Reversible dissociation of zinc in bovine carbonic anhydrase

Vol.‘2, No. 3 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REVERSIBLE

DISSOCIATION

OF ZINC IN BOVINE CARBONIC ANHYDRASE

Sven Lindskog Institute

March 1960

and Bo G. Malmstrijm

of Biochemistry, Uppsala,

University Sweden

of Uppsala,

Received February 24, 1960 Keilin

and Mann (1940)

(CA) is a zinc rious

laboratories

ters,

1959)

mogeneous

varies

but recent

since

Zn at neutral

Watts

and Wormall,

sible

on destruction

ters,

1959).

within

reversibly

chelating

agents,

of the protein

at

remove

it

(Tupper,

has only

possible

been posand Pe-

to dissociate

enzyme at pH 5 in the presence

a loss

it

radioac-

low pH (see Keller

now found

of

attached

with

of a month

such as l,lO-phenanthroline

by addition

presence

is firmly

is no exchange

of the metal

from the

the

and Peon a ho-

does not

1951).

however,

1960)

The metal

a period

of Zn causes

regained

(Lindskog,

pH even during Removal

from va-

(see Keller

electrodialysis

and there

anhydrase

reported

has revealed

of enzyme.

1941),

carbonic

a wide range work

extensive

We have,

the metal

be fully

content

of the protein

and Mendive,

The removal

The zinc

analytical

preparation

to the protein

tive

that

metalloenzyme.

one atom of Zn per molecule

(Scott

demonstrated

of

(OP) or citrate.

of activity,

which,

however,

can

of Zn. Experimental

Bovine skog

(1960).

by dialysis zinc

erythrocyte The protein

against

(see Lindskog,

the calorimetric

CA was prepared was freed

OP at neutral 1960).

procedure

by the method

of extraneous pH, which

The enzyme activity described 213

previously

metals

causes

of Lind(CU, Fe)

no loss

was estimated (Lindskog,

of by 1960)

Vol. 2, No. 3 BIOCHEMICAL

except

that

of Beers

a two-channel

que used for

stopped-flow

was employed.

(1956),

scribed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.exchange

(Malmstrom,

estimated

with

the Y-ray

activity

1953).

scintillation

tions

of known concentration,

stock

solution

prepared

The loss of activity 4O against

in

Fig.

acetate 1. At given

activity in

solution

Since

cent of

2.10 -3 fi,

as the control added

metal

cannot

could

In

to the

to reactivate

the

removal

the

addition,

taking ments, enzyme,

against

when standing buffer

without

in a final

inactivated

metal

containing

to the same activity (Co +2 , Ni +2,

ions

enzyme under

of OP, acting

concentration

CA samples

reactivated

as per

the

Cd+2)

same conditions,

as an inhibitor,

by the

be involved. loss

of activity

be due to factors

direct

slowly

and the CA

have been expressed

cases Other

is a strong

give

activity

When Zn++,

at

10 -3 3 OP is shown

were removed

dialysis

1).

a simple

While

tion

(Fig.

failed

so that

CA loses

enzyme was in all

same

of CA is dialyzed

aliquots

activity.

was added

of the

experiments.

containing

and the activities

of the control

OP, the

pH 5.0,

at pH 5, a control

OP was included,

of Ln 65 solu-

and Discussion

intervals,

measured.

by comparing

a series

when a solution

buffer,

of in 65 was

by dilution

the exchange

Results

has been de-

(Baird-Atomic) with

to that techni-

The concentration

samples

as used in

dialysis

measurements

counter

of l-ml.

similar

The equilibrium

the radioactive

earlier

apparatus,

March 1960

evidence

place

in

extraneous details

other

indication

exchange

in the than that

studies that

removal

a loss

of chelating

Zn 65 was added of concentrations

of Zn,

removal

is involved. in Table

I,

of the metal is

agents.

given

described

the reactiva-

of the metal

to exchange being

214

just

Zn 65 , summarized

with

a reversible

the presence

experiment

with

In these

experi-

the Zn in the

in the table.

