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

DS1314

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

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

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

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.

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

D E D I C A T E D

TO

My Parents* Slstara «nd Brother.

v l l

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

Page

HI. RESULTS AND DISCUSSION 37

BIBLIOORAPHY 58

BIOORAPMY 67

LIST OP PUBLICATIONS/PRESENTATIONS 68

ix

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

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

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

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

ISOLATION AND CHARACTERIKATXON OP SEKD PROTEINS

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).

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

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«

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

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

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

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

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

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.

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

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.

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

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

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-

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).

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.

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).

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.

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

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.

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

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 #

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.

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,

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)

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).

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

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

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.

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.

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.

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)

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

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.

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

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

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

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

> 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

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.

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 .

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 .

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.

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).

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*

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.

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.

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

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^ )•

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

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.

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*

%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.

&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).

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*

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

BZBLXOGRAPHY

1. Goldstein* Z»J*« Hughes* R«C.* Monslgny* M«* Osawa* T, and Sharon, N. (1980) Nature* 285* 66.

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.

7. Sharon* N. and Lis* H. (1972) Science* 177* 949

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.

12. Ashwell* O. and Morell* A.O, (1977) Trends Biochem.Sci., 2* "' ^

13. Monsigny* M.,Kieda*C, and Roche*A,C.(1979) Biol.Cell* 3[6* 289.

14. Ashwell* o. and Harford*J. (1982) Mn.Rev.Biochon., 5JL* 531.

15. Monsigny* M.* Kieda* C. and Roche*A.C. (1983) Biol.Cell* 47* 95.

16. Reitherman* R.v}«, Rosen* s.D. and Barondes* s.H. (1974)Nature* 248* 599.

17. Nowak*T.P. and Baronde8*S,H.(1975) Biochim.Biophys.Acta.* 393* 115.

18. Etzler, M.E. and Kabat* E«A. (1970) Biochemistry* 9, 869.

19. Oalbraith, w, and Goldstein* I.J. (1972) Biochemistry* 11* 3976.

58

59

20. HaiiBnar8trcxn# S., HammarsfcMn^M.L., Sundb^ld, O,, Amarp« J. and Lonngren, J. (1982) P«sc« Natl.Acad.Set• USA, T^, 1611.

21. Horejal, v. and KocourelcJ. (1978) Blochltn.Blophys.Acta», 532« 92.

22. Allen, A.K. and Neubarger, A. (1973) Bioch«n. J.* 135# 307.

23. Kilpatrick, D.c. and Yeoman, Mi.M. (1978) Biochem. J., 175, 1151.

24. Allen,A.K., Neuberger,A« and Sharon,N. (1973) Biocham.J., 131, 155.

25. Pereira, M.E.A. and Kabat, E.A. (1974) Blochamlatry, JL3# 3184.

26. So,L.L. and Goldstein, l.J. (1967) J.Inaminol., 99, 158.

27. VanWauve.J.P., Loontlai8,P.Gi. and Dfe Bruyne,C.K. (1975) Bioehim. Biophys.Acta. , 379, 456.

i 28. Kor t t , A.A, (1984) Eu r . J . Biochem., 138, 519.

29. Nicol«on,o.L., Blau8teln,vT. and Staler,M.E. (1974) Biochemistry,^ 196.

i 30. To/o3hiroa,3., Oeawa,T. and Tcwonura, A. (1970) Blochim. Biophys, Acta., 221, 514.

31. Allen,A.K., Desai,N.N. and Neuberger,A. (1976) Bioch«»n. J., 155,127

32. Pre8snt«C.A. and Romfeld, S. (1972) J.Biol.Cheiu., 247, 6937.

|3. ljia,H. and Sharon,N. (1977) in The Antigens (Sela,M. ed.) Vol.4 pp 429-529, Academic Press, NeWYorX.

34. De Boeck,H,, i:ioontien8,F.O., Lio,H., Sharon,t7. (1984) Arch.Biochem. Biophys., 234, 297.

35. Roberta,D.D. and Goldstein,X.J. (1983) Chemical Taxonany, Molecular* Biology and Function of Plant Lectins. Progress in Clinical and Biological Research. Vol. 138, NewYorki Lias, pp 131-141.

