Page 1
ISOLATION AND CHARACTERIZATION OF SEED PROTEINS
By SEEMA HASAN
PROTEIN HESEARCH LABORATORY DEPARTMENT OF BIOCHEMISTRY
J. N. MEDICAL COLLEGE A. M. U., ALIGARH-202002
Date Approved :
A. Salahuddin, Supervisor
A Dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Philosophy in
Biochemistry in the Faculty of Medicine of the Aligarh Muslim University
ALIGARH 1988
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CERTIFICATE
I c e r t i f y t h a t thc» worV pr<^aented in t h e fo l lowing
pages has been c&cci-yd ou t by Miss Se(ana Ha^an and t h a t i t i s
s u i t a b l e fo r tivi s'./ard of M . P h i l , d e g r e e in B i o c h e n i a t r y of
t h e A l i g j r h i-'usliir. J n l v e r s i t y , A l j g a r h .
( A, SAI AHUDDlfj' ) Professor and Chairman Department of Sioehemistry J.N. Medical College, Aligarh Muslim University,
Aiigarh
il
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ABSTRACT
Cajanua cajan lectin interacts specifically with D(+) • mannose^
D(+)-.glucose and N(-»-)-acetyl-D-glucoaaBnine, similar to that of concwia-
valin A, The hemagglutinating activity of Ca ianus calan pulse horaoge-
nate was checked using trypainlaed and untrypsinized erythrocytes of
rabbit* goat« sheep and human* The homogoiate was found to agglutinate
only trypsinized rabbit red blood cells.
For the isolation of lectin* pulse was soaked overnight in lOnW
sodium phosphate l»iffered saline (PBS)* pH7.5 containing 0*02^ sodium
azide. Then it was homogenized and centrifuged* The supernatant show
ing positive agglutination with trypsinized rabbit erythrocytes was
made 30< with respect to ammonium sulfate and the salt concentration of
the supernatant was raised to 50'i»
For further purification ion exchange chromatography of 50< pre
cipitate was performed on DEAE-cellulose column* Bound protein was
eluted with increasing concentration of sodium chloride* Protein elu-
ted with 0.15M NaCl showed positive agglutination with trypsinized rab
bit erythrocytes and this active protein gave four bands on SDS-polyae*-
rylamide gel electrophoresis* having Rtn values of 0*49* 0*58* 0*68 and
0.77/ indicating that the preparation was not pure. Therefore affinity
chromatography of 50^ ammcmium sulfate fraction: was done on Sephadex
3-200 column* After specific elutlon with 0*5M glucose* protein was
concentrated and dialyzed* Then it was applied on HPLC-Shim Pack Diol-
150 gel filtration coluirai* Five major peaks were detected indicating
that the prefwration was not pure* so repetitive affinity chrwnatogra-
phy on Owwiucoid Sepharose 4B column was carried out. The bound protiin
ill
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was specifically eluted with 0.5M glucose In lOniM PBS, pH7.5 • Peak
fraction after reanoval of glucose showed hemagglutination with trypsini««
zed rabbit erythrocytes and only a single peak with 100^ purity with
respect to concentration was detected on H' LC-Shira Pack Diol-150 gel fil
tration colunui. The retention time was found to be 6.39 minutes*
Then 50^ precipitate was directly applied on Ovomucoid Sepharose
43 column* After specific elutlon with glucose the peak fraction was
dialyzed* It showed positive honagglutinatlon with trypsinized rabbit
erythrocytes* It was then applied on HPLC-Shim Pack Dlol-150 gel filt
ration column. One major peak with 80-5 purity with respect to concen
tration was detected* The retention time was found to be 6*37 minutes*
Molecular weight, calculated using both retoition times was found to be
identical indicating that both proteins are same* Molecular weight and
stokes* radius were found to be about 66,000 daltons and 3*17 ran resp
ectively*
Iv
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I wish to express my deep gratitude to my honourable
Supervisor« Professor A, Salahuddln« Chairman, Department of
Biochemistry, J.N. Medical College, Allgarh Muslim ttolverslty,
Allgarh. His able guidance, regular encouragement. Inspiration
and Intelligent criticism helped me in the fulfilment of this
task. I am also thankful for his Invaluable advice and provision
of necessary facilities.
I express my gratefulness to my teachers Prof. M.A.
Slddiqui, Dr. M. Saleemuddln; Dr. S.M# Hadl; Dr. Masood Almvad;
Dr. BHquees Bano and Dr. Naheed Beno Naqvi for their encourage
ment in attaining the ultimate success.
Z thank Dr. Sudhlr Kumar Agarwal; Dr. (Mrs.) Rasheedunnisa
Begum and Mrs. Khushtar Salman for their co-operation and kind help.
I owe my special thanks to Mr. Krlshnan Hajelay Mrs* Nausheen Ahmad;
Miss Najma All; Mrs. Renu Tyagl; Miss Parveen Salahuddin, Mrs. Manjeet
Kaur, Mr. Mir Muzaffar, Mr. Khalid Fazli, Mrs. Sadhana Shaima and Dr.
Partha Sarkar for their skilful help, valuable suggestions and encou
ragement at every stage of my work.
My affectionate thanks are due to Miss Sabiha, Miss Zia Afroz
Hasan, Miss Reshma Naheetd, Miss Azra Abid and Mrs. 2k>ya Oalzie.
Page 7
I also extend ray thanks to Mr* Tameezuddin, Mr* All
Manzar, Mr* Lai tohB Bnad« Mr* Shamshad* Mr* Milr* Mr* Behzad Khan*
Mr* Kafeel and other ncw»-teachlng staff m««nber« of th« Department
for their co-operation* Z also thank Mr* Sanjeev Sehgal for typing
the manuscript skilfully*
In this research project* financial assistance from
PX*-4G0 is greatly acknowledged.
( SECMA HASAN )
Vi
Page 8
D E D I C A T E D
TO
My Parents* Slstara «nd Brother.
v l l
Page 9
cONTrarrs
Page
ABSTRACT ill
ACKNOWLEDOEMENTS V
DEDICATION vil
LIST OP TABLES X
LIST OP PIOURES Xl
LIST CP ABBREVIATIONS xlll
I. INTROrUCTI N 2
II. E CPEniM-lNTAL 20
A. Matoriala 20
F. Methoutf 22
!• Eatimatlcm of Protein Conner-ration 22
2. Measurement of pH 24
3. Her I agglutination e :-9ay 24
4* Salt Induced solubilization of proteins 26
5. laolatlon of lectin frcmi Calanua caWn 27
6. Ion-exchange chromatography 2^
7» Affinity Chromatography on S€oaad€»x O-^OO 2'
8. Isolation of Ovoaueold t-rom egg v/hlte ?^
9. Preparation of Ovomucoid Sephar^ S€i-4B column 3'"
10. Affinity Chr< to'raph/ on Ovat»i.col<l Sepharo« r- 31 o4B column
11. SDS-polyacrylamide gel electrophoresis 32
12. High performance liquid chromatoqraphy 32
vili
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Page
HI. RESULTS AND DISCUSSION 37
BIBLIOORAPHY 58
BIOORAPMY 67
LIST OP PUBLICATIONS/PRESENTATIONS 68
ix
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LIST OP TABLES
TABLE CcHimcHriXy used a f f i n i t y chromatography media for t h e p u r i f i c a t i o n of l e c t i n s
3
TABLE I I Marker p ro t e ins used for t h e c a l i b e r a t i o n of HPLC Shim Pack DioI-150 ge l f i l t r a t i o n column.
34
TABLE I I I
TABLE rV
TABLF.
Hemagglutinatlng Act iv i ty of pu l se hcMnogenate fr«n Ca ianus ca1an»
Sa l t induced s o l u b i l i z a t i o n of p ro t e ins from Ca Ianus ca1an»
Retention t ime, area and r e l a t i v e concent ra t ion of peaks obtained on ••^I/'Shim Pack Diol-150 gel f i l t r a t i o n colvunwi
38
40
49
TABl£ VI Percantage y ie ld a t each pu» i-Eicatins-ster)
57
Page 12
LIST OP FIOURES
PIOURE 1,
PIOURB 2.
FIGURE 3.
PIOURE 4.
PIOURE 5.
FIGURE 6.
FIGURE 7.
FIGURE 8.
FIGURE 9.
FIGURE 10,
Schematic represcmtatlon of circular homology of lectins
Calibration curve for the estimaticm of protein ccmcentration by Lowry'a method using BSA as the standard protein
Calibration curve for the estimation of protein concentration by dye binding method using BSA aa the standard protein
Calibration curve for the determination of molecular weight of proteins by standard curve of VejVo Vs. log M,
Calibration curve for the deterroinati(»i of stokes radius by the method of Ackers.
Effect of sodium chloride concentration on solubilization of proteins in distilled water.
Effect of sodium chloride concentration on solubilization of proteins in lOrnM Sodium phosphate buffer« pH7.5 •
Effect of sodium chloride concentration on solubilization of proteins in IftriM Tris-HcL buffer, pH7.5 •
Ion-exchange chromatography of 50^ ammonium sulfate fracticm of Calanus calan homogenate on DEAE-cellulose coluirant """
SDS-polyacrylamide gel electrophoretogram of active protein fraction obtained after ion-exchange chronatography.
23
25
35
36
41
42
43
45
46
Xl
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FIOURE IX,
PIOURE 12 .
