Cobalamin catalyzed oxidation of sulfhydryl groups

Cobalamin catalyzed oxidation of sulfhydryl groups

Vol. 8, No. 6, 1962 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS COBALAMIN CATALYZED Department Bacteriology, of OXIDATION The OF SULFH...

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Vol. 8, No. 6, 1962

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COBALAMIN CATALYZED Department

Bacteriology,

of

OXIDATION

The

OF SULFHYDRYL

Hebrew

GROUPS

Medical

University-Hadassah

School,

Jerusalem,Israel J.Aronovitch

and

N.Gmssowicr

Received June 29,1962 Vitamin enzymatic

El12

B12

Peel

(1962)

were

has

tion

describes

of

(TTC).

Calibretion

with

dithionite,

gmups

were

Q951). DPN+

of

curves

Aquocobalemin

with method

ximetely oxidation

equel

rates.

the of

of

mercaptans

of

(Eggarar

the mle

acceleof vitamin

homocysteine

both

This by

and

communica-

vitamin

R12

deri-

nucleotide(DPNH),

measuring of

were

oxygen

the

prepared

by

chloride

reduction

of

of cobelamin.

method of Grunert

alcohol

dis-

uptake,

tetrarolium

triphenyl

fmm cyanocobalamin

Reeker

TTC

Sulfhydryl

and Phillips

by photolysis

dthydmgenase

systsm

at

pH

4.

(ADHI

(1950).

B catalyzed

In the absence 1).

the oxidstion

of

homocysteine,

and 2,3-dimerceptopmpanol

Zmercaptoethanol,

was very low (Table

that

in presence

mercaptan

aid

or factor

studying

aquocobalamin.

by

TTC

prepared

of

greatly

cobalamins

of

reduction

reduced

excess

a number

complex.

followed

or

of

was

glutathione,

that

diphosphopyridine

by the nitmprusside

Aquocobalamin cysteine,

reduced

was

groups,

or with

the

oxidation

in

and Abeles,lY60),

noticed

presence

of

a coenzyme

While we

the

msrceptans

determined

to

in

sulfhydryl

was identified

according

reported

of a mercaptan-cobalemin

Oxidation appearance

as

(Bmwnstein

honocysteine,

oxidation

the

formation

function

racently

the non-enzymatic

mediating the

of

oxidized

rapidly

to

of mercaptoethanol.

in the methyletion

vetives,

shown

reactions

the auto-oxidation

DPNH

end

been

oxidation-reduction

et a&1960). rate

has

of any cobelamin

The oxidation 416

rate

the

rate

at appmof

auto-

was found to be directly

Vol. 8, No. 6, 1962

BIOCHEMICAL

AND BIOPHYSICAL

Table

Oxidation Cobalamin added

Factor B Aquocobalamin Cyanocobelemin None

as

air)

and

ions

acceptors flavins

oxidation

12

17

, Co

3+

of

and

similar

RS’I of

is

B or

the

were

inhibited

the

uptake

form

B12 deriva-

(1.5~10-~#,

with

2x10%

was considerably , Fe3+)

pH 4;

oxidizable

less

inactive

at aquocobale-

(DBCC) was almost

2+

substrete at

vitamin

the

buffer

pH dependence

honucyeteine

wenzyme

, Fe

phosphate 37oC.

hornocysteine

A

anion

factor

cobamide

2+

of

active

rate

1~10%

completely

in the

presence adenine

flavin

uptake,

suggesting

at

450

dinucleotide

a rapid rate

not

of

Direct

as active

active(Table at

the

by of

DPNH

mercaptan,

cobelamins.

thus

lowered

1).

concentrations

of

air.

confirmed

however, DPNH

suggesting 417

changes

that

in

addition

free

the

reduc-

mononucleotide rate

of

as electron

this

significantly

and

the

mey act anaerobically

conditions

in

oxygen

Flavin

(2x10W4M)

measurements

anaerobic

oxidized

oxidation

(FAD)

flevins

that

mu under

of

1x10 -4 M aquocobalamin.

of

from cobalamins.

DPNH was caused

with

the

cyanocobalamin

(Co

(2xlCl~M)

TTC

and

oxygen

of

200

5~1Ll-~M.

of

(FMN)

)

(oxidant)

to 0.5.

most

oxidation

benzimidazole

while

that

was the

B

was obtained

iron

KCN tion

)

( uM 100

30 umoles; in air, et

catalyst

both

pH up

indicating

maximal

aquocobalamin,

up to

min.

