Purification of wheat germ agglutinin by affinity chromatography on a Sepharose-bound N-acetylglucosamine derivative

Vol.

52, No.

BIOCHEMICAL

2, 1973

PURIFICATION OF WHEAT GERM ON A SEPHAROSE-BOUND

R.

Lotan,

A.E.S.

April

9,

BIOPHYSICAL

RESEARCH

AGGLUTlNlN BY AFFINITY N-ACETYLGLUCOSAMINE

Gussin:

H.

Lis

and

Department of Biophysics Weizmann institute of Rehovoth, Israel

The

Received

AND

N.

COMMUNICATIONS

CHROMATOGRAPHY DERIVATIVE

Sharon

Science

1973

SUMMARY: Sepharose-Z-acetamido-N-(E-aminocaproyl)-2-deoxy-~-D-glucopyranosylamine was prepared by a react ion of 2-acetamido-3,4,6-t ri -0-acetyl-2-deoxy-R-D-91 ucopyranosylamine and N-(benzyloxycarboxyl)-c-aminocaproic acid, removal of the Oacetyl and the benzyloxycarboxyl groups and coupling to Sepharose. The product was used for the purification of wheat germ agglutinin, by adsorption from a The puri f ied crude wheat germ extract and elution with O.lM acetic acid. agglutinin was homogeneous on SDS-polyacrylamide gel electrophoresis and had a specific hemagglutinating activity of 3000 u/mg when tested on trypsinized rabbit erythrocytes. It was rich in cysteine, cystine and glycine, and contained no sugar.

INTRODUCTION: can

be

Cell-agglutinating

purified

either

affinity

chromatography

reported

the

and

its

and

by

conventional (for

preparation

use

for

the

purification

D-g1

ucosami

specific

ne,

and

wheat The

germ

of

its

columns

of

has

been

previously

use

the

agglutinin

protein

I and

2).

as

lectins,

chemistry,

or

by

We have

recently

WGR

agglutinin,

a lectin

the

analogous

purification (4-7)

from

ovomucoid,

Science 01060,

Copyright 0 19 73 by Academic Press, 1~. All rights of’ reproduction in my form reserved.

for

of

N-acetyl-

wheat

of from germ

the

derivative N-acetyl-D-glucosamine-

commercial lipase

a glycoprotein

wheat by rich

germ.

affinity in

Center, U.S.A.

Smith

656

College,

chromatography

N-acetyl-D-glucosamine

(5.61.

described,

specific

(3). of

(WGA) of

Clark Mass.

soybean

synthesis

for

insolubilized

address:

refs.

D-galactose

the

purification

on

“Permanent

describe

see

of

known

Sepharose-e-aminocaproyl-B-D-galactopyranosylamine

and we

proteins,

methods

reviews

of

N-acetyl-D-galactosamine Here

sugar-specific

Northampton,

Vol.

52,

BIOCHEMICAL

No. 2, 1973

EXPERIMENTAL

:

a gift

Pfizer

Inc.

(New

(Uppsala,

Sweden),

and

cyanogen

Commercial

wheat

from

N-Acetyl-D-glucosamine

germ

Melting were

determined,

i .r.

spectra

KBr

silica heating

used

as

pellets

(1

laboratory

of

Weizmann

Institute

microanalysis

the

rabbit

-5”

at

and

(12

ml),

filtered.

The

N,N-dimethylformamide)

evaporated

and to

Yield

-

i.r.

data:

C=O)

and

62%

dryness. (3.68

v,“,“: 1745

(11, then

with

Organic

with

a Varian

on

were

of

by

and

was

20%

a

T-60 below

precoated

plates

sulfuric

performed

acid

by

themistry

at

the

the

a spectrophotometric

method

-

triethylamine

(12

at

allowed The

to residue

m.p.

203”-205”;

1530

cm-l

(peptide n.m.r.

acid -5”.

filtrate

12)].

g),

(0-acetyl);

t.1.c.

ions

Polarimeter;

temperature

spraying

determined

from

The

1 ized + 11.9”

[c,]:’ Amide

1), 2.72

657

1655 (5H,

which twice (c,

(CONH), phenyl),

the from

1.0,

mmoles) for

tlD

a solution

( 10

mnoles

D-g1 at

were

stirred

added

stirred

16 h after

crystal

(12

was

immediately

was

for

was

mixture

mnoles)

2-acetamido-2-deoxy

mixture

stand

data:r

The was

[synthesized

literature

hours

ascending

Microanalyses

kept

ml,

two

bath

141

recorded

with

N-(benzyloxycarbonyl)-e-aminocaproic

20

for

the

by

were

spectra

vacua,

mmoles),

2-acetamido-3,~,6-tri-O-acetyl-2-deoxy-~-D-glucopyranosylamine,

to

mg KBr)

n.m.r.

detected

No.