FlGfURE 1 3 ,
Gel chromatograptiy on flPLC Shim Pack Dio l -150 column o$ l e c t i n i s o l a t e d a £ t e r a f f i n i t y chi-<»natography on Sephadex <3-.200 column*
Gel chromatography of Ovomucoid on Sephadex ou ioo column*
A f f i n i t y chrcMnatoQraj^iy on Ovomucoid-Sepharose-4B column of p r o t e i n s p e c i f i c a l l y e l u t e d frctm Sephadex 0*200 column*
riams 14, Oel chroatatography o£ purified lectin from Ca 1anua'*ca Ian on HPW:-Shlra Pack D i ^ i ^ i g o column*
FIGURE 1 5 . A f f i n i t y chrcMnato^raphy of 504 ammonium s u l f a t e f r a c t i o n of Ca lanua c a l a n homo-g e n a t e on Ov<»nucotd Sepharose-^B column*
FIGURE 16 . Gel chromatography of p u r i f i e d l e c t i n from Ca lanua ca la i i on HPLC Shim Pack Dio l -150 column.
Page
48
50
52
53
54
56
xii
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LIST OP ABBREVIATIONS
BAPNA - oi, •N-benzoyl-QL-arglnine-p-nltroanllide.
3SA - Bovine serum albtimin.
Ca - Calcium*
c laiA « Complementary deoxyribonucleic acid.
Con A - Concanavalin A,
DEAE - Diethyl amino ethyl*
EDTA - Ethyl^ie diamine tetraacetic acid.
Gal - Galactose.
oic - Glucose*
Olc NAc - N-acetyl glucosamine.
HPLC - High performance liqaiA chromatography.
Man - Mannose.
Mn - Manganese.
m RNA - Messenger ribonucleic acid.
PBS - Sodium phosphate buffered saline*
PHA - Phytohemagglutinin.
SDS - SodixMn dodecyl sulfate
TCA - Trichloroacetic acid*
xiii
Page 15
ISOLATION AND CHARACTERIKATXON OP SEKD PROTEINS
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INTROmiCTION
Studies on seed proteins include their identifications, purifi
cation # characterization, modifications during the seed life and their
biological role. The seed proteins include enzymes, inhibitors, phy-
tohemagglutinins ani storage proteins. Since legume seeds are a rich
source of lectins, therefore I have isolated and partially characteri
zed l»2tin frc»n Cajanus caian, a plant belonging to the Leguminoseae
family.
Occurance and isolatJCTi of lectins*
Lectin is a multivalait carbohydrate binding protein or a glyco
protein of non-iiwnune origin which agglutinates cells and/or precipit
ates glycoconjugates (1)
The richest source of lectin in plants is the Leguminoseae
family. Lectins have also been discovered in other families as well,
like Euphorbiaceae, Verbenaceae, Malvaceae, Polygonaceae, Solanaceae
and Acanthaceae (2). The best characterized lectin from monocots is
wheat germ agglutinin (3). Although lectins were first discovered in
plant seeds, they have also been detected in other plant tissues such
as roots, leaves and bark. Lectins have also beai found in bacteria,
fungi, invertebrates and vertebrates including mammals (3-15).
Page 17
H
I
g
•H
« CM lA
• o> M
"• • * f4
1
1" «
• «0 «• •^
* tn «*
«-« in r<
1
f^ «*
1
U
«
s
i (0
«
8 «
< ^ r<«
I I
m
0» c
•o n 0
o
«
CM
in CM
in
in
tn
o o UTS
If.
«n
«
s « •a
in
10
s I
Page 18
e:
in lA
e to
"0
M
«
0 H
i « 0 tj (0 H a o> » *-4 tt S 0
f «9 S5 O
o 5 S m
P Q 5 H
- *> « r^ « N 8 ^ 9 0
<t» n 0 tJ
o
u
T
O
1 4J
> ^ tiS « o 1 U >•«
8'a
• 00
8 1 ^ §& •M W r-l
Sin »H +1 >*« c > •H ^
•H O
• 0«
Page 19
For Isolation and purification of lectins, affinity chromatogra
phy technique is employed that exploits the specific sugar binding
property of the lectins. Differwit chromatography media are used for
purification of lectins. Some of th«n are aiuramariaed in Table I. 'Gen
erally specific ligands are linked to an isoluble matrix for affinity
purification of lectins. Sephadex and Sepharose, which are cross-link
ed polymers of glucose and galactose respectively, have also been used
as affinity media for the isolation and purification of lectins. Ery
throcytes treated with formaldehyde or glutaraldehyde have also servwd
as affinity adsorbents for lectin purification (16, 17).
Carbohydrate specificity of lectinst
Carbohydrate specificity is determine:! by inhibition of aggluti
nation or precipitation between lectin and a glycoconJugate, Sugar,
which may be in the form of monosaccharide, disaccharide, oligosaccha
ride or glycoprotein, which is required in minimal concentration for
inhibition reaction is n»8t specific for that particular lectin. On
the basi«, that which monosaccharide is the most effective inhibitor of
agglutination, lectins are classified into specificity groups like glu
cose, mannose, galactose, lactose, fucose, N-acetylglucosamine, N-ace-
tylgalactosamine and N-acetylneararainic acid specific lectins
Generally lectins bind to a monosaccharide unit but PolIchos
biflorus lectin (IR) and lima bean lectin (19) bind to di-and trisacc-
harides more strongly than alkyl glycosides. There are several lectlts
Page 20
which have complex sugar specificity for example the pentasaccharide
Gal i 1-401C NAc (^ 1-2-(Oal P 1-4 01c NAc 1-6) Man is the moat pot
ent inhibitor of Phaseolus vulgaris lectin* although it is also inhi
bited by the disaccharide 01c NAc ^ 1-2 Man (20). Lectins from Solan-
aceae family for example potato and Datura stramonium are inhibited by
N-acetyl glucosamine oligc»ners (21,22*23). There are several classes
of lectins which are inhibited by the Internal glycosyl units. Lentil
lectin, pea lectin and concanavalin A belong to this class. They int
eract with internal 2-0- oi, -D-mannopyranosyl residues, wheat germ
agglutinin too reacts with internal 4-0-substituted N-acetylglucosam-
ine residues (24). The and ancwneric forms also play a role in
determining carbohydrate specificity. There are some lectins like
concanavalin A, pea lectin and Lotus tetraqonolobua lectin which eith
er bind to form or to f' form of sugar (25-28). Whereas soybean lec
tin and castor bean lectin do not differentiate between the two anorae-
rlc forms. Molecular shape also determines lectln-carbohydrate inter
actions. For example, Ulex europeua lectin binds two oligosaccharid
es which are structurally differait but have similar shape (57). The
two oligosaccharides are L Puc ^c 1-2 Oal P 1-4 Glc NAc and L-Puc<-
1-2 Gal P' 1-3 Glc NHj. If the latter trlsaccharlde is acetylated to
L-Fuc 1-2 Gal ^ 1-3 OlcNAc, then it is not bound by the Ulex europ-
JS45. lectin because of change in topography. Clerodendlon trichotomum
lectin is a glycoorotein. Its predominant oligosaccharide structure
was determined and the reactivity of this carbohydrate molet/ towards
various lectins like concanavalin A, lentil lectin, pea lectin, Vicla
Page 21
faba lectin, Ulex europeua agglutinin and wheat germ agglutinin were
studied and it was found that all lectins except wheat germ agglutinin
bound to the above carbonhydrate moiety (58), This shows that (a) af
-xylosyl residue substituted at C-2 of th® P -raannosyl residue of N-
1inked oligosaccharide does not affect the binding with mannose-spocl-
fic lectins, (b) lentil lectin, pea lectin and Vjcia faba lectin can
bind to N-llnked oligosaccharides containing an<< -L~fucosyl residue
attached to the C-3 group of the asparaglne linked N-acetyl-D-glucosa-
mine residue and (c) Ulex europeus agqlutlnin I can bind to the ( °C 1-
3)-llnked fucose r' sldue of tho N-llnked oligosaccharide (58).
The critical position in the carbohydrate moiety is the C-4 hyd-
roxyl group. Glucose and mannose specific lectins usually do not bind
to galactose and vice versa (3, 29-31). Lectins which are glucose
specific also Interact with mannose, indicating that variation at C-2
position of the carbohydrate moiety does not make much of a difference
on binding es in the case of concanavalln A. Sugars which Interact
with the lectins are generally in the pyranose form. Exception to this
fact is concanavalln A, which binds D-fructose and D-arabinose, both
being in furanose form (3). There are few lectins which also require
the amino acid to which the carbohydrate moiety is linked, for the
interaction as is ex5^1ified by the mushrocwn, Agaricus bisr>orus lectin
(37). The association constants for binding of lectins and monosacch-
3 4 —1 arldes ilea in tho range of 1x10 to 5x10 M *(33). Lectin carbohydrate interactions can be studied by changes in fluorescence or ultra-violet
Page 22
8
absorption o£ the ligand or the protein molecule*
Lectin from Erythrlna crlstaqalll binds to N-dansylgalactosandne,
a hydrophobic sugar derivative, more strongly than N-acetylgalactosam-
Ine (34) suggesting the presence of a hydrophobic region near the bin
ding site* Binding of hydrophobic compounds like ^ -Indoleacetic acld«
1, 8-anllinonaphthalenesulfonic acid and 2, 6-toluldonylnaphthaIene
sulfonic acid (35) Is not Inhibited by specific sugars* This observa
tion suggests that carbohydrate binding site and site for the binding
of hydrophobic compounds are distinct* In lima bean the two binding
sites are 28A°apart (36) and in the case of concanavalln A, they are
35A°apart*
Usually there is one binding site per subunlt as in concanavalln
A which has four binding sites per tetramer (37,38)* Similarly soy
bean agglutinin and peanut agglutinin also have four binding sites per
tetramer (39,40). Lentil lectin has two sugar binding sites per dlmer
(41) and Helipc pomatia lectin has six binding sites per h€»camer (42).