36. Kella, N.K.D., Roberts, D.D. Shafe, J.A. and Goldstein, l.J. (1984) J. Biol.Chem., 259, 4777.

37. So,L.L. and ^Idstein, l.J. (19G8) Bioehim.Bloph/a.Acta., 165, 398..

38. Yari«,J., Kalb,A.J. and LevitzHi,A. (1963) Bioehim.Biophys.Acta., 165, 303.

39. Neurohr,K.K., Young,W.M. and Mantsch,H.H. (1980) J.Biol.Chem., 255 i 9205.

60

40. De aoeck^H., Iiis,H,, Van Tilbeurgh,H., Sharon,N. and Loontlen8,F.O. (1984) J.Blol.Chem., 259, 7067.

41. Stein/ M.D,^ Howard,I.K. and Sage,H.J. (1971) Arch.Blochero.Biophys*^ 146, 353.

42. HamraarStrom, S. and Kabat, E.A. (1971) Blochanistry, 10, 1684.

43. Van Wauve, J.P., Loontiens, P.O. and De Bruyne, C.K. (1973) BiodhlB« Biophys. Acta, 313> 99.

44. Podder, S.K., Surolla, A, and Bachhawat, B.K. (1974) Eur. J. Blo-chem., jjA, 151.

45. Olsnes, S,, Saltvedt, E. and Phil., A (1974) J. Biol. Chera., 249, 803.

46. Khan, M.I., Surolia, N., Mathew, M.K., Balarara, P. and Surolia,A. (1981) Eur. J.Biochem., 115, 149.

47. Crowley, J.F., Goldstein, I.J., Amarp, J. and haongren* J.(1984) Arch. Biochem. Biophys., 231, 524.

48. Appukuttan, P.S., and Basu, D, (1985) FEBS Lett., 180, 331.

49. Etzler, M.E., Oupta, S. and Borrebaeck, C. (1981) J.Biol. Chom., 256, 2367-70.

50. Privat, J.P., Delraotte, F. and Monsigny, M. (1974) FEBS Lett., 46, 224.

51. Nagata, Y. and Burger, M.M. (1974) J.Biol.Chem., 249, 3116.

52. Olson, M.O.J, and Liener# I.J. (1967) Biochemistry, 6j 105.

53. Lotan, R., Skutelsky, E., Dannon, D, and Sharon, N. (1975) J.Biol. Chem., 250, 8518.

54. Peumans, w.j,^ Stinisaen, H.M. and earlier, A.R. (1982) Biocyiem J., 203, 239.

55. Ashford, D.Desai, N.N., Allen, A.K., Neuberger, A.,O'Neill, M.A. and Selvendran, R.R. (1982) Biochom. J., 201, 199.

56. Nachbar, M.S., Oppenheim, J.D,, and Thomas, J.O. (1980) J.Biol. Chem., 255j 2056.

57. Hindsgaul, 0., Norberg, T., Le Pendn., J. and Lemleux, R.U.,(1982) Carbohydr. Res., 109, 109-42.

61

58. Kltagaki->Ogawa« Matsumoto, Z.«Scmo« N, Takahashl^ N.«Endo« s, and Arata« Y, (1986) Eur. J.Blochara., 161, 779.

59. Lis. H. and Sharon# N. (1984) In Biology of Carbohydrates, («d. v. Ginsburg and P.w. Robbins) Vol. 2, pp. 1-85, New YorH» Wiley,

60. Borland. L,, Van Halbeek, H., Vliegenthart, J.F.O., Lis, H. and Sharon, N. (1981) J. Biol, Cham., 256, 7708.

61. Ohtani, K. and MisaXi, A, (1980) Carbohydr. Res., 87,275.

62. Misaki, A. and Goldstein, I.J.(1977) J.Biol.Chem., 252, 6995.

63. Priqent, M.J., Kontreui, J. and Streeker, G. (1984) Carbohydr. Res., 131, 83.

64. Sl-iaron, N, and Lis, H. (1982) in The Protein a (Neurath, H. and Hills, R.L. eds.) Vol. 5, pp.l'-l44. Academic Press, New Yori.