Rotat

( 1) .

of

according

100

by

apparatus.

erythrocytes.

(20 then

N.Y.).

6-tri-O-acetyZ-N-[~-lbenzyZoxycarbonyI!-~-aminocaproyZ]

N,N-dimethylformamide

min.

in

in

was

lsobutylchloroformate

in

(Rochester,

a Perkin-Elmer

Department

activity

of

Co.

Pharmacia

Science.

trypsinized

a solution

Kodak

from

Fisher-Johns

with

minutes.

2-deoxy-B-D-glucopyranoeylamine

to

a

237,and

were

of

2-Acetamido-3,4,

added

with

tested

was

WGA.

tubes,

was

a few

Hemagglutinating using

of

performed

Spots for

COMMUNICATIONS

obtained

Eastman

mg compound

compounds

of

source

Model were

200”

RESEARCH

4E was

from

determined

semimicro

G (Merck). at

Sepharose bromide

I-dm,

of

gel

(8,g)

York).

Spectrophotometer

Homogeneity

BIOPHYSICAL

(Z-acetamido-Z-deoxy-D-glucopyranose)

were

Evaporations

50”.

and

points

of

apparatus.

of

was

in

Perkin-Elmer

AND

room

(10) 20

in

ucopyranose

temperature

solvent

was

methanol

:ether.

N,N-dimethylformamide); 1700 3.05,

(benzyloxycarbonyl 3.55

(2

NH),

7.96,

Vol. 52, No. 2,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ve, ml

Figure

I.

8.05 H,

Affinity chromatography of WGA on a column of Sepharose-2-acetamidoN-(c-aminocaproyl)-2-deoxy-8-D-glucopyranosylamine. An extract from 100 g defatted wheat germ (90 ml) was applied to a column (2.8 x I7 cm) and after washing with 600 ml saline (I5 ml.fractions), elution (arrow) was performed with O.lM acetic acid (6 ml -fractions). The flow rate was 100 ml/h and chromatography was carried out at 4”. HA - Hemagglutinating activity.

(gH,

three

6.51;

N,

acetoxy

7.15.

groups),

C28H3g01

8.12

lN3

(3H,

requires

acetamido

C,

group).

56.68;

H,

6.57;

Found: N,

C,

56.85;

7.08.

2-Acetamido-N-[N-(benzyloxycarbonyZ!-~-aminocaproyZ]-2-deoxy-B-DgZucopyranosy

Zmine

Freshly to

a solution

The

mixture

was

monitored

The

solution

was by was to

go% (2.1 data:

prepared of

evaporated

9);

O.lM

compound for

t.1.c.

in

C,

56.67;

The

50

room

the

ml

was

[al:’

N,

+ I);

9.17.

(13)

absolute

methanol) The

@:I

v/v)

of

solid

CO2 from

(c, (CONH)

C22H3308N3

was

to

Rfs. and

the

, 1705

(benzyloxycarbonyl C,

56.52;

hydrogenated pressure

of in

at

room

the

compound presence

temperature

LI of for

(4.5 10% 8 h.

mmoles

in

658

catalyst

I). was Yield

-

1.r. C=O) H,

7.12;

N,

. 8.95.

(1 I I).

75 ml

palladium-on-charcoal The

Table

:ether.

2-Acetamido-N-(~-aminocaproyZI-2-deo~-B-D-gluaopyranosy~amine A solution

pH 8.

deacetylation

solvent

methanol

N,N-dimethylformamide);

dropwise

give

of see

1.0,

requires

added

progress

(for

crystallized

12.9”

1655

methoxide

temperature.

addition

residue

amide

7.07;

in

acetone-methanol

218~-220~;

H,

mmoles

by

(peptide

sodium

5 h at

neutralized

m.p. cm-l

methanolic I (5

stirred

dryness.

1530

Found:

( f I) .

methanol:water, (50

was

f i I tered

mg)

8~2 at off

v/v)

atmospheric and

the

was

Vol.

52, No.

2, 1973

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE Rf

values

of

Compound*

performed iez) .