Lectins with non-identical subunits like Ricinus ccmimunls agglutinin
(43,44). Abxnis agglutinin (45,46) and fava bean lectin (11) have only
<Mie sugar binding site in two sulMinits. Tetramerlc lectins like Datum
stramonium agglutinin (47) and jack fruit agglutinin (48) have two
sugar binding sites. Lectins from Dollchos biflorus is a tetramerlc
lectin which is composed of two closely related subunits but only cme
kind of subunlt appears to have sugar binding site (49). Wheat germ
Page 23
agglutinin is the only lectin which has two sugar-binding sites per
subunit (50« 51)•
Carbohydrate content of lectins
Lectins are generally glycoproteins which have varying amount of
carbohydrate content* For example carbohydrate cont«)t in potato and
tomato lectin is as high as 50% (55,56) whereas in Phaseolus lunatus
lectin it is Just 3 to 54 (3,10). Mannose/glucose, galactose, glucos
amine, galactosamine, fucose, xylose and arabinose are the ccwnmon car
bohydrate moieties found in lectins. On the basis of carbohydrate moi
eties lectins can be divided into two classest (i) those containing
mannose and N-acetylglucosamine and (ii) those containing galactose and
L-arabinose, Xylose and L-fucose may also be present in certain lectts
belcmging to the former class (59). Lectins from soybean (60), tora
bean (61) and lima bean (62) belong to the first class while lectins
from potato and Qatura straraonium (55), both from Solanaceae family,
belong to the second class of lectins. Soybean lectin (60), tora bean
lectin (61) and Vicia qraminea lectin (63) are all N-linked glycopro
teins. Their core oligosaccharide regions are similar to those of
animal glycoproteins which are asparagina linked. On the other hand
potato lectin and Datura stramonium lectin, both have carbohydrates.
0-1inked to serine (55). Carbohydrate moieties present in glycoprot
ein lectins are not required for their biological activity as is indi
cated by the fact that when these sugar residues are modified as in
Page 24
10
the case of soybean lectin (10) and Dolichoa blfflorus lectin (64),
there ia no change in their h«nagglutinating activity. There are some
lectins like concanavalln A (52), wheat germ agglutinin (24), peanut
agglutinin (53) and barley lectin (54) which are devoid of carbohydrate
moieties.
Molecular properties of lectinst
Generally^ molecular weights of the lectins range between 36,000
and 270,000 (3,9,10). Molecular weight of wheat germ agglutinin is
36,000 (65), whereas that of lima bean lectin is 269,000 (f>6). Some
lectins require metal ions for their biological activity lilce concana-
2+ 2+ valin A requires Mn and Ca (3), Dolichos biflorua lectin requires
2+ only calcium ion (Ca ) for its activity (67) while lima bean lectin
2+
requires only manganese ion (Mn ) for binding of caccharide unit8(3).
Effect of metal ions on the biological activity of lectins is bost stu
died for concanavalln A. Kalb and Leirtzski (68) founJ that concanava
lln A binds bivalent metals at two differait sites-31 and S2. SI site 2+ **+
binds Mn while S2 site binda Ca' • It was found that SI sita was occ-2+ upied first and th«i 32 site could bind Ca and occupation of both
these sites was necessary for the binding of carbohydrate residues.
Reffloval of raetal ions, in concanavalln A, results in an unlocked state
or conformation which is devoid of any activity towards saccharides.
When both metal binding sites are occupied, there is a conformational
transition from unlocked state to locked state. Demetallization causMi
a change in the configuration, from cis to trans, of a peptide bond
beti^en alanine 207 and aspazrtic acid 208. The two metal binding sites
are 4.3 to 4.6A**apart.
Page 25
11
There are some lectins with unusual characteristics. For exemtpie
lectins frcxn potato, tomato and Datura 8trara<»ii\Bn all belonging to
Solanaceae family, have high content of L.arabinose and they also con
tain hydroxyproline, a rarely occuring amino acid. (55,56) Wheat germ
agglutinin has an unusual property that it contains large number of
disulfide bonds. (69). There are sc»ne plant seeds which have isolec-
tins (3,9,10,70)r.These iaole^tins differ in some properties like asso
ciation constant for the specific sugar and electrophoretic mobilities.
Some isolectins consists of subunits with different sugar specificities.
(3,10).
Structure of lectinst
A number of lectins have been conpletely sequenced, some of which
are concanavalin A (71), fava bean lectin (72), lentil lectin (73),
soybean lectin (74) etc. There are still some lectins espCKjially the
two-chain lectins frcxn the plants of the Vicieae tribe which are yet to
be sequenced (10). Lectins can also be divided into two classes on the
basis of nximber of polypeptide chains (a) «ie chain lectins and (b) ttfo
chain lectins, those having the light oC chain and the heavy P chain
(75). Two chain lectins occur in Vicieae tribe only (10). Neverthe
less there is extensive sequence homology between the one chain lectin
and the two chain lectins when they are aligned in such a way that the
P chains of the two chain lectins correspond to the NH2-terminal sequ
ences of the one chain lectins, followed by the <?(, chains (76). Sequeneis
Page 26
II.
homology of concanavalin A with two chain lectins can be obtained by
aligning / with a few del suctions/ the amino ends of the two chain lectins
with residue 123 of the concanavalin A lectin, proceeding to the carb-
oxyl terminal of the latter lectin and continuing along the amino ter
minal region (74). This is termed as circular homology (Flg.l). The
extensive homologies obsezrved between different lectins suggests that
these are conserved proteins and that they have a common genetic origin.
Only wheat gerra agglutinin has been completely sequenced from the Gra-
mlneae family (77). Rye and barley lectins, belonging to the Oramineae
family, have similar properties to that of wheat germ agglutinin and
their subunlts can be exchanged (78). This indicates that these lect
ins too are conserved and arise from a cormnon ancestor. This fact may
also hold true for lectins from Solanaceae family, such as potato and
Datura stramonium lectins (73,79). They are similar in their carbohy
drate specificity, amino acid composition and carbohydrate composition.
The most commonly occurlng secondary stiructure in several lectins is
therpleated sheet structure.(80-82), Lectin from Vlcia faba (broad
bean) also has extensiverstructure. Its structure is similar to that
of concanavalin A. (83). It is an ellipsoidal dlmer and has two metal
24- 2+ ion binding sites (Hn and Ca ) and a saccharide binding site per protomer.
Biosynthesis of lectins»
Plant lectins are synthesized In nearly the same way as animal
glycoproteins. Prom the site of their synthesis they are transported.
Page 27
13
s/'^/y/zA Concanavalin A IllUllllllllllllllllttll S 0 l | > € a 7 l i^ctlTL
I I f a v l n
fIGURR It Sc'h«aatic represent at Ion showing circular homology by
aligninent of fava bean lectin °^ and P chains, soybean
agglutinin and concanavalin A sequences
Page 28
14
to endoplasmic reticultim through a •l ntl peptide where N-glycosylat-
ion via the dolichol phosphate pathway takes place* Modification of
carbohydrate moieties proceeds in the golgi apparatus fr'->m where they
are transported to theri final destination* Pava bean lectin consists
of a signal sequence* followed by the r chain and then the =<!, chain,
suggesting the structure NH2-signal- r chain-'^ chain-Cf>OH. (84). vSig-
nal sequence is removed in the endoplasmic reticulum* Then the protein
is cleaved to yield the at and the ^ chains in the protein bodies.
Lectin from pea plant is synthesized in the same way (85)* Both two
chain lectins are derived from a single polypeptide precursor. ct)NA of
concanavalin A (one chain lectin) contains a region corresponding to 29
residues of the signal sequence, followed by a coding region correspo
nding to amino acids 119-237, « region coding for 15 amino acids not
found in the mature lectin and finally a region of amino acids 1-llB,
During the formation of mature Con A, it is suggested that there is a
transposition and ligation (between residues 118 and 119) of two pep
tides produced from the precursor polypeptide (75)* The genes encoding
the subunits of phytohemagglutinin isolectins E-PHA and L-PHA, are
located on the same chromosome which are 4 Xilobases apart (B6). 3ra-
mineae lectins are synthesized in an unusual way because removal of
signal sequence from 23,000-dalton precursor proteins to give the
18,000 dalton mature lectin subunits does not take place in endoplas
mic reticulum but it is a post-translational event ( 87, 88). There
Page 29
15
is an additional post-translational modification in the developing ri<B
embryos and that is splitting of 18^000 dalton subunits into two frag
ments (88). The Dolichos biflorus seed lectin contains two structura
lly related subunits, but there is single oolypeptide precursor for
both subunit types and this suggests that differences between the
subunit types arise by post-translational processing (B9). There is
maximal production of seed lectins during the midraaturation stage,
after which the lectin content declines i.e. in the late maturation
stage as is seen in lectins of soybean (90) and pea (85). There is
also an incease in roRNA in the midraaturation stage, indicating lectin
content regulation at the transcriptional level.
Role of lectins!
2ole of lectin in mediating symbiosis between Rhizobium and leg
ume is attracting most attention. This suggestion was first put for
ward by Hamblin and Kent (91) after their observation that bacteria
that nodulate beans were agglutinated by same bean extracts and that
erythrocytes that could be agglutinated by the bean lectin bound to
root hairs. Much work has been done on syybean-Rhizobium ^aponicum
interaction and the clover-Rhizobium trifolii interaction but the most
convincing evidence for the symbiotic role of lectins are present for
the clover-Rhizobium trifolii interaction (92). White clover lectin
is present in seeds and in roots. It was found that only specific
strains of Rhizobium were agglutinated by white clover lectin (93,94)
and that 2-deoxyglucose specifically inhibited both, Rhizobium attach-
Page 30
16
raent and lectin binding (93). Relationship between seed and root lec-in
tins from soybean and their possible role/medlatlng symbiosis is yet to
be determined. The other proposed functions for lectins In plants are
storage and defcmce. Presence of s<Mne lectins in protein bodiosCgs^^'e)
suggests the storage function of lectins. Role of lectin In the defen
ce of plants against bacterial, vlraland fungal pathogens (11,97-99)
have he&n proposed. Addition of barley lectin reduces considerably the
growth of barley mosaic virus in tissue ealture (100). This observati
on too suggests the protective function of lectins. Pres«ice of lect
ins at the invasion site of Infectious agents (101) and inhibition in
growth of these agents by lectin binding (102,103) supports the defence
mechanism.
