To this 100 ml.of
85% (V/V) phosphoric acid and 50 ml. distilled water
was added. Final concentration in the reagent were 0.05%
(W/vt Commassie brilliant blue G-250, 23.5% (W/V)
ethanol, and 42.5% (W/V) phosphoric acid.
0.6 E c
C3' 0.5· ~
LI1
r-« 0.4 >-r-(/) 0.3 z w 0 --l 0.2 « LJ
r- 0.1 a.. 0
0.2 0.4 0·6 '0 .. 8 1.0 1.2 1.4 (0 nc.o f N - Ace t y 1- N e u r Q min i C Q C i d ()1m 0 Ie s 1
Fig. 8 Standard Curve for N-acetyl-neurarnic acid.
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Assay Procedure:
. Protein standard containing 1 to 20 JIg in a volume
of 0.1 ml. then diluted the sample to 0.8 ml. with
distilled water and 0.2"ml. in Bradford reagent was
added to it and mixed several times by gentle inversion
of the test 'tube. ,The absorbance of 595 nin was measured
after 5 miri. to one hou~ against a reagent bl~nk prepared
from 0.8 ml. of the appropriate buffer water anq 0.2 ml.
of Bradford reagent~
ELECTROPHORESIS
a) Polyacrylamide gel Electrophoresis (PAGE),:
The purity of the enzyme was checked by disc gel
~lectrophoresis at pH 8.9 by the method of Davis (1491.
Elettrophoresis was carried out at 4o C. The separation
gel was made of 7.5% aorylamide and 0.27% N, N-methyl
bi~-acrylamide coritained 0.375M Tris-Hcl buffer, pH
8.9. 'The stacking gel. of 3% acrylamide and 0.08% N,- N
methyl bis-acrylamide contained 0.067 M,. Tris-Hol pH 6.1. '"'
Then;the solution was polymerised b"y adding 0.050%
ammonium persulfate and 0.0"50% TEMED. The gels, were
poured in glass tube ( 6 x 100 mm). The sample protein
(10~40 ng) with 10% glycerol was loaded. The running
buffer containedO.05M Tris-Hcl and G.3 M glycine at .
pH 8.3 and electrophoresis was commenced by applying 1
rnA current per gel. After 30 min. the current was , .
raised to 4 rnA per gel, till" the marker dye reached about
0.5 cm from the bottom of the gel tube.
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After electrophoresis, the gels were sta~ned for 4
hours for protein with 0.2% corrunassie brilliant blue
R-250 in methano~, glacial-acetic acid and water (25 : 10
65) and then destained in methanol, acetic acid and water
(20: 10: 70).
b)SDS - Polyacrylamide gel'Electrophoresis(SDS-PAGE):
"',',;
SDS-PAGE was done according to the method of Laemmli
(150). The ~eparating gel was made of 10% acrylamide 0.27%
N~N' methylene bis - acrylamide, 0.1% SDS and 0.375 M tris
Hcl buffer (pH 8.8). 'l'he various_ components were mixed
deaerated-and polymerization was initi'!l-ted by adding \
0.025% ammunlum persulfate. The, solution was quickly
poured into 6 x 130mm gel tubes, and water layered on
top. After polymerization was over,stacking gel contain
ing 3% acrylamide, 0.08% N-N' ..:. methylene - bisacrylamide
O~l% SDS and 0.125 M Tris-Hcl (pH 6.8) was poured and
allowed to polymerize. The separation gel of 100 rom were
prepared. The running buffer was composed of Tris(0.025M)
glyCine (0~192 M), buffer, pH 8.3 ~nd 0.1% SDS~
The sample buffer for SDS-PAGE had the following
composition:
, Tris-Hcl
SDS
'Glycerol
2- Mercaptoethanol
Bromophenol blue
0.0645 M(pH 6.8)
2 %
10%
5%
0.001%
The samples were irrunersed for 2 min. in boiling
water bath cooled and loaded onto the gels. Standard
proteins were also run each time alongwith the sample.
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After loading the samples, stacking was carried out at
1 rnA per gel in constant current mode and electrophoresis was
donee; by ·using a current of 4mA per gel at 30oe, till the
marker dye reached about I em from the bottom of the gel
tube. The gels were stained overnight after the run in a
staining solution and detained as mentioned in neutral
gel electrophoresis.