Ethyl

derivatives

precoated

on

t.1.c.

systems

silica

gel

plates

(20x20

Acetone:Methanol (9:1 v/v)

Methanol (9:l

I I

0

0.57

0.95

III

0

0

0.3

description

was

of

evaporated

Yield

80%

data:

1530

12.78.

I,

II

to

(1.2

and

dryness.

9);

m.p.

cm-l

(pept

i de

Cl 4H2706N3

rev

Ii res:

of the ligand (III)

Binding

Sepharose

49

was

Ill,

see

text.

The

residue

193”-195” Amide C,

was

(dec.).

I),

1655

50.43;

crystallized

[a]:’

(CONH).

H,

8.17;

from

+ 23.3” Found

N,

C,

(c,

Axen

conjugated unreacted

bound

to

the

bound

(14)

with

cyanogen

of The

1 igand

bromide

per

ml

the

for

and

3 h in

by

was

The

acid

glucosamine

resin

procedure

Sepharose.

determined

packed

detailed

gently on

liberated

conjugated

Purification Extraction

was

the

the

stirred groups

Sepharose

H,

7.93;

then

reacted

found

to

of of

2 umoles

(3).

pH 8, ligand

the

to

covalently

conjugated

a Beckman-Spinco

contain

general before

IM ethanolamine,

hydrolysis on

the

described

amount

with

amino of

resin acid

covalently

Sepharose.

of WGA. of

the

;

12.6.

by

following

was

I .O water)

to Sepharose 4B

activated

active

estimation

analyzer.

al

resin

block

and

et

methanol:

50.32;

2-acetamido-N-(E-aminocaproyl)-2-deoxy-B-D-glucopyranosylamine, of

CAMAG

:Acetone v/v)

1

ether.

The

cm)

1.0

f i 1 trate

method

in

0.55

For

N,

solvent

Acetate:Acetone (4:1 v/v)

I

i .r.

on

COMMUNICATIONS

I

N-acetylglucosamine different

T.1.c. was (Mut tenz/Schw

RESEARCH

agglutinin

from

defatted

659

commercial

wheat

germ,

and

Vol.

52,

No. 2, 1973

isolation by

of

Allen

et

obtained

(2.8

1 i brated

100

ml/h,

0.1

with

M acetic

acid

eluted

pooled,

AND

water

with

(O.lM,

50%

had in

the

was

sugars

SDS.

of

a column

which

had

been

the

rate

of

amount

(A280

addition

~0.05))

of and

monitored

U.V. of

by

then

its

absorbing

IM

NaOH,

dialyzed

only

three

steps:

lyophilized.

Allen

bulk

and

of

these

proteins

column

could

affinity in

the

the

was be

with

described

in

chromatography

a b-fold

proteins

to

not

eluted

that

involves

resulted

adsorbed

however

to

(~0.01%)

on tested

not al.

a single

column

eluted acetic

the

literature

in

(Fig.

I), The

with

0.1M

purification

present

negligible.

band

analysis

that

of

reported

the

amino

by

the

No acid

the

active

but

the

agglutinin

N-acetyl-D-glucosamine acid.

The (7),

but

it

This contradicts

660

IO%

final

and

product

was

obtained

polyacrylamide of

WGA gave

literature;

it

was

indication

for

analyser

trace, acid

result other

gel

hydrolysates

the

phenol-sulfuric

a glycoprotein.

(7)

in

acid in

(6,7,15).

glycine

observed

et

acid

similar

WGA is

as

Amino

and was

that

to

significant

containing

above

not

WGA migrated

very

showing

supernatant

at

was

described

yield.

0.1%

sugar

report

the

as

purified

neutral

4”

effluent

fractionation

The

of

cysteine,cystine

amino

yield.

activity

a composition in

no

as

clear

eluted

effluent

dropwise

precipitation

activity

it

a comparable

containing

was

and

and

sulfate

was

to

ml);

same

The

acid

described

fraction

adsorbed

200

the

The

by

sulfate

about

sulfate

was

in

centrifuged

ammonium

hemagglutinating that

at

until

acetic

procedure

Ammonium

ammonium

essentially

Sepharose 4”.