Several lectins can distinguish between normal and malignant
cells and thus they can be used in cancer chemotherapy. For example,
wheat germ agglutinin agglutinates tumor cells at concentrations which
fails to agglutinate normal cells(104). Plant lectins can also be uae3
as an antitumor agent. For example, when Agarlcus blsporus is injec
ted into mice bearing tumor, it inhibits tumor growth by 39X in 3
weeHs time (105). Lectins like concanavalln A and phytohenagTlutinln
are mitogenic I.e. they proliferate lymphocytes. R«noval of lectins
by their specific sugars leads to inhibition of the mitogenic activity
(106-109). This indicates that lectin binds to the carbohydrate moie-
tias present on lymphocyte surfaces and cause their proliferation.
Microfilaments may also play a role in lymphocyte activation (110).
Page 31
17
Lectins are of use in the identification of various claaaea of cells.
For example, peanut agglutinin can distinguish between inunature and
mature human and mouse thymocytes (111,112)• It can also distinguish
between immature and mature chicken lymphocytes and thus they can be
fractionated using peanut ag lutinin. Peanut, soybean and wheat germ
agglutinins have been used to separate mouse spleen T and 3 cells
(113-115). Lectins can discriminate between different microbial speci
es and also pathogenic and non-pathogenic strains. Fox example, wheat
germ agglutinin has beCTi used to separate Neisseria qonnorrhoeae frcxn
other Neisseriae and related bacteria (116)*
Affinity chromatography on Immobiliaed lectins have be«i used to
Isolate and purify glycoproteins like immunoglobulins (117), interferons
(118), rhodopsin (119) enzymes (120) and viralglycoproteins (121). 011-
gosachharides can also be separated on lectin affinity column (122).
Carbohydrate moieties present on the surface of glycoproteins can also
be analysed using lectins. There are certain lectins which agglutinate
human erythrocytes. Lectins from Phaseolus Ixinatus, Vic la cracca and
Dollchos biflorus are specific for h blood ty'>e cells (2,3). Blood
group O specific lectins are obtained frcsn Lotus tetraqonolobus and
Ul^c europeus (123,124). A lectin specific for B group cells h^s he&n
isolated from Qriffonla simplicifolia (125). The lectin from Sophora
japonlca agglutinates both A and B blood type cells. (126). This fun
ction of lectins is used in blood typing.
Page 32
19
hectin from aources other than plants»
Although lectins were first isolated from plants, now they are
being purified frcmi other sources as well« liXe micro-organisms, inver
tebrates and vertebrates* There are many bacteria that can agglutinate
erythrocytes and other types of cells and this agglutination may be
inhibited by sugars (127,128). This suggests the presence of lectins on
the surface of bacteria. There is eviderice that these bacterial surface
lectins are involved in their adherence to 0pith«iiai cells, for exam
ple, in the urinary and gastro intestinal tracts (128,129), during inf
ection. Pew lectins have also been isolated and characterized from
invertebrates, for example, the lectins of the snail Helix pomatja (3),
of the lobster Homarus americanus (130) and of the crabs Limulus poly-
phaaus (131). Most of the invertebrate lectins are sialic acid speci
fic except for Helix poraatia lectin which is blood-type specific. Lectin
from h«nolymph of crab Cancer antennariua (132) is 9-0 and 4-0 acetyl-
ated sialic acid specific. Vertebrate lectins are d/6ivided into two
classes (a) integral monbrane lectins and (b) soluble lectins (11,133).
Oenerally the vertebrate lectins are galactose/mannose specific. These
lectins bind to galactose residues of aged glycoproteins, which have
lost their terminal sialic acid residues and are thus removed fr<wn the
circulation (14). -galactoside specific lectin from the eel Electri-
cus electricus was the first soluble lectin to be purified (134, 135).
Vertebrate lectins have been isolated from other sources as well, with
different sugar specificities (133), Soluble lectin from chick embryo
which is ^ -galactoside specific has heen completely sequenced (136).
Page 33
19
Much work has to be done to understand the role of lectins in
mediating symbiosis between legume and its specific Rhizobium. Ca janus
cajan is a legume and therefore I have isolated and partially charact
erized the lectin from the pulse of Ca lanus ca jan. Its relationship
to the root lectin and its role in mediating symbiosis is to be stud
ied.
Page 34
I I , EXPERIMENTAL
A. Materials
1. Proteins
Transferrin (lot No.l4P-9425), bovine serum albumin (lot Mo.
lOF-033), ovalbumin (lot No.l05C-802l), ehymotrypslnogen A (lot No.
40P-8050), rlbonuclease A (lot No. 140-0266), cytochrcMtte £ (lot No.
09C-0088) and trypsin (lot No. 65F-.8005) were purchased from Sigpma
Chemical Company, U.S.A.
2. Sugars
D(+)-glucose, D(-»-)-manno8e and D(+)-lacto8e were obtained from
mm, India. D(4) galactose was purchased from Loba Ch«nle, India and
N( + )-.Acetyl D-glucosaralne was obtained frcwn Pierce Chemical, U.S.A.
3. Reagents used in sodium dodecvl sulfate Polvacrvlanide gel electrot" 'I!3333.
Reagents used in SDS polyaery1amide gel electrophoresis with
their sources in parenthesis were acrylamide (BDH, England)« N-N'-
methylene bisacrylamide (Reanal, Hungary), N,N,N*,N•-tetramethyethy-
lenedlamlne (Ferak, Berlin), ansnonlvunpersulfate (E.MercIc, India),
bromophenol blue (BD«, England), sodium dodecyl sulfate (Glaxo, India),
trls-hydroxymethylamlnanethane (Sigma Chemical Company, U.S.A.), acetic
acid (Glaxo, India), cooroassle brilliant blue R (Sigma Chemical Company,
U.S.A.), glycerol, methanol, chloroform and dichlorodimethylsilane were
purchased from BDH, India.
20
Page 35
21
4, ChrcHnatoqraphic media
Diethylerainoethyi (DEAE) cellulose (Sigma Chemical Company, ush),
Sepharose-413 (Pharmacia, Upoaala^ Sweden), blue iextran, Sophadiex '' KP
flknvl Sephadex C5-200 were purchased from Sigma Chemical Company, J.s.A.,
•: anogeo bromide waa obtained fran Sisco Research Laboratories, India.
S* Reagents u»ed in iaolation off lectin
Sodium dihydrogen phosphate, sodium monohydrog«n phosphate,
sodium chloride and anwnonjlum sulfate were purchased frc»n Sarabhai 'i
Chemicals, India» Sodium bicarbonate, sodium carbonate, ethylendiam-
inetetra acetic acid (EDTA) and sodium hydroxide were obtained from
BDH, India. Dialysis bag were obtained from Sigma Chemical Cc n' any,
U.S.A. Other reagents were sodium azide (Pluka, Swltserland), glycine
(Loba Chemie, India) and trichloroacetic acid (nw, England).
Cajanus calan pulse was purchased from a local market.
Page 36
22
B. Methods
!• Estimaticm of protein ccmoentraticm
Protein concentration was estimated by the method of Lowry et
al« (K8) and by the dye binding method (159).
(a) lowry'a method
To 1 ml of pxroteln solution« 5 ml of freshly prepared copper
reagent (4 5 aodliun carbonate (w/v), 4-i sodium potassium tartarate(w/v)
and 2% copper sulfate (w/v) in the ratio of lOOtltl) was added in this
sequence. It was Incubated for 10 minutes at room tanperature. Then
1 ml of diluted (li4) phenol reagent« prepared by the method of Folin
and Ciocalteu (160)^ was added. The solution was allowed to stand at
ro<»n temperature for 30 minutes. The colour intensity was read at 700
nm against the blank on AIMIL photochem«8 colorimeter.
A curve between protein concentration in micrograms and abaor-
bance at 700 nm was obtained by the method of least squares. The
curve (Pig.2) fits the equation.
(CD. ) QQ • 1.79x(rag protein) + 0.061 — (1)
(b) Dye binding method
For the preparation of dye 100 rag of coomassie brilliant blue
0-250 was dissolved JLn 50 ml of ethanol* Then 100 ml of 85 4 (v/v) t
orthophosphoric acid was added and the volume was made to one litre \ i
with distilled water. To 1 ml of protein solution was added 5 ml of
Page 37
23
e c
O O
5 >-H to Z UJ a
< o
o
'00 200 300 400
PROTEIN CONCENTRATION nq
.-J 500
TICJURS 1» C8kLtbr&t.ton curve for t h e est imat ion of ricoteln concBn"
tratic»5 by X«owry*s method (158) using BSA ag the sc^nJarJ
^ ro te ln . The s t r a i g h t i i n o was drawn by the method of
i^iast squares and f l t a the 0qua.tiont
(p.D.)^QQ«l»7^(n^, proteinJ+0.061 #
Page 38
24
dye solution* The colour intensity was read after 5 minutes at 580 ran
on AIMIL photochapa • 8 colorimeter.
