Vol. 91, No. 3, 1979 December

BtOCHEMICAl

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

14, 1979

A Co(III)

DERIVATIVE

Received

October

4,

I.

G. R. I#unske, Legg

in Biochemistry and Biophysics the Department of Chemistry Washington State University Pullman, WA 99164

1979

050

OF CONCANAVALIN Al

M. S. Urdea', D. J. Ehristie3, 3. A. Magnuson , and J. Program

1045-l

and

SUMMARY

Co(II1) has been stoichiometrically incorporated into jack bean concanavalin A. The Co(II1) protein still possesses a binding site for an additional divalent transition metal ion which together with Ca(I1) can induce the sugar binding ability. No H202 oxidation of Co(I1) occurs with demetallized concanavalin A activated with Ca(I1) and Co(I1) unless Co(I1) is present in a stoichiometric excess. Evidence is presented to indicate that kinetically stable Co(III) is bound to a completely different location than the thermodynamically stable Co(I1) protein site. The jack variety

bean lectin,

of carbohydrates

Until

recently,

it

[e.g.,

Mn(II),

Ni(II),

subunit have

shown

that

of

that

capacity

The exact

1 This grants

metal

4

activity role

a transition

activity.

metals

does

of

the

transition not

(2).

is alone

capable

ions

is

capable and that

the

mono-

investigations of

have

conferring

bind

ions to its

remains site

unclear.

mono-

The hypothesis

Sl so that by United

a calcium

States

Public

California,

address: Department of Biochemistry and Biophysics, San Francisco, San Francisco, CA 94143.

Present

address:

Milwaukee

To whom correspondence

and co-workers

alter

Other

ion

to each

complement

significantly

protein

Ca(II),

metal

(1). metal

Christie Ca(I1)

a

to Con A (3,4,5).

of metal

must

the

research was supported in part GM 23081 and CA 14496.

2 Present 3

like

that

pH Con A containing

without

binds

ion must be bound

saccharide-binding

transition

saccharide-binding

transition

the

Mn(II),

specifically macromolecules

accepted and Ca(II)

at physiological

saccharide-binding indicated

generally

or Co(II)]

monosaccharides

addition

A (Con A),

and carbohydrate-containing

has been

of Con A for

of binding the

concanavalin

Blood

and reprint

Center, requests

Milwaukee, should

ion

that

a

can bind

Health

to

Service

University

of

WI. be addressed. 0006-291X/79/231045-06$01.00/0

1045

Copyrighr All rights

@ 1979 by Academic Press. Inc. ofreproduction in anyform reserved.

Vol. 91, No. 3, 1979

its

site

S2 and induce

To better

define

oxidize

the

Co(I1)

Unexpectedly metal

BIOCHEMICAL

at

role

is

presented

site

at

the

binding

the

S2 site,

can be formed

in proteins

stable

complexes.

needs

metal

ion,

and suggests sites

for

which saccharide

that

the

that

kinetically

other

further

examination.

we attempted

to

Co(III)-Con

derivative

indicates

at

RESEARCH COMMUNICATIONS

a substitution-inert

and Mn(II),

which

activity

transition

a Co(II1)

such as Ca(I1)

Evidence

Co(I1)

of

Sl to produce

we produced

ions,

nor

saccharide

AND BIOPHYSICAL

still

than

those

required

binding

Co(II1)

is

additional

activity.

neither

inert that

A derivative.

at the

Co(III)

form

Sl

complexes

thermodynamically

MATERIALS AND METHODS 1~ Con A was isolated from jack bean meal as previously E2& of 13.7 was used for the protein concentrations (7). was prepared as described by Kalb and Levitzki (8). Ca(I1) the various derivatives was checked by ato .c absorption. trations were determined by counting of " Co (New England Beckman Biogamma II spectrometer. A specific activity of 10,000 cpm per nmol of Co was used. The concentration of tive was determined by biuret assay.

described (6). An Demetallized Con A contamination in The cobalt concenNuclear) on a approximately the Co(II1) deriva-

-4 The Co(II1) derivative was prepared as follows. To an 8.08 x 10 M demetallized Con A in 1 M NaCl, pH 6.5, 4"C, was added 3 equivaAfter 2 h, 1.1 equivalents of CaC12 were added and the protein was in&bated for an additional 2 h. The4solution was diluted with M Ca(II), Co(I1) Con A, buffer to produce a solution which was 4.04 x 10 0.02 M barbital, 0.5 M NaCl, 0.04 M phenol, pH 7.5 at 4'C. Phenol was added to prevent metal-catalyzed free radical damage that can occur upon Co(I1) to The oxidation was initiated by the addition Co(II1) oxidation with H202 (9). solution. of enough freshly prepared H202 stock to give a 4 mM reaction After an appropriate time (see Results) the reaction was quenched with After 30 min at catalase (1 pg/ml; Sigma) and acidified to pH 2.0 with HCl. 4'C, the solution was chromatographed on a G-25M Sephadex PD-10 disposable Fe(II)EDTA reduction of the column (Pharmacia) equilibrated with 10 mM HCl. Co(II1) Con A derivative was carried out in 10 mM HCl with a 50-fold molar The Fe(II)EDTA was prepared according excess of the iron complex for 30 min. to Urdea and Legg (9). The fluorescent sugar derivative, 4-methylumbelliferyl u-D-mannopyranoside from Koch-Light Limited. The fluorescence saccharide OluM) , was purchased binding assay of Christie et al. (2) was used for all Con A binding activities A Perkin-Elmer MPF-3L fluorimeter was used for the binding studies. reported. RESULTS Figure various Co(I1)

times

1 summarizes under

was acidified

the

a number for

cobalt

to Con A stoichiometries

of conditions.

30 min

(pH 2.0),

1046

If

obtained

Con A containing

the metal

ions

could

at

Ca(I1) be removed

and by

Vol. 91, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Addition of com pH7.5

co(m) con A pn 2.0

Fe (II) EDTA Reduction (30mln)

I

L

I

1

I

I

2

3 Time

I

4 5 ( hn 1

opo-Con , pli 2.0

A

6

1: Cobalt stoichiometry of Con A after various treatments and chromatography on a PD-10 colunm. Con A with Ca(II) and excess Co(I1) acidified with HCl and chromatographed at pH 2.0 (a-m). Con A with Ca(II) and excess Co(I1) treated with H 0 acidified, and chromatographed at pH 2.0 (o-o). Con A 2' with Ca(I1) an a excess Co(I1) chromatographed with pH 7.5 buffer containing 0.02 M barbital and 0.5 MNaCl (e-a). Con A with Ca(I1) and excess Co(I1) treated with H202 and chromatographed with pH 7.5 buffer (o-o). The upward arrow shows that a second cobalt can be bound to Con A containing Co(II1) at pH 2.0. The downward arrow shows the demetallation step induced upon reduction. Figure

chromatographing protein remove

on a PD-10

and metal the

metal

following

25,500-dalton H202 oxidation pH 7.5,

If

is

amount

with of

with

the

acid,

an equivalent cobalt

(Figure

the

cobalt

cobalt

acidified of

of 1).

before cobalt

derivative

chromatographed Co(II),

cobalt

cobalt,

were

per

bound

protein

(o-o). monomeric

per

after

as described,

In addition,

a

chromatography

obtained

the

the

not

was found

of acid-stable

was not

If

at pH 7.5 did

to oxidize

of acid-stable

two equivalents If

10 mM HCl (m-m).

chromatography

complement

protein

(o-e).

treated

and provided

stoichiometric

amount

oxidation,

subunit

with

When H202 was added

a full the

equilibrated

acidified,

in the

4 h,

Con A was detected.

not

(e-w).

increase

approximately

at pH 7.5

were

ions

time-dependent After

ions

column

6 h of brought

binds

when Co(I1)

another is

to

Vol. 91, No. 3, 1979

oxidized

in

where

the

Co(I1)

to each

presence

experiments

of Co(I1)

with

ions

detected

were

not

(not

in the

50-fold

cobalt presence

molar

95% of

the

reduction

reverses

cobalt

is

required

Co(II1)

active.

is

Co(III)-Con is also

bound

With

excess cobalt

a stoichiometric

of Ca(II),

less

excess

than

0.1

of H202 did

was present.

Ca(I1)

be removed

When Co(I1)

and Ni(II),

by treating

followed was removed

for

was carried

of it

the

is

acid-stable

protein

by column

chromatography

in 30 min

(Figure

acid-stable

is most

plot

protein

MUM binding out

prior

shown in

Co(I.1)

Co(I1)

a two-fold

acid-stable

Even a 50-fold

incorporation

assay

A Scatchard

Ca(II),

cobalt

derivative,

and Ni(I1)

is

1).

cobalt

likely

that

with

a

with

10

Since

H202

and Fe(I1) the

acid-stable

(9).

activity,

The Co(II1)

1.03

or absence

excess

modification,

The fluorescence binding

could

for the

conditions

per monomer).

of Fe(II)EDTA

is

under cobalt

with

was used.

Con A containing

(0.96

out

Only

excess

unless

of

carried

be found.

excess

oxidation

were

presence

cobalt

Over

and Ni(II)

one acid-stable

monomers.

stable

DIM HCl.

tive

could

can be detected The acid

above

in the

ions

RESEARCH COMMUNICATIONS

shown).

when a five-fold either

Ca(II)

to exchange,

to protein

acid-stable

oxidized

slow

mentioned

cobalt

give

cobalt

are

respect

of Co(II),

acid-stable

AND BIOPHYSICAL

of Con A containing

and Ni(II)

Con A monomer

All

amount

BiOCHEMICAL

to

according the

2.

prior Ca(II)

activated

by Mn(I1)

of

other

are

metal

nearly the

(not

shown).

or by Ca(I1)

saccharide

ions,

identical

(2).

is not

A containing

and for

and Co(II)

of

and co-workers

to Co(III)-Con

The results

to oxidation

A containing

to Christie

addition

of MUM binding

Figure

to Con A, a measure

Ca(I1) for

the

Fe(II)EDTA-reduced The Co(II1)

deriva-

and Mn(I1).

DISCUSSION It Sl site Ca(I1)

is evident

from

of Con A.

The production

and Ni(I1)

which

the

binds

data

presented

that

of a Co(II1) MUM strongly

1048

Co(II1)

is not

derivative

of

supports

this

bound

to the

Con A containing

contention.

It

is

Vol. 91, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Scatchard plot for MUM binding to Con A containing Ca(II), Ni(II), Figure 2: and Co(II1). Lfree represents free MUM and v represents fraction of carbohydrate sites occupied per 25,500-dalton subunit of Con A. also

unlikely

is not

that

capable

binding

Co(I.11)

of binding

activity

Co(I1) are

in

strict

substitution-inert field

ligands

(10).

Either

the

are ligand

another

the

Ca(I1)

the

Co(II1)

form

site

has been

kinetically

inert

the

Co(I1)

that

not

probe

a protein

introduced

into

assumed

that

Co(I1)

to Co(II1)

complex

strong

in the

bind

are not

is

Co(II1)

the

conditions

a stable

six

relatively geometry enough

Sl site.

of coordinating

that

in a few cases

Under

to stabilize There

is,

and stabilizing

to a position Whether

other or not

than

this

is

investigation.

as a biological

complexes

saccharide-

in an octahedral is

capable

When Co(I1)

inert

metal

evidently

under

initiates

derivative

to H202 oxidation.

essential

in Con A (12,13).

Co(II1)

a kinetically

is

site

metal

Co(II1)

must be met to obtain

lanthanides

of

occurred

susceptible

that

The utility

has probably

not

the

(2).

reported

presently

generally

protein

protein

is

(10).

addition

to the

reach

since

on the

or transition site

Ca(I1)

It

composition

however,

site

which

provided

not

Ca(I1)

native is

complex.

or H202 may simply

It

Sl site

requirements

Co(III),

Co(II1).

as in the

the

Co(II1)

strong

the

MUM, although

at pH 7.2,

investigated, There

occupies

subject

is produced

from

its

to ligand

metal-binding

oxidation

(14,lS).

1049

stems

will In the at

present other

to

exchange site,

occur

a site

capacity

it

is

--in situ. study, than

This however, the

Vol. 91, No. 3, 1979

BIOCHEMICAL

thermodynamically phatase, binding

stable

Co(II) site

Co(II)

evidently

binding

migrates

oxidation

is

therefore

seriously

considered

site.

from

to H202 oxidation

prior

AND BIOPHYSICAL

questionable

the

(14).

Similarly,

in alkaline

structural

to

The generality

and alternate

in any Co(I1)

RESEARCH COMMUNICATIONS

to Co(II1)

sites

the

phos-

catalytic

of --in situ of oxidation

must be

oxidation.

Acknowledgment We would studies

like

and for

to thank

helpful

Dr.

J. A. Wells

for

his

aid

in preliminary

discussions. REFERENCES

1. 2.

Goldstein, I. .I., and Hayes, C. E. (1978), Adv. Carbohydr. 25, 127-340. Christie, D. J., Alter, G. M., and Magnuson, J. A. (1978), 17,

3. 4. 5. 6.

Brewer, C. F., Marcus, D. M., Grollman, A. P., and Sternlicht, H. (1974), J. Biol. Chem., 249, 4614-4617. R. G. (1978), Biochemistry, 17, 4245-4250. Harrington, P. C., and Wilkins, Christie, D. J., Munske, G. R., and Magnuson, J. A., Biochemistry, in press. Agrawal, B. B. L., and Goldstein, I. J. (1967), Biochim. Biophys. Acta, Yariv, 165,

a. 9. 10. 11. 12.

Biochemistry,

4425-4430.

147,

7.

Chem. Biochem.,

262-271.

J.,

Kalb,

A. J.,

and Levitzki,

A.

(196g),

Biochim.

Biophys.

Acta,

303-305.

A. (1968), Biochem. J., 2, 669-672. Kalb, A. J., and Levitzki, Urdea, M. S., and Legg, J. I., Biochemistry, in press. Legg, J. I. (197S), Coord. Chem. Rev., 3, 103-132. Becker, J. W., Reeke, G. N., Jr., Wang, J. L., Cunningham, B. A., Edelman, G. M. (1975), J. Biol. Chem., 250, 1513-1524. Arch. Biochem. Biophys., Sherry, A. D., and Cottam, G. L. (1973),

and 156,

665-572.

13. 14. 15.

Barber, B. H., Fuhr, B., and Carver, J. P. (1975), Biochemistry, 16, 4075-4082. Anderson, R. A., and Vallee, B. L. (1977), Biochemistry, 16, 4388-4342. B. L. (1978), Biochemistry, 17, 3385-3394. Van Wart, H. E., and Vallee,

1050