65. Rice, R.H, and Etzler, M.E, (1974) Blochero. Biophys. Res. Ccwnmun., 59, 414.

66. Salbraith, W. and Goldstein, I.J. (1972) Methods Enaymol., 28, part B, 318.

67. Borrebaeck, C.A.K., Lonnerdal, B. and Etzler, M.E. (1981) Biochem­istry, , 4119.

68. Kalb, A.J. and Levitzki, A. (1968) Biochem. J., 109, 669.

69. Wright, C.S. (1977) J. Mol. Biol., Jll, 439.

70. Makela, o. and Makela, P. (1965) Ann. Med. Exp. Biol. Penn., 34,402

71. Cunningham, B.A. (1975) Pure Appl. Chem., 41 , 31.

72. Kopp, T.P., Hemperly, J.J. and Cunningham, B.A. (1982) J. Biol. 1 Ch«a., 257, 4473.

73. Foriers, A., Lebrun, E., Van Rapenbuach, R., Oe Nave, R, and Strosberg, A.D, (1981) J, Biol. Cham., 256, 5550.

i 74. Kemperly, J.J. and Cunningham, E.A. (1983) Trends. Biochem. Sci. g, 100.

75. Lis, H. and Sharon, N. (1986) Ann. Rev. Biochem., , 35-67.

76. Strosberg, A.D., Lauwereys, M. and Foriers, A.(19R3) in Chemical Taxonomy, Molecular Biology and Function of Plant Lectins. Progress in Clinical and Biological Research(Goldstein, I.J. and Stsler,N.B, eds.) V(^.138, pp, 7-20, New Yorkt Llss.

62

77* Wright* C*S«« Oavilane«« F. and Peterson* D«l.(1984) Biochemistry, • 13, 280.

i 78* Peumans, Vf.j., Stinissoi, H«M« and Cariier, A.R. (1982) Planta, 154, 568,

j 79. Desai, N«N«, Allen, A.K. and Neuberger, A,(1981) Biochem* J«, 197, i 345. i I 80, Hermann, M.S*« Richardson, C«E«, Setzlar, L,H., Behnke, W.D, and

Thompson, R«E. (1978) Biopolymers, 17, 2107*

^ 81. Jirgenaons, B. (1979) Biochim. Biophys. Acta., 577, 307.

82. JirgenscHis, B. (1980) Biochim. Biophys. Acta., 623« 69.

83. Recke, G.N. and Becker, J.W. (1986) Science, 234 (4780), 1108.

84. Hemperly, J.J., Mostov, K.E. and Cunningham, B.A.(1982) J. Biol. Chem., 257, 7903.

85. Higgins, T.J.V., Chrispeels, M.J., Chandler, P.M. and Spencer, D. (1983) J. Biol. Chem., 258, 9550.

86. Hoffman, L.M. and Donaldson, D.D. (19B5) EMBO. J., j , 883.

87. Stinisnen, H.M., Peumans, W.j. and Carlier, A.R. (1982) Plant Mol. Biol., i, 277.

: 88. Stinisseiv H.M., Peumans, .J. and Carlier, A.R. (1983) Plant Molb. Biol., 2, 33.

89. Sehnell, D.j.« Alexander, D.C., Williams, B.o. and Etzler, M.E. (1987) Bur. J. Biochsm., 167, 227-231.

! 90. Goldberg, R.B., Hoschek, o. and Vodkin, L.O.(1983) Cell, 32* ^S.

I 91. Hamblin, J. «»d Kent, S.p. (1973) Nature, 245, 28.

92. Daszo, F.B. (1981) J. Suprarool. Struct., , 29.

j 93. Dazzo, P.B., Yanke, w.B. and Brill, w.j.(l978} Biochim. Biophys. Acta., 539, 276.