A curve betwe«i protein concentration in micrograms and absor-
bance at 580 nm was obtained by the method of least squares. The curve
(Pig.3) fits the equation.
(O.D.)ggQ • 5.187x(mg protein) * 0.017 — (2)
2. Measurement of PH
Elico digital pH meter, model LI-.120, was used for pH measureme
nts in conjuction with Ellco ccwibined electrode, type 51. The pH meter
was standardized in the acidic range by 0.05 M potassium hydrogen pth-
alate, pH 4.0 and in the basic range by 0.01 M sodium tetra borate
buffer, pH 9.2.
3. Hemagglutination assay
Rabbit, goat, sheep and human erythrocytes were isolated by the
method of Hoebeke (165). Four millilitres of blood in 0.5 ml of 2.8 5
EDTA was centrifuged at 1000 rpm for 10 minutes. Tha aupezmatant was
discarded and the pellet of red blood cells was washed four times with
0.15 M. sodium chloride. For trypsinisation, 10 mg of trypsin dissol
ved in 0.5 ml of 10 nW sodium phosphate buffered saline, pH 7.5 (PBS),
was incubated with 1.5 ml of v ashed red blood cells for 10 minutes at
37* 0. Then trypsinlzed erythrocytes were washed four times with lOmM
PBS, pH 7.5 and pellet of red blood cells was collected.
Page 39
25
20 40 60 80
PROTEIN CONCENTRATION 100
ug
PT3URE 3J Calibration cuirve for the estimation of protein concen
tration by dye binding method (129) using '=«SA as the
standard protein. The straight line was drawn by the
method of least squares and fits the equation*
(O.D,)ggQ»5.187x(mq» protein)+0.017,
Page 40
26
To Check hemaggiutlnating activity of Ca ianus caian homogenate,
0.1 ml of trypsinized or untrypsiniaswa erythrocytes from various spe
cies were taken on a glass slide. Then 0.1 ml of protein solution
was added. Agglutination was checked visually and time for onset of
agglutination was recorded. Sugar specificity was checked by incuba
ting 1 ml of 0.1 M sugar solutions with equal volume of protein solu
tion for 15 minutes at ro<wn t«nperature. Then 0.1 ml of this mixture
and equal volume of erythrocytes were taken on a glass slide and time
for onset of agglutination was recorded.
4. Salt induced solubiligatiwi of proteins
Effect of increasing salt concentration on solubilization of
proteins was studied. Sodium chloride concentration was increased in
three solvent systems, i.e.« distilled water« 10 TCM sodium phosphate
buffer, pH 7.5 and 10 raM Tris-HCl buffer, liH 7.5 .
Twenty grams of Ca Ianus caian (pigeon pea) pilse was soaked
overnight in 100 ml of each buffer. After homogenization for 5 minu
tes, the susp^ision was filtered through a cheesecloth and the filtr
ate was centrifuged on Beckman L8-55M Ultracentrifuge at 40,000 rpni
for 50 minutes at 25°C. Supernatant was collected and protein concen
tration was estimated in each case by the method of Lowry et al, (15R)
Page 41
27
5» Isolation of lectin from Calanus calan
Twenty grams of pigeon pea pulae was soakei overnight in 100 ml
of 10 mM sodium phosphate buffered saline (PBS), pH 7.5 contiainini
0.02'''> sodium azide. Then it was homogenized in mixer for 5 minutes and
the suspension was filtered through a ch-asecloth. The filtrate was
centrifuged on Remi T-23 centrifuge at 6,000 ri»n for 30 minutes, at
25*'c. The supernatant showing positive agglutination with trypsinized
rabbit erythrocytes was made 30% with respect to ammoni'um sulfate and
the salt concentration of the supernatant was raised to 50'., The pro
tein thus orecioitated was dissolved in minimal volume of lOrm ^^s, -SH
7.5 and dialyze 1 extensively to renove ammonium sulfate.
Lectin was further purified by affinity chromatography on Seoha-
dex G-lOO column (1.5 x 14 cms). The column was equilibrated with lOrM
PBS, pH 7,5 • About 84 mg of protein in 7 ml was applied. The column
was washed with buffer whose volume was equal to the total volume of
the column. The bound protein was specifically eluted with 0.5 M glu
cose in equilibrating buffer. Protein was collected in 5 ml fraction
each at the flo'>/ rate of 20 mlA»r. All the fractions were pooled,
concentrated in air and then dialyzetJ to remove glucose. Protein was
estimated by the method of Lowry et al; (158) and sodium dodecyl sul
fate polyacrylamide gel electrophoresis was done by the methoc of
Laemmli (164).
Page 42
28
6. Ion esKzhanqe chrcMnatoqraphv
Ion exchange chromatography waa performed using dieth/lamlnoe-
th/-l cellulose resin. After swelling* the resin was treated with 200
ml of 0.1 M sodium hydroxide for two hours. Then it was washed v.'ith
distilled water till the pH became neutral. It was then treateJ with
200 ml of 0.1 N hydrochloric acid for two hours followed by washing
with distilled water to ronove excess of acid. The regenerated resin
was packed in the column(2.4 x 8 ons) and was equilibrated with lOim
sodium phosphate buffer, pH 7.5 • Two and a half millilitres of 50<
precipitate containing 50 mg protein was applied on the column. Bound
protein was eluted with incr asing concentration of sodium chloride.
Protein was collected in 10 ml fractions at a flov; rate of 40 ml/hr
and the fractions were monitored by the dye binding method (159). The
fractions showing positive agglutination with trypsinized rabbit eryt
hrocytes were pooled and concentrated in air. Sodi'jm dodecyl sulfate
polyaery1amide slab gel electrophoresis was performed by the method of
Laenmli (164).
7. Affinity chromatography on Sephadex Q»200
Sephadex O-200 column ( 5 x 12.5 cms) was equilibrated with 10a !
PBS, pH 7.5 . Thirty eight millilitres of 50^ ammonium sulfate preci
pitate containing about 1 gm protein was applied. After washing the
column with equilibrating buffer whose volume was equal to the total
volume of the column/the bound protein was specifically elJted with
0.5 M glucose in the same buffer. All the fractions were oooled, con
centrated in air and dialyzed to ranove glucose and they were
Page 43
29
monitored by the method of Lowry et al; (15B). Hemagglatinating acti
vity was checked with trypsinlzed rabbit erythrocytes. Then gel filt
ration on high performance liquid chromatography (HPLC) column was
carried out*
8. Isolation of ovomucoid frcwn egg white
Ovomucoid was isolated by the method of Waheed and Salahuddin
(161). To 100 ml of egg white equal amount of BO mM sodi'jm phosphate
buffer pH 7.5 was added. Then 10 ml of 100- trichloroacetic acid (TCA)
was added to the solution to bring the final concentration of TCA to 5'<.
After incubating for tvra hours the solution was filtered. Filtrate vma
dialyzed against 0.06 M sodium phosphate buffer, pH 7.5 to r«nove tric
hloroacetic acid. To dialyzed filtrate 90 5 ammonium sulfate was added
and kept overnight. Then it was centrifuged on Sorvall 5 C-5C centri
fuge at 12,000 rpm for 15 minutes at 25®C. Precipitate was dissolved
in 60 ntfl sodium phosphate buffer, pH 7.5 and then dialyzed extensively
against the same buffer to remove ammonium sulfate. Antl-tryptic acti
vity of ovcMiiucoid was estimated using trypsin as enzyme and a6 -N -ben-
zoyl-DL-agrinine p-nitroanilide (BAPNA) as substrate. To different
cone aitrations of ovomucoid 0.1 ml of trypsin (1 mg/ral) was added and
it was allowed to incubate for 10 minutes at 37®C. Then 1 ml of subs
trate BAPNA (1 mg/ml) was added to the mixture. After incubating for
10 minutes at room temperature the reaction was stopped with 1 ml of
30i acetic acid. The colour was read at 410 nm. Purity was checked by-
gel filtration on Sephadex O-lOO column equilibrated with 0.06 M phos
phate buffer pH 7.5. The coltjmuid.l x 35 eras) was packed according to
Page 44
30
the method described earlier by Ansari and Salahuddin (163). The
homogeneity of the packing was checked by passing blue dextran. Five
millilitres of protein solution containing 88 mg of protein in 5 ml
was applied to the colunm and fractions were monitored by the method
of Lowry et al; (158).
9, Preparation of Ovomucoid Sepharose«»4B coXuron
Ovomucoid Sepharose - 4B column was prepared by the method of
Cuatrecasas (162). Ten millilitres of Sepharo8e-4B gel was first was
hed with 100 ml of distilled water and then was suspended overnight in
20 ml of 0.15 M sodium chloride. To decrease the temperature, ice fla
kes prepared from distilled water were added to the gel. Then 3.3 qms.
(330 n^/ml) of solid cyanogen bromide was added and the pH was adjusted
to 11 by adding 4 M sodium hydroxide. The activated Sepharose-4B gel
was washed inmiediately with chilled distilled water followed by chilled
0.2 M sodium carbonate buffer, pH 8.2 containing 0.15 M sodium chloride.
Then about 184 mgs (11 ml) of ovomucoid in carbonate buffer was mixed
with the gel in a conical flask and incubated overnight at 4°C. Gel
was washed eight times with approximately 20 ml of carbonate buffer and
protein was assayed in the supernatant each time to estimate the unbo
und protein. Then washed gel was incubated in 0.2 M sodium carbonate
buffer, pH 8.2 containing 0.2 M glycine for two hours at room tempera
ture to block the free activated groups.
Page 45
31
10. Affinity chromatography on OVCTnucoid Sepharoee - 4B column
The protein isolated by affinity chromatography on Sephadex
O-200 column was rechronatographed on Ov«tiucoid Senharose-4B column
(0.8 X 5.8 cms) equilibrated with IttnM sodium phosphate buffered salinf
pH 7,5 » Six millilitre of sample containing 6 mg protein was applied.