34 4

no oxidation

increasing

TTC,

The

of

essentially

Factor

Dimethyl

Cobalt

( u1./5

57 19 3

homocysteine, 2 ml. Incubated

volula~,

with

with

tested.

min.

contained:

was

increased

6, in

48 8

concentration

mercaptan.

tives pH

the

There

observed the

a.4

flasks total

to

(reductant).

of

consumed

1

Warburg 50 umoles;

the rate

B12 derivatives

Cobalamin concentration 2 10 50

28

proportional

was

vitamin

by

Oxvoen

0.0

pH 8,

1

homocysteine

of

RESEARCH COMMUNICATIONS

the

ebsorbancy

assumption, of

a mercapten

decreesed

radicals

the of

the

Vol. 8, No. 6, 1962

substrate

BIOCHEMICAL

were

formed

as

titatively

oxidized

absorbancy

to

Fig.1.

Oxidation

of

DPNH, phosphate

mM); buffer

1.

to

the

oxidation with 2.2

and

the

2.8

:

DPNH

of

molar

ratio

indicated

a molar

ratio

catelase,

suggesting

B

factor

upon

8qUOCOb818min ethanol

while

6 a

(at new

no

spectrum 355, band

500

with

of

of

4.

that

peroxide

change

in

with B were changed

02

system

and

was

the

was

incubated

at

the

ebout

418

reduced

475

mM);

as

was

found

affected

intensity

in mu

addition

appeared

of

product.

or dicyano-

However,

when

or

absorption

be

oxidation by

homocysteine main

to

oxidetion

final

the

In asseys

group

mercaptan.

530 mu) decreased

a maximum

at

cyanocobalsmin

with

rapidly;

aquocobalamin.

(0.02 25OC.

added.

not

of

oxidizable

(fig.11,

respectively.

the

spectrum

system

and

sulfhydryl

not

in

chrom8tographically

TTC

ratio

ADH

aquocobalamin, in air

ADH

to

uptake

This

an

the

homocysteine, added

increase

homocysteine

(2 mM); incubated 2.

DPNH ~8s quan-

instantaneous

addition

mM);

thiol

of

and

the

presence

and

of

factor

activity.

were identified

of

incubetion or

their

factor

was

the

homocysteine, pH 8, (5

RESEARCH COMMUNICATIONS

cobalamin

by

upon

cysteine

Comparison

There

in

the

shown

level

1.

-

as

homocystine

products

TTC

DPN+

of

of homocysteine.

Addition

Cystine

a result

original

(0.1

AND BIOPHYSKAL

with

band and (fig.2).

mercaptomaxima

disappeared, Analogous

for

BIOCHEMICAL

Vol. 8, No. 6, 1962

spectral

changes

original

spectrum

or

were

Abaprption ( -), -

b

occurred

upon

-),

after

of by

followed

of

the

by

Helgeland

(1961)‘

Williams

(1961)

complex

No

aquocobalamin. addition

aeration,

here

is

stable

involve anion the

absorption

conjugated The products, relative

On

of

the

reversion

to

hydmgen

basic

aquocobalamin

inactivity

560

the

mercaptan-factor buffer pH

with

minutes

the

pemxide,

in

the

photolyzed

cobamide

and

of

formation

of

followed

by

coordination

shorter

coenzyme,

lower

wavelengths

the

spectrum

isolated by Pratt and

complex complex

indicate

the a

Barker, described may

to and

mM).

can

The

described

the

intensity may

mu.

(Brady

The

the

color)

pigments

coenzyme

light.

hand,

355

complex

cobamide

atom

(2

yellow

the

the

cobalt other

et that

mbalamin

to

(of

absorbancy

mu resembles

complex.

B 8.

mercaptoethanol

complex

- picoline

to

of

be

the

1

the

bands

to

-

air

chain

seem

30

decrease

contrast

of

RS-.

for

the 330

towards

B and phosphate

in

mercaptan-cobalamin

and

reduction

tide

the

the

in

factor mM)

incubation

between

However,

1961).

of (0.02

measuring

be

and

spectra B

factor

Formation

the

with

RESEARCH COMMUNICATIONS

cyanide.

Fig.2,

of

observed

AND BIOPHYSICAL

the

mercep-

displacement rupture

in

the

vitamin. or

the

active of

B cations,

factor forms

of

cyanocobalamin

the and

419

catalyst. the

or

their This

inhibition

reduction is

suggested

by

KCN.

by The

Vol. 8, No. 6, 1962 coordinated placed

BIOCHEMICAL uater

molecules

by anionic

may presumably

ligands function