COMMUNICATIONS

DISCUSSION

purification

II). WGA,

applied

at

fractions).

with

water,

extraction,

(Table

kept

detected

neutralized

against

The

was

ml.fractions),

(6 ml.

done

the

conjugated

column,

(I5

were

centrifugation,

germ the

RESEARCH

280 nm.

were

extensively

The

of

280 nm was

fractions,

material

ml

saline at

at

RESULTS

ine.

and wheat

100

absorbing

The

defatted

BIOPHYSICAL

fraction

dialysis

containing sal

AND

sulfate

After g of

with

absorbance

of

100

first

material

ammonium

(7).

17 cm)

equi

55%

al.

from x

with

the

BIOCHEMICAL

is

the and

presence there

reaction in

reports

rich

agreement

(4,6,15).

of was

no

(16), with

the

Vol.

52, No.

BIOCHEMICAL

2, 1973

AND

TABLE Purification

Fraction

Total

Water extract 100 g defatted germ

sulfate (55%) and centri-

Purified affinity

WGA after chromatog-

RESEARCH

COMMUNICATIONS

I I

WGA from

protein ( 9 1

from wheat

Ammonium precipitate dialyzed f uged

of

BIOPHYSICAL

Wheat

Germ

Total activity (hemaggl ut i nating units)

Specific (uni

activity ts/mg)

Yield &

28

280,000

10

100

3.5

140,000

40

50

yo,ooo

0.03

3000

32

why

Table

II.

Ion

Protein et al. agglutinin. metrically

exchange

according

to

the

same

peak

was

other

is

now

chromatography

the

method

different in

The

the first two fractions was determined according to Lowry and in the last step by direct weighing of the lyophilized Hemagglutinating activity was determined spectrophoto(8,9).

of

Allen

hemagglutinating

differences

of

in

(17)

use

of

activity from

the

wheat

the

ACKNOWLEDGEHEIJT

REFERENCES: 1. Sharon,N. 2. Lls; H.

purified

et

al.

(7)

reported

used

for

described

N-acetylglucosamine-specific being

the

(~3000

that

co1 umn

of

WGA on gave

three

u/mg).

the

isolation in

this

protein

The

previously

a SP-Sephadex

of

peaks

amount

(7). the

of

This

each

protein

may

in

reflect

This research from a friend Argentina.

ication

for

(e.g.

from

UZem

and Lis, and Sharon,

H.,

Science, N. ,Ann.

was supported of the Weizmann lJJ, 949 Rev’.. Biochem.

661

in

part Institute

by

the europeus

a contribution in Buenos

(1972). 1972

(in

each

strain

isolation and

tested. :

having

agglutinin.

commun

lectins

column

press).

Aires,

potato)

Vol.

3.

52, No.

Gordon,

(1972) 4. 5.

6. 7. 8. 8. IO. II. 12. 13. 14. 15. 16.

17.

2, 1973

J.A.,

BIOCHEMICAL

Blumberg,

S.,

Lis,

AND

H.

and

BIOPHYSICAL

Sharon,

RESEARCH

N.,FEBS

Letters,

COMMUNICATIONS

2,

133

*

Burger, M,M. and Goldberg, A.R.,Proc. Nat. Acad. Sci. U.S.A., 57, 359 (1367). Burger, M-M., Proc. Nat. Acad. Sci. U.S.A., 62, 994 (1969). Levine, D. Kaplan, M.J. and Greenaway, P.J.,Biochem. J., 129, 847 (1972). Allen, A.K., Neuberger, A. and Sharon, N.,Biochem. J., 131,755 (1973). Liener, I.E. ,Arch. Biochem. Biophys., 2, 223 (1355). Lis, H. and Sharon, N.,Methods in Enzymology, Vol. 28 (Ginsburg,V. ed.) Academic Press, New York 1972. p. 360. Schwyzer, R., Iselin, B.M. and Feurer, M., Chem. Abst., 56, 4864 (1962). Horton, D. in Organic Syntheses, Vol. 46 (Corey, E.J. en, John Wiley and Sons, Inc., 1366, p.1. Bolton, C.H. and Jeanloz, R.W., J.Org. Chem., 28,3228 (1962). Zemplen, G. and Pacsu, E., Bet-., 62, 1613 (192g, Ax&, R., Porath, J. and Ernback, S.,Nature, 214, I302 (1967). Nagata, Y. and Burger, M.M., J. Biol. Chem., m, 2248 (1972). Dubois, M., Gilles, K.A., Hamilton, J.K., Rebx, P.A. and Smith, F., Anal. Chem., 28, 350 (1956). Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randal 1, R.J., J.Biol .Chem., J&j, 265 (1951).

662