After washing the colunui thre« times its total volume by equilibrating
buffer, the bound protein was specifically eluted by 0.5 M glucose in
the same buffer. The column was monitored spectrophotometrlcally at
280 nm. Active peak fraction was dialyaed to remove glucose and than
it was analyzed by gel filtration on VtPhC column.
Dialyzed 50' precipitate, starting with 20 gms. of pulse« was
then applied on Ovomucoid Sephairose«>4B column (0.8 x 5.8 ORIS). The
column was equilibrated with lOtnM sodium phosphate buffered saline, pH
7.5 . About 12 mg of protein in 0.6 ml was applied. The column was
washed three times the total volume of the column. The bound protein
was specifically eluted with 0.5 M glucose in equilibrating buffer.
Protein was collected in 3 ml fractions at a flow rate of 20 ml/hr and
the fractions were monitored spectrophotcwnetrically at 280 nm. Peak
fraction was dialyzed to renove glucose and then its hemagglutinating
activity was checked. Gel filtration on HPLC column was then perfor
med of the fraction which showed positive agglutination with tr/nsini'*
zed rabbit erythrocytes.
Page 46
32
11» Sodium dodecyl sulfate polYacrvrlamide gel electrophoresjg
Sodium dodecyl sulfate polyacrylaralde gel electrophoresis was
performed in Trls-glycine buffer pR 8.3 (0.025 M Tris, 0.192 M glycine
and 0.14 SDS) according to the method of La«T»nli (164). For 10 < cross-
linking the small oore gel was prepared by mixing 6 ml of solution A
(29'4 acr/laraide and 0.8 4 N,N* -methylenebisacrylamide) and 4.5 ml of
solution B (1.5 M Tris-HCl buffer, pH 8.8 containing 0.4S sns). To
this mixture was added 7.5 ml of distilled water, 70 ul of 10 4 ammonium
persulfate and 10 ul of N,N,N',N'-tetramethylethylenediamine. It was
allowed to polymerize in presiliconized slab assembly for one hour at
room temperature after which stacking gel was added. For the prepara
tion of stacking gel 0.9 ml of solution A (29 S acrylamide and 0.8'4 H^-
methylenebisacrylamide) and 1.5 ml of solution C (0.5 M Tris-HCl buffati
pH 6.8 containing 0.4' SOS) were mixed. To this mixture vjere added 3.6
ml of distilled water, 20 ul of 10 4 ammonixim per sulfate and 10 ul of
N,N,N',N*-tetraraethylethylenediaraine. The solution was poured above
small pore gel and the crab was fixed. After polymerization was comp
lete comb was taken out and sample was applied. Electrophoresis was
carried out for thr e hours at 50mA current flow per slab. The gel
was stained with 0.2'5 cocxnassie brilliant blue R in 48 4 methanol ani
42^ acetic acii and destained mechanically with 10 4 acetic a«id.
12. High performance liauM chrOTaatoqraphy
HPLC Shim Pack Diol-150 column(a79xi2cms.) was equilibrated with
O.IM sodium phosphate buffer, 0H 7.5 containing 0.2M sodium chloride.
Page 47
33
Plow rate was maintained 1 ml/min. by Shimadzu L6CA liquid chrcwnatogra-
phy pump and the protein peaks were detected by Shimadau RF-540 spectto-
fluor<»neter. The excitation wavelength was 28") nm and onission wavele
ngth was 354nm. For cytochrcxne £ the excitation wavelength was 276 nm.
and onisaion wavelength was 306 nm. The slit width for both eoccitatlon
and emission was lOnm. The peaks were recorded automatically byShimadai
CR3A integrator. Marker proteins used for the calibration of HPLC col
umn are listed in Table II. Calibration curves obtained for molecular
weight and strokes* radius are shown in Pig.4 and Pig.5 respectively.
Molecular weight and stokes* radius were calculated by the equations:
Ve/Vo- -0.4522 log M+3.5007 — (3)
Stokes* radius (nm)-4.7597 erfc"(l-Kd)-0.8785 ~ (4)
Page 48
34
TABLE • II
MARKER PROTEINS USED FOR THE CALIBRATION OF HPLC
SHIM PACK DIOL - 150 OEL FILTRATION COLUMN {Vo«4,83)
s.No. Proteins ^ e Molecular stokes Weight Radius erfc~(l-Kd)
SmL 1. Transferin 6.213 81«000
2. Bovine serum albumin 6.252 69,000
3. Ovalbumin 6.693 43*000
4. Chymotrypsinogai A 7.603 25,000
5. Ribonuclease A 7.81 13,700
6. Cytochrane £ 7.79 13,400
3.60
3.06
2.80
1.81
1.75
1.70
0.89
0.88
0.77
0.58
0.54
0.54
Page 49
35
160
150
1.40
130
120
UO
Xs 6 \
••
•"
• •
< , . 4—^
4 V (5
1 . ..
X
i- ^ -
— - ' ^ - ^ — — " • ' • ' — • • • • " ' • • • ' — - I ' • • . ' • — •'• • ^ • - w —
\ 1
i 1 .
40 5.0 42 44 4S 48 LOG MOLECULAR WEIGHT
.X..m. 4. C«U.rati.n curve .or the < ^ ^ - - ^ - ^ ^ ^ " ; ; X ^ ^ r U we-iant. of proteins by standard curve of Ve/Vo s. Ic^
molecular weight.
Marker oroteln. uso. were. (D Tran^ferin <^^^;^-'^
«crum alb.«n5.n 3) Ovalbumin (4) Chymotrypsinogen A (5)
Ribonucleaae A (6) C/tochrore c
T^e straight line, drawn by the method of leaat
squares, fits the equationj
Ve/Vo«-0.4i>22 log M^3.5007.
Page 50
36
0 0.2 0.4 ae OB IJO
FIGijrRE St Calibration curve for the determination o£ stokes radius
by the method of Ackers <167).
Hackee orotelns used wer«« (1) Transferin (2)
Bovine a©rm^ albumin (1) Ovalb\»nln (4) Chyp ot-cypsinogen A
(5) Rlbcnuclesse A (6) Cytochnwie c.
The straight iine^ drawn by the method of laast
squares^ fits tue liquation«
Stokes Radius (nm)«4.7597 erfc"*<i-Kd)-0.8785
Page 51
111. RESULTS AND DISCUSSION
1. Determination of Sugar Specificityt
Hemagglutinatlng activity of Ca janus calan homogenate was checked
using trypsinized and untxrypsinlzed erythrocytes from various species
like rabbit, goat, sheep and human* Homogenate was found to agglutinate
only trypsinized rabbit exrythrocytes as indicated by earlier reports
(166). So trypsinized rabbit red blood cells were used to check hema
gglutinatlng activity in further experiments. Time of onset of agglutin
ation for 500 ug protein was recorded to be 30 seconds (Table III).
Sugar specificity was checked by inhibition of agglutination. Five sug
ars were used for the inhibition study, which were D(+)-glucose, D(+)-
mannose, D( + )-galactose, D( + )-.lactose and M(4-)-acetyl-i>-glucosamlne.
Out of these five sugars only three i.e. D(+)-glucose, D(+)-mannose and
N(+)»acetyl-D-glucosamlne were able to Inhibit hemagglutination of try
psinized rabbit erythrocytes (Table III). The above results indicate
that Ca lanus cajaai lectin is glucose, mannose and N-acetyl-glucosamine
specific, similar to concanavalin A.
2« Salt induced solubiligtation of protein
Salt Induced solubilization of protein from Ca janus cajan was
studied by increasing the sodium chloride concentration in * '"®® solvent
syst^ns i.e. water, which has no buffering capacity; IftnM sodium phos
phate buffer pHT.S; and lOmM Tris-Hcl buffer, pH7.5 • Sodium chloride
concentrations used tirere 0.15M, 0.3M, 0.5M and l.OM. It was found that
/?'' - - "a 4. • 3/'ci-/y. Ay
Page 52
33
TABLE - 111
HEMAOOLUTINATINO ACTIVITY OP PULSE HOMOOENATE PROM CAJANtJS CAJAN
S u g a r s Time of onset of hemagglutination (O.IM) (Seconds)
• 30
D(+) GalactopyranosiJe • 30
D(+) Lactopyranoside - 32
D(+) Olucopyranoside - 180
N(+) Acetyl-D-Olucosarolne - 330
D(+) Mannopyranoside - 441
Page 53
39
there was increase in solubility with increase in salt concentration
in all three solvent systems. In case of water there was sharp incre
ase in protein concentration upto O.SM sodium chloride, above which it
decreased. In case of sodium phosphate buffer too there was sharp inc-
rease in solubility upto O.SM sodium chloride but at l.C»l salt concen
tration* solubility decreased. In Tris-Hcl buffer there was gradual
increase protein conc«itratlon upto 0.5M sodium chloride. At l.OM salt
concentration, solubility decreased. In case of Tris-Hcl buffer, the
total protein solubilized was less as compared to phosphate buffer and
water (Table IV; Pig.6-8). This result indicates that Tris-Hcl buffer
has salting out effect cm the system used rather than its usual salting
-in effect on proteins. The best solubilizing agent was found to be
sodium phosphate buffer.
3» ^finity chromatography on Sephadax O^IOO columnt
Sephadex is cross-linked dextran, wh^ch is the polymer of glucose.
Since Cajanus cajan lectin is glucose specific, therefore Se]^adex O-lOO
and Sephadex O-200 were used as an affinity media, as in the isolation
of concanavalin A.