Molecular, Weight Determination:
Molecular weight determination of Hyaluronic acid
binding protein was determined by gel filteration on a
Sephadex G-100 column ( 1 x 57cms) according to the
method of Determan and Michel (151). The column was first
equilibrated with PBS (0.01 M Phosphate buffer pH 7.2 Nacl
8 gm/L) by passing at least five column volumes of the -
buffer through the column at a constant pressure and
flow rate. After equilibration, the void volume of the
column was determined by passing 2 rnl (2 mg/ml) Blue
Dextran. The position of the maximum of the peak in ;the
elution volume was taken as the elution volume of blue
dextran as well as the void volume (Vo) of the column
as in Fig. 9. Fo:!;, calibration of the column four prot'eins
of known mole6ular weight (ribonuclease, 13,700);
Chymotrypsinogen, (25 i 000); ovalbumin (43,00 0) and bovine
serum alb}lmin 6 mg/2ml (67,000) were run and from the
elution volume of each proteinKav and from the elution
volume was determined with the help of the following
relationship:
0.30
0'.25
0.20 0 co 0.l5 ("oJ
<!
0.10
0.05
0, 5 10 15 20 25 30 35 40 F R A[ T ION N U M B E R
Elution rofileof Blue-Dextran on se hadex G-100 column: 2 ml Blue dextran (2 mg mJ) in 100 mM Sodium phosphate buffer containing 0.8% NaCI, pH 7.2, was applied to a Sephadex G-100 column (1x54cm) equilibrated previously with the 'same buffer. Elution was done with' the same buffer at a constant flow rate of 16 ml/hr.
K " av
K av
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=
=
=
=
=
v - V e 0
Vt - Vo
elution volume of standard proteins.
Void volume.
Total volume of the gel bed.
Partition coefficient betwee
the liquld phase and the gel
phase.
By plotting K values against logarithm of mole~ av cular weights (in a semi-logarithmic plot) a standard
curve was obtained. Purified HBP and low mol.wt.protein
markers were run through the column and from their
elution volumes (V ) molecular weight was determined , e using the calibration curve.
AMINO ACID ANALYSIS
Amino acid analysis was done according to Moo~e
et ai. (152) ~n the Technicon Sequential multisample(TSM)
amino acid analyzer. It is an automated instrument
designed to separate, detect and quantitate amino acids.
Principle:
The rate of travel of amino acids on a column of
a sulfonated polystyrene resin is a function of both the
charges possessed by an am~no acid and the riature of its
side chain, it is a result of the affinity of the resin
for both the ionic and'non-ionic portions of the molecule.
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Thus" an amino acid with a branched side chain will be
~luted earlier than a straight sid~ chain, likewise,
an amino acid, with the presence of an acidic group on
side chain will be eluted earlier than the basic group
on side.chain.
Reagents Used:
1. Buffer No.1 - 100 mM Citrate Buffer, pH 3.25
To 3 litre of 100 mM citrate buffer in IN NaOH
was added,
a) 240 ml of methyl cellosolve
b) 1.0 ml thiodiglycol
c) adjusted pH 3.25 with 6N HCl
d) added 40 ml 6f Brij-35.
Volume made to 4 litres with distilled water.
2. Buffer No.2 - 100 roM Citrate Buffer, pH 4.25
To 3 litres of 100 mM citrate buffer in IN
NaOH was added.
a) 1. Q ml of thiodiglycol
b) pH adjusted to 4.'25
c) added 4'0 ml of Brij-35.
Volume made upto 4 litres.
3. Buffer No.3 - 100 mM Citrate Buffer, pH 5.25
q'o·3 litres of 100 mM citrate buffer in IN
NaOH added
a.) 1.0 ml of thiodiglycol
b) pH adjusted to 5.25
c) added 40 ml of Brij-35
d) added 40.0 gm of NaCl.
Volume made upto 4 litres with distilled water.
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4. Ninhydrin Reagent
1% Ninhydrin reagent is used in AN sodium
,acetate buffer and methyl cellosolve in 4 litres of
total buffer.
5. Hydrazine Solution
2 mM solution of hydrazine sulfate was made in
.distilledwater and 30% of Brij-3S, making the volume
to 4 litres.
6. Methyl Cellosolve
50% of methyl cellosolve was made in distilled
water.
PROCESSING OF THE SAMPLE
a) Hydrolysis
Hydrolysis of the sample. was 'done according to
Moore et al.(lS2) and as modified by Chatterjee 'and
Abrol (NRL, IARI., New D'elhi, unpublished) .• (153) .
One mg of HBP (purified) was taken in hydrolysing
tubes (18 x 150· mm) and 12.0 ml of 6 N HCl was added.
The sample in the tubes were chilled and evacuated for
30 min. The hydrolyzing tubes were sealed under vacuum
and kept at 1100 + lCC for 24 hrs. After the hydrolysis,
the tubes are brought to room temperature (If the
analysis is not to be performed immediately, the hydro
lysis tubes can be stored in the deep freeze) .