94. Dazzo, F.B., and Brill, w,j, (1977) Appl. Environ. Microbiol., 33, 132.

95. Man«), J.F. and Miege, M.N. (1981) Physiol. Veg., 19. 45.

96. Manon* J.P. and Pusztai, A* (1982) PXanta^ 155, 328.

97. Boyd, w,c. (1963) Vox. Sang., £, 1.

98. Etzler, M.S. (1981) Phytopath., 71,, 744.

99. Leach, J.E,, Cantrell, M.A. and Soqueira, L. (1982) Physiol. Plant Pathol., , 319.

00. Patrldge, J., Shanncm, L. and Gunof, D.(1976) Biochlm. Biophys. Acta., 451, 470.

01. Mlshkind, M., Rallchol, M.V,, Palevitas, B.A. and Keegstra, K.(1982) J. Call. Biol., 92, 753.

02. Mirelman, D., Oalun, E., Sharon, H. and Lotan, R.(197S) Nature, 256, 414.

03. Barkai, O.R., Mirelman, D. and Sharcm, N.(1978) Arch. Kicrobiol., 116, 119.

04. Auta, J.C., Sanford, B.H. and Wang, L.H. (1965) Proc. Natl. Acad. Sci.. USA. 54, 400.

05. Tak««hi, S. and Rikabo, C. (1986) Jpn. KoXaki Tokkyo Koho JP 61, 205.

06. Allen, N.K. and Brilliantine, L. (1969) J. Inwiunol., 102, 1295.

07. Sharon, N. (1976) in Mitogens in Iiwntinology (oppOTiheiro, J.J. and Rosenstreich, D.L. eds.) pp. 31*41, Academic Press, New York.

08. Novogrodosky, A. and Katchalski, &• (1971) Bioehim. Bioj^ys., Acta*, 22p, 579.

,09. Toyoshima, s., Akiyama, Y., Nakano, K. T<Miomura, A, and Osawa,T. (1971) Biochemistry, JIO, 4457.

.10. Rao, K.M.K. (1982) J. Theor. Biol.. 98, 61.

.11. Reisner, Y., Linker-Israeli, M. and Sharcm, N.(1976) Cell Immunol.* 25, 129.

.12. Reisner, Y., Biniamnov, M., Rosenthal, E., Sharon^ N. and Ranot,B. (1979) Proc. Natl. Acad. Sci. USA, 76, 447.

13. Reisner, Y., Oaehelin, C3,, Dubois. P., Nicolas, J.P. and Sharon, N. (1978) Proc. Natl. Acad. Sci. USA, 75, 2933.

14. Bourguignon, L.Y.M., Rader, R.L. and Mc Mohan, J.J. (1979) J.Cell Physiol., 99, 95.

CI

Relatmw, Y*« Ravld* A, and Sharon* N. (1976) Biochom. Biophys* Res. ConOTun»# 2i» 15S5»

Braun A.a. and Oalley^ J.P*(1981) Blochem* Btophys* Res. Coiamun., 98, 1029.

Leon« M.A, (1967) Science* 158* 1325.

Domer, P., Scriba, M. and Weil, R. (1973) Proc. Natl. Acad. Sci.

USA, 22' 1981.

De Grip* w,j, (1982) Methods Enz/mol., 8^, 197.

Biahayee, s. and Bachhawat, B.K* (1974) NeurobioX., £, 48.

Ronald, B. and Michael* M. (1986) Biosei. Rep», , 591.

Donelly, E.H. and Goldstein, l.J.(1970) Biochem* J., lie. 679.

r aXela, O. (1957) Ann. Med. Exp. Biol* £kippl.« 11. 35, 1.

Boyd, w.c and Shapliegh, E. (1954) Blood, £« 1195.

Hayes, C.E. and Goldstein, I.J.(1975) J. Biol. Chem., 250. 6837. Flory, L.L. (1966) Vox. Sang*, H , 137.

Mirelman, D., ed., (1986) Microbial Lectins and Aggluttoins, New Yorlcs Wiley.

s-harcm* N.(1986) in The Lectinsi Properties, Punctimis and ppli<4 cations in Biology and Medicine (Liener, I.e., Sharon, N. and Gold­stein, I.J. eds.j New YorXt Academic.

Beaehy, E.H., ed. (1980) Bacterial Adherencet Receptors and Recog<# nitlon, Ser. B, Vol. 6, Lcmdcmt Chapman and Hall.

M>el, C.A., Campbell, P.A., Vanderwall, J. and Hartrnm, A.L.( 1984) in Recognition Proteins, Receptors and Probest Invertebrates. Pro* grass in Clinical and Biological Reseaxrch (Cohen, E. ed.) Vol.l57« pp. 103«14, New Yorki Lias.

Berondes, S.H. and Nowak, T.P.(1978) Methods Ensyim>l., , 302.

Ravindranath, M.H., Higa, H.H., Cooper, E.L. and Paulson, J.C. (1985) J. Biol. Chem., 260, 8850.