Seven millilitres of 504 precipitate containing 84 mg. of protein
was applied on Sephadex <5-l00 column (1.5 x 14 cms.). Bound protein
was specifically eluted with 0.5 M glucose in lOmM phosphate buffered
saline, pH7.5. All fractions were pooled, concentrated in air ani dia*
lyzed to remove glucose. Protein conceaitration was estimated to be 106
Page 54
> M
m <
Ck
§ *-i
6 to & g K < o
o B: u. to g H t ' E-O
0 .
E>. O
Z c H
e-g H •J H p i
t> c w n w o D n H
t4
:3 w
i o m »-i •
r» 1
H
u
CO r-4
!L. y
r- » >j-«^ C i W E-t
g Btn
• o t * f4
H " H U
(!) CJ «M
t^ 2 «/) 45 > CO 0}
+» t- 10 • 5 ^ W 0 . > « J 0 o x
u
:< 1
H
to to
f-l % &3 >
to 1
c
0 N - ^
a r - ( 03
P 0 ^ | to
C O 0 8
0 «j V4 41 c - * *^ C O S^
OiS c C«W U 0
c •H
4 j i ; 0 N ' - " ' W-iH • O-iH CP
•H 6
(8 !5 "w * ) r - l P 0 (-« n
C O 0 O • H ' O ^JTS « 10 u - M r ^ * ^ C O E 0) ( O - ^ vz c o<«
0 0
c © • 0 4J & 0 N * - * W "H •
a^H n «-l jQ 0»
V'-* P C)
C O 0 «
10 «
4J »-l*-« C O S
c 0 H4
0 0
' * tf) • •
in *-t
0 0
CO « • •
f* CM
in <-* •
0 0
ON «-t • •
i-i CM
m r-*
• 0 0
10
•
n • 0
r* •
CM
m • 0
H I •
CM
m • 0
^ •
in •
0
rH
• r j
tfl • 0
0
•
in • 0
«o •
0
• «-4
0 •
M
0
• • - I
0» •
CM
0
• r-#
40
Page 55
41
^ 3.0 zf o < en
UJ
O z o o
z
o CL
2.0
1.0 .
0 02 0.4 0.6 OB 1.0
SODIUM CHLORIDE CONCENTRATION, M
FIGURE 6t Effect of sodium chloricle concert rat Ion on solubilization
of proteins of Ca janus oajan* Pulse was hcanogenlzed in
distilled water containing different concentrations of
sodium chloride.
Page 56
42
6
r-Z.O
I -Z LLl
o
i o UJ I -O a.
0 a2 0.4 0.6 0.8 UO SODIUM CHLORIDE CONCENTRATION, M
PIC5TJRS 7? Rfcff^-t of ccxliur. ch lo r ide concent ra t ion on s o l u b i l i z a t i o n
of p ro t e in s of Cajanus caian* Pulae was homogenizedl in
\'3iaM sodium phosphate bufcfer, ^ 7 . S , containing d i f fe ren t
concent r a t ion 8 of sodiiam c h l o r i d e .
Page 57
43
0 0.2 0.4 0.6 a s 1.0 SODIUM CHLORIDE CONCENTRATION, M
FIGURE 8r Effect of sodivun ch lo r ide cnncontra t ion on so?.ub5,llzatlon
of p ro t e in s of Calanua ca|an« Pulse wac honogonJ-^orl in
lOmM Trls-Hcl buf fe r , pH7»5, containing d i f f e r en t concen
t r a t i o n s of sodltim c h l o r i d e .
Page 58
44
ui per ml. Total protein concentration was 530 ug In 5ml solution.
The yield was found to be 0.02" .
The protein solution gave positive agglutination with trypslnlzed
rabbit erythrocytes. Hundred microlitre of this sample containlnq lOug
protein was applied on SDS-polyacrylamide slab gel electrophoresis but
no band was detected. The reason could be overestimatlon of the proteJn
and hence protein applied for electrophoresis waa not enough for the
detection of the band. So ion exchange cbromato'iraph^ vgs perform(?<U
4. lon-'exchanqe chromatography!
Dieth/laminoethyl (DEAE) resin is an anion exchanqer. Ca janus
cajan lectin was found to bind the DEAE resin which indicates that pro
tein carries net negative charge at pH7.5 •
Fractions eluted with 0.15M sodium chloride (Flg.«>) showed posi
tive ag,|lutination with trypsinlzed rabbit red blood cells. All these
fractions were pooled, concentrated in air and dialyzei against lOmM
sodium ohosohate buffered saline* pll7.5 . Protein concentration as
estimated by dye binding method was calculated to be 100 ua. -jpr ml.
Total oroteln concentration waa 500 ug in 5 ml solution. Han^rei micro-
litre of sample containing lOug protein was applied on sns~-ol/acrTlarn-
ide slab gel electrophoresis with 10 i crosslin'<'lng. Foir '-»nP5 v r?"
detected (Pig.10) having Rm values of 0.49, 0.58, 0.63 and 0.77, conclu
ding that the -;rcp5racion was not pure, so affinity chrorato^ra->h7 on
Sephadex G-200 vras performed.
Page 59
45
£ c O
s H <
> •
h-(/) Z UJ o -J < o H-O. O
OB
Oj&
0.4
02
0 80 160 240 320 400 480 560
ELUTION VOLUME, m)
FIGURE 9t Ion exchange chrcanatography of 504 ersnoniurn sulphp.te
fraction of Caianus caIan horaogenate on ^EAK-collulose
colxann (2.4 x 9 cana.)
About 50 mg« protein in IQmM sodium phosphate, buffer,
PH7.5, was applied on DSAE-cellulose column equilibrated
with the same buffer. The bound protein was elutel with
increasing concentration of sodium chloride, indicated by
arrows. Protein was coHecte<3 in 10 ml fractions anJ moni
tored by the dye binding method (159).
Page 60
46
©
©
PIOURS 10: SDS-polyacr/lamlde gel elactrophoretocjram of ac t ive
protein fraction obtained after lon-eocchange chrc»na«
tography.
T<5n mlcro^jram o€ p ro te in wa3 applied on c;:i;-
polyacrylaanlie s l ab gel e l ec t rophores i s with 104
c roas - i Ink ing using 5mA cur ren t per well*
Page 61
47
5. Affinity chrOTnatoqra|)hy on Sephadcpc <3-200 colunmtt
All the fractions which were specifically elated with 0.5M glucose
in lOm? aocliun phosphate buffered saline, pH 7.5 were pooled, concentra-
I ted in air anJ. dial /zed to remove glucose. Protein concentration was
J estimated to be 10 ing per I0ml» soluticm and this solution showed hema
gglutination with tr/osinized rabbit erythrocytes. Then 25 ul of the
sample containing 4 uj o€ protein was applied on HPLC Shim Pack Diol-150
column (0.79 x 12 cms.) equilibrated with O.IM sodium chloride. Five
major peaks vere detected (Fig.11; Table V> indicating that the prepara*
tion was not p^re- So repetitive affinity chromatography on Ovomucoii
Sepharose-4^ column was performed,
6« Isolation of oyomucoid from egg white* — — • — • — W M i » i i i i n n i — I I I • • • • ^ • • • • • • • • • • ^ • • • • • • ^ • • • • • w i a i M a
OvcamucoiJ is a glycoprotein consisting of about 25' carbohydrate
by weight. Carbohydrate moities present are N-acetyl-D-glucosamine, ru
mannose, D-galactose and N-acetyl neuraminic aciJ in unequal amounts.
The content of N-acetyl-T>-glucosamine ani D-mannose is more than n-gala-
ctose and sialic acid. Since Ca janus ca Ian lectin is D-mannose an J J-
acetyl-D-glucosamine specific, therefore ovomucoid was used as a liqanJ
in the oreparntion of tho affinity column.
For different concentrations of ovomucoid, percent inhibition of
tryT tic activity ranged from ^6 to 100. A single oeak was obtained on
Sephadex Gt-ioo column (Fig. 12) which indicated that the preparation 'Aas
homogeneous on the basis of size.
Page 62
LU O 2 UJ O CO LU QC O
UJ >
-J UJ 1 J
1
11 A
, u 1
'
5CM=0.I25RF
f
L 1
43
4 8 12 MINUTES
FIGURE 111 Qel f i l t r a t i o n chromatography on HPLC Shim P?ck nioJ-150 coliitnn (0.79 x 12 cms.) of l e c t i n i s o l a t e d a f t e r a f f i n i t y chx-omatography on Sephadejc G-'200 column.
About 4 ug p ro te in in 25 u l so lu t ion wea applied on MPLC Shim Peck Dloi-150 coiupin ecrui l lbrsted with O.IM sodium phosphate buf fe r , pH7.5, conta in ing 0.:>M nodliBn c h l o r i d e . HPLC was c a r r i e d out a t a flow r a t e o€ 1 ml/ mln. The column was monitored by f luorescence measurements.
Page 63
TABLE - V
RETENTION TIME, AREA AND REIJATIVE CONCENTRATION OP PBAKS OITAINSD ON HPLC SHXM PACK DIOL - 150 OEL FILTRATION COLUMN.
49
Fig. No.
11.
14.
16.
Peak No.
1. 2. 3. 4. 5. 6. 7. a.
1.
1. 2. 3. 4. 5. 6. 7.
Retention time (minutes)
5.470 6.425 7.082 7.712 9.707 10.758 11,457 14.075
6.393
5.05 5.357 5.715 6.372 7.905 9.857 11.447
Area
20S75 17749 38089 33167 1603 1151 15760 250
71434
31796 10453 38573 440727 1898 4645
242782
Relative concentratIon
16.0314 13.8291 29.6771 25.0426 1.2487 0.8967 12.2799 0.1^46
100.00
5.751 1.8907 6.9768
79.7157 0.3433 0.8401 4.4823
Page 64
sn
0 20 40 60 80 ELUTION VOLUME, ml
FXOURE 12. Gel chron.atc..r.phy of Cvomucold on Sephadax C-100
Goluain (1.1 X ^5 cms.).