- 47 -
b) Concentration by Flash Evaporation
After hydrolysis, the seal was broken and the
contents filtered through a sintere4 glass funnel. The
hydrolysing tube and the funnel were washed several
times with double distilled water to ensure qualitative
recovery of the amino acids. The filtrate was quanti
tatively transferred to a round bottomed flask of a
rotary flash evaporator. The sample was evaporated to
dryness at 50-550 C under 'reduced pressure. The residue
was washed 5-6 times with small volumes of double
distilled water to get rid of the excess HCl. Finally,
the residue was dissolved in one ml of sample buffer
(0.1 M sodium citrate" pH 2.0). This was the stock
supply of amino 'acids.
c) Loading of Sample for Analysis on TSM
An aliquot, of stock' (100 ul) was loaded on 'to two
sample cartridges, care, was taken not to leave sample
on the side walls of the, cartridges.
While keeping it vertical, a vacUum was applied
to the bottom of the cartridge. As soon as the sample
was drawn from the top filter, vacuum was stopped and
a Teflon plug was inserted. Norleucine was used as
an internal standard.
d) Chromatographic Ana!'ysi~
The automatic recording equipment is used in
conjunction with 2 ion-exchange columns of sulfonated
polystyrene resins. The long column is for the separa
tion of neutral and acidic amino acids while;shorter
- 48 -.
for basic amino acids. Buffers'are delivered to the
columns-through the sampler, from two high pressure
pumps. The sequence of these buffers from the storage
reservoirs is controlled bi a peristalic valve. This'
valv~sequence~ the buffer, as well as th~ flow of
effluent to the analytical system or waste. It is
electromechanically controlled.
The column effluent that flows into the analytical
system are mixed with a segmented stream of reagents
into the' system through the proportioning pump. The
combined streams are then passed through a heating bath
equipped on its output side, with a finned cooler. The
separated amino acids react to form a coloured product
in the reagent stream, whose absorbance is monitored
continuously in a colorimeter and recorded by a two-pen
recorder as a linear trace of each amino acid.
To calculate the amount of each amino acid
present in an·unknown sample the following formula is
used
A, )l moles = ~p x NE x )lMsp.
).m sp
where, A = Area unknown peak )lP
Aun = Norleucine area from the same chromatogram
NE = Norleucine equivalent to the corresponding sp peak from a standard chromatogram
)lMsp = )lffioles in the standard sample (0.025 value
was used for calculations).
- 49 -
IODINATION OF PROTEIN
Protein Iodination by Iodo-Gen
a) Principle
Iodo-Gen was first described by Fraker and J •.
Speck'as a reagent for the iodinatio? of proteins and
cell membranes. 1,3,4, 6-Tetrachloro-30\,,6 G(.-diphenyl
glycoluril (Iodo-gen) is extremely stable and insoluble
,in water allowing rapid iodination in the solid phase
with aqueous solutions of I and proteins. The incidence
of side chain reactions with this method is negligible.
Iodo-gen dissolved in chloroforms are plated on walls of
the test-tubes. Iodinations are terminated by decanting
the r~action solution from the solid Iodo-Gen. There
fore, no reducing agent is needed. This reagent can
iodinate micrograms of protein.
I
b) Iodination of hyaluronic acid binding protein
The Iodination of hyaluronic acid binding protein
was performed according to. Fraker et ai. (154) by using
a sparingly soluble chloramide, 1,3,4,6, tetrachloro-3oc.,
6 OC-dipenylg lyco11.lril, . called as Iodo-gen. A thin
film of Iodo"':gen was made by taking 10 ).1g of Iodo-gen
in 20 »1 of.chloroform in a test tube (10 x 75 mm).
The chloroform waS evaporated by rotating th~ tube in
a 370 C bath so that a thin film of Iodo-gen is formed
at the bottom. This is done, because of the fact that
Iodo-gen reacts rapidly in solid phase with aqueous
mixtures of iodine and proteins to yield iodinated pro
teins.
- 50 -
To 20 pI of chloroform containing 10 pg of Iodo
gen (23 x 10-25 mole) was placed in a 10 x 75 nun test
tube, the chloroform was evaporated by rotating the tube
in a 370 bath so that a thin film of IOdo-gen is formed
at the bottom. To this Iodo-gen ~oated tube 20 pI (6 pg)
of HBP in 0.01 M phosphate buf~er, pH 7.2, was added.
20 ul of Iodination buffer (0.1 M phosphate buffer, pH
7.2, with 0.1% BSA and 0.1 sodium azide) :5 )..lCi of (NaI*)
of 125 t and 20 ul of KI (50 ug). The reaction was
allowed to proceed for 5-10 min at 42 0 C with gentle stir
ring; the reaction was terminated by loading this iodi
nated,mixture on G-25 column which has been previously
packed on disposable 10 ml pipette and equilibrated with
0.1 M phos'phate buffer, pH 7.2, containing 0.1% BSA and
0.1% sodium azide. Eluates of 500)..11 were collected,
counted, iodinated proteins were seperated from ·free
1 125 and were pooled and then used for bi~ding assays.