133. Barondes, S.H. (1984) Science, 221» ^259.

65

134. Toiehberg, V.l., SiliMui !•# BoltBch, D.D, and Reaheff, 0.(1975) Proc. Matl. Acad. Sci. trSA, 21* 1383.

135. Levi, O. and Telchberg, V,i,(l981) J. Biol. Chem., 256, 5735.

136. Hlrabayaah.1, J., KawaaaXi, H., Susuki, K. and Kaaal, K. (1987) T. of BloGhaza., lOl, 775.

137. Agarwal, B.B.L. ani Ooldateln, I.J. (1967) Blochira. Blophya. Acta, 147, 262.

138. Aaberg, K., Holman, H. and Porath, J. (1968) Blochloi. Blophya. Acta., 1§2, 116.

139. Wang, J.L., BacXoor, J.w,, Reeke, Jr., <3.N. and Edelman, a.M.(1974) J. Mol. Biol., 88, 259.

140. Tlcha, M., Entlicher, O., Kostir, J.v. and Kocourek, J. (1970) Biochim. Biophya. Acta., 221, 282.

141. Baumann, CM,, Stroaberg, A.D. and Ruediger, H.(1982) J.Biochera., 122, 105.

142. Olanea, S. and Phil, A. (1973) Eur. J. Biochew., 25» 179.

143. Allen, H.J. and Johnson, 2.A.Z, (1976) Carbohydr. Rea., S^, 121.

144. Yokoaawa, H., Sawada, H., Abe, V., Numakunai, T. and Ishii, s.i. (1982) Biochem. Biophya. Res. Conwnina., 106, 451.

145. Shanker, I.P.N., Wilkinson, K.D, and Ooldstein, l.J. (1976) Arch., Biochem. Bioi^ya., 177, 330.

146. Bloch, R. and Burger, M.M. (1974) Biochem. Biophya. Res. CoiTirr.jn., 58, 13.

147. Lonngren, J., cJoldsteln, I.J. and Bywater, R. (1976) FEB8 Lett., ii, 31.

148. Appulcuttan, P.S., Surolia, A. and Bachhawat, B.K.(1978) Ind. j. Biochem. Biophya., iA, 382.

149. Majiimder, T. and Surolia, A. (1978) Prep. Biochem., £, 119.

150. Majurader, T. and Surolia, A. (1979) Ind. J. Biochem. Biophya. Ife 200.

151. N8imba.»Lubaki, N., Peumans, w.j. and earlier, A.R. (1983) Bioehsm* J.# 215, 141.

66

152. Ph.D. Thesis of Mohd. Islam Khan (1986).

153. Oppenheira, J.D., Nachbar* M.S., Salton, M.R.J, and Aull, P. (1974) Biochon. Biophys, Res. Coffimun.# 58, 1127.

154. Sela, B.A., Wang, J.L. and Edelman, O.M. (1975) J.Biol. Ghem., 25Q. 7535. ^

155. Felsted, R.L., Leavltt, R.D., and Bachur, N.R., (1975) Biochlm, Biophys, Acta., 405/ 72.

156. Mohan, S., Doral, D.T., Srimal, S. and Bachhawat, B.K. (1982) Bio-chero. J., 203, 253.

157. Vretbald, P. (1976) Biochim, Biophys, Acta, 434, 57.

158. Lcwry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chera., 193, 265-275.

159. Bradford, M.M. (1976). Anal Biochera., 72 , 240-254.

160. Polin, O. and Clocalteu, V. (1927) J.Biol Chem., Jl* 627-649.

161. Waheed, A. and Salahuddln, A. (1975). Blochem. J., 147, 139.

162. Cuatrecasas, P. (1970). J. Biol. Chem. 245, 3059-3065.

163. Ansari, A.A. and Salahuddln, A. (1973). Blochem. J. 135, 705-711.

164. Laernrall, U.K. (1970) Nature (Lond) 227, 680-685.

165. Hoebeka, J./ Porier, A., Schreiber, A. and Strosberg, D.A., (1978). Blochem. J. 92# 5000-5005.

166. Agarwal, P. and Mahadevan, A. (1984) Acta Phytopathologica Acade-mial Scientlarun Hungarical, JL9, 219-235.

167. Ackers, G.K. (1967) J. Biol. Chem., 242, 3237-3238.

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

68

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.