Five ..iUilitires of protein aolutton containing
B9 mg^. protein 0.06?'! sodium phosphate buffer, t: 7.5,
vas applied to taa coiu.r iIlbrat.o.i v.lth tn^ .ame
buffer, protein was collected in 5 ml fract.lon^ at a
flow rate of 30 ml/br. anc fractions were monitored by
Lovrry's method (15^ )•
Page 65
SI
7. Preparation of Ovorauooid»S«phTOfic 4B Column i
One hundred and eighty four milligrams of protein was mixed with
the gel for the preparation of Ovomucoid Sepharose-4B column. One hun
dred and sixtynine milligrams of protein remained unbound. Therefore,
[ protein bound to the gel was calculated to be ISmg per 10 ml gel or 1.5
mg per ml of gel.
8. Affinity chrcHnatocrraphy on Ovomucoid s©pharose-4g Column:
The protein isolated by specific elution on Sephadex G-200 column
was applied on Ovomucoid Sepharoae-4B column (0.8 x 5.3 cms.), six
millilitre of sample containing 6 mg protein was aprjlied. Fractirms
eluted specifically with 0.5M glucose in lOmM ohosphate baff^rei saline,
pH7.5 v/ere monitored spectrophotwnetrically at 200 nm (Fig. 13). Peak
fraction which showed herojKagglutination with trypslnizeJ rabbit erythr
ocytes after removal of gluoose was applied on HPLC column. Twenty
microlitre of sample containing 2 ug of protein was <ippii:jc!. / s:-n:3le
pea}ii was detected (Fig, 14) having retention time of G.30 riiinuter,,
(Table V),
Then 0,6 ml of 50 i precipitate containing 12 UKJ. prc/toln ;;::'; aop~
lied on the Ovomucoid Seph3rose-4B column (0.0 x 5,o cr:iB). The, -jound
orotein was specifically eluted with O.Sn glucose in I'DmM e<;(Uilibrating
buffer ani fractions were monitored spectrophotomet:riGdlI/ «•- 2ro nn
(Fig,15). Peak fraction after removal o^ glucose snovjei he?iiri.3-!lutin-
ation with trynslnized rabbit erythrocytes. 'I'his fraction \,an than
Page 66
5?
0.06 .
a o:bi o
5 10 15 ELUTION VOLUME m!
PIGTTKE 13, ?.ff .nity chrorrntography on Ovonucold-Sepharo^e 4n col amn
(Q.ii X S.J cms.) of protein speciflcall/ elutecl from
Sephadex <^?00 col'iitJi.
six mlllil^tre of ss' rjle containing 6 mg. protein
-as IpoUed to the column equilibrated with lOmM sodium
phosphate buffered saline, pH7.5. The bound protein was
specifically eiuted with 0.5M glucose in equilibrating
buffer in 3 ml. fractions at a flow rate of 20 inlA»r.
Fractions were monitored spectrophotometrlcally at 280 nm.
Page 67
53
at U 2 UJ U
O -J
>
!5
(I
^ 1
1 1
_ , j 1 1 1 - 1
4 8 MINUTES
5CM=:.0.125RF
•
— — - - . ^ i ^ — ^ — . 1-
^xauRE 14. oal chror^atographr of pur i f ied l e c t i n from Calanu« c a i s a
o,, HPU:-.Shlia pack DloX-lSO column (0.79 x 12 cms.)-
Twenty m l c r o l l t r e of sample cont^inim about 2 uof pro te in
. a s applied on i^'U: Shlxn Pack Diol-150 column e q u i l i b r a t e
v^tth O.m sodium ^hosphat^r buf fe r , pH-^-S. containing 0.2M
.-, :»« i oTjr ij»8 ca r r i ed out a t t h e flow r a t e of GOviiUin chloi:iii©» UVI/Z was* carrier* vv*w
1 ,.1/min. 'l^« coluim was aionitor^a by f l u o r e s c ^ c e
in«>asur€SEn€nta*
Page 68
%i
5 10 (5 20
ELUTION VOLUME, ml
25
>,^-^*i.«arai>nv of 50^ araioonium s u l f a t e f rac t ion Tjom^ 15» Aff in i ty ehromatogrepny ox aw
/«,« K«r«/-^enate on Ovomucold-S^^haxoae 4B of Ca ^anus ca <ati hor«cgen«ce ^ ^
colamn (O.T x S.3 «m».) .
A^.t 12 m ccoteln In 0.6 t.1 cf lOnM sodium
pho.pnate buffered .aline, |^7.5, vaa appiiod on th%
col>^. uill-orat^ with th. s ^ l uffer. The hound
protein was specifically eluted with 0.5M lucos« In
eauillbratlnci buffer in 3 ml fractions at a flow rate
of 20 «.l/nr. Fractions w«re c»onitcr^ sp^ctroohoto-
metriCAlly at 280 fim.
Page 69
&5
applied on HPLC column. Twcmty mlcrolitre of sample containing about
20 ug protein was applisvi. One major symmetrical peak was obtained
having retention t'rae of 6.37 minutes (Pig.16; Table V) which was foind
to be nearly same fcr the protein isolated by repetitive affinity chro-
matograpny on Sephadex. O-200 followed by Ovomucoid Sepharose-4B column*
This shows that both proteins are same. The molecular weight and fteHcf^
radius calctilatetJ frcsm eguations (3 and 4) were found to be 66,000 iinl*
tons and 3.17 n.m. respectively. The relative concentration of the
major oeak was founci tc be 80'i. This shows thot our preparation is
homogeneous on the basis of size. The yield of the protein vras found
to be 0,07-', (f able VT).
Page 70
56
UJ o z UJ o (/) liJ
o
IJJ >
<
1
4 8 MINUTES
i i
5CM=4RF
t
• ^
12
FIGURE 16: Gel ehrofnatography of pur i f i ed l e c t i n from Ca lanua calan
on HPLC-Shim Pack Diol-150 column (0.79 x 12 cms . ) .
Tw«»nt;r m i c r e l l t r e of sample conta in ing about 20 ug
prct.<iu was appiiev.1 on HPlXi colunan e q u i l i b r a t e ! with O.IH
sodiujr. phosphate buf fer , pH7.5, conta in ing 0.2M sodium
c h l o r i d e . n^LC was c a r r i e d out a t a flow r a t e of 1 ml/rain.
"-'he colvwn wae .nonitored b / f luorsecence measuremaits*
Page 71
57
TABXJS • VZ
PIRCSNTAOS VXELD AT SACH FimXFXCATlOM 8TRP
S.No. Purification step Total Proteins '< Yield ( ITKfS. )
1 • Homogenate .
2 . Supettiatant of 30^ ammcmlum s u l f a t e s a tu r a t i on
3 . P r e c i p i t a t e of 50'4 ainmonium s u l f a t e s a tu ra t i on
4. Pro te in e lu ted a f t e r a f f i n i t y chranatography
2677
1296
306
2
-
48
11
0.07
Page 72
BZBLXOGRAPHY
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2* Tom* G.C, and Western* A.(1971) in Chemotaxonomy of the legumlnoc-eae (Harbonne* J.B., Boulter* D» and Turner* <3,L. eds.) pp 367-462* Academic Press* London*
3. Ooldstein* I.J. and Hayes* C.E. (1978) Adv. Carbohydr. Chem.BSoctMm., 35* 127.
4. Lis* H. and Sharon*N, (1973) Annu. Rev. Biochem.* 42* 541.
5. Leiner* I.E. (1976) Ann. Rev. Plant Physiol.* 27, 291.
6. Rapin* A.M.C. and Burger* M.M.(1974) Adv. Cancer Res., 22* 1.
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8. Bourrillicm* R.(1973) Exp. Ann. Biochiin. Med.* 32 serie* pp 59-91* Masson et ci<»* Paris.
9. Lis*H. and Sharon*N. (1981) in Biochemistry of plants(Marcu8*A,ed.) Vol.6* pp 371-447, Academic Press, NewYork.
10. Lis*H. and Sharon*N. (1984) in Biology of Carbohydrates (Oinsburg, V, and Robbins* P.H, eds.) Vol.2 pp 1-85* Wiley-lnterscience* NewYorX.
11. Bar(»ides* s,H. (1981) Ann. Re«. Biochero., 5£, 207.
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58
Page 73
59
20. HaiiBnar8trcxn# S., HammarsfcMn^M.L., Sundb^ld, O,, Amarp« J. and Lonngren, J. (1982) P«sc« Natl.Acad.Set• USA, T^, 1611.
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BXOORAPHy
Seflma Hasan was bom at Shahjahanpur on December
21, 1962. She passed High SehcK>l Examinaticm in 1979 from
Our Lady of Fatima^ Aligarh. She received her B.So* (HOTIS*)
degree in Choaistry in 1983 and M.So. degree in Biochemistry
in 1985 from Aligarh Muslim University* Aligarh. She was
enrolled as Ph.D. student in November* 1985« in the
Department o€ Biochemistry* Jawaharlal Nehru Medical College,
Aligarh Muslim University* Aligarh. She is a member of the
Society of Biological Chemists (Xndia).
67
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LIST OP PUBLICATICWS/PRESENTATIONS
"Xsolation of leetln from C«lanus calan*
Seeraa Ha««n
Proceedings of the 56th Annual Meeting of the
Society of Biological Chemists (India) held at
Tirupati from Decaiaber 28-30, 1987.