An

Vol. 2, No. 3 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

70 time

Dialysis

Fig. 1. Loss and regain of CA activity OP. Three ml. of a 0.07 $ CA solution

March 1960

20 (dcrys) during dialyzed

dialysis against at 4' against

100 ml. of 0.05 i acetate buffer, pH 5.0, containing 10 -3 -M OP, with change of dialysis liquid every 24 hrs. The lower curve (dialyzed): activity as a function of dialysis time. The upper curve (reactivated): activity after addition of Zn ++ in a final concentration of 2*10e3 -'M

uptake

of radioactive

compartments

Zn was observed

of the dialysis

about 2 weeks while The concentrations the radioactivity corrected However,

for

dialysis

cells.

given

of the extraneous dilution

Zn analyses

thus demonstrating

This

equilibrium

at equilibrium

in the protein-containing uptake

is established

exchange,

in 48 brs.

Zn added and are thus not

such a dilution

and a correction 215

for

in Table I are based on

from the non-radioactive showed that

continued

Zn in the enzyme. had taken

place,

has been applied

Vol. 2, No. 3 BIOCHEMICAL

in calculating of enzyme; neous

the average a complete

albumin

proteins

of varying

complex;

(metalloenzyme), could

tidase

zinc

be detected

1953)

under

to albumin

with

(1958).

equilibrium

was established

namely

and enolase very

1952),

as fast

while

serum enolase

No bin-

carboxypep-

similar

to that

of Vallee,

in contrast

Table

bovine

the same conditions.

of binding

However,

to a number

and carboxypeptidase

the observations

and Neurath

and extra-

Zn++ binding

affinity,

Malmstrdm,

(CP) showed a degree

intrinsic

Gurd and Goodman,

was measured

CA, in accordance

between

binding;

March 1960

(0) of Zn++ bound per molecule

For comparison,

(non-specific

(metal-enzyme

number

equilibration

Zn was assumed.

of other

ding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Rupley,

to the case with

as the

of

dialysis

Coombs CA,

equilibrium.

I

Equilibrium dialysis of CA In the presence of chelating agents at pH 5.0 and 4'

Dialysis

medium

0.1 5 nitrate buffer, 1 M NaCl 0.05 E acetate buffer, 2.5010 -4 E OP

The experiments conditions,

Enzyme cont. (molar)

of the metal

versible

dissociation

3

10.10'5

5.5.10-5

2.5.10~~

0.68

6.g.10-5

9.4.10-5

7.2-10~~

0.45

described

demonstrate

Zn can be reversibly

removal

Extraneous Ln'+ cont. (molar) Protein Non-protein camp. cow

results

that,

dissociated in a loss

216

from

of activity.

of Zn in a metalloenzyme

under

certain

CA, and that Such a re-

has previously

Vol. 2, No. 3 BIOCHEMICAL

been accomplished ding

strength

appears

by Vallee in the

in the rate

of various

than

very

The studies

with

March 1960

CP. While

similar,

the bin-

the Zn++ in CP

in CA, as evidenced

of equilibration

chelating

published

(1958)

--et al.

two enzymesis

more “available”

rences

and in the

by the diffeinhibiting

effects

agents. are being

extended

and a complete

report

will

be

elsewhere.

This Swedish tutes

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

investigation

has been supported

Natural

Science

of Health,

U.S.

Research Public

by grants

Council

Health

from

the

and the National

Service

Insti-

(RG-6542).

References Beers,

R.F.,

Gurd,

F.R.N.,

(1952

1

Jr.,

Biochem.

and Goodman,

D.,

and Mann,

Keller,

H.,

and Peters,

S.,

Malmstrijm,

T.,

J. Am. Chem. Sot.,

Biochem. U.H.,

Biochim.

B.G.,

Scott,

D.A.,

Tupper,

R.,

Vallee,

D.S.,

492 (1956). 74,

670

J.,

34,

1163 (1940).

Hoppe-Seyler's

Z. physiol.

Chem.,

228 (1959).

Lindskog,

429

62,

l

Keilin,

317,

J.,

Arch.

Biophys.

Acta,

Biochem.

and Biophys.,

and Mendive, Watts,

(1951)

B.L.,

Chem. Sot.,

J.R.,

in press

J. Biol.

and Wormall,

R.W.E.,

5,

Chem., A.,

(1960). 345 (1953). I&,

Biochem.

445 (1941). J.,

50,

l

Rupley, -80,

J. A., 4750

Coombs,

(1958).

217

T.L.,

and Neurath,

H.,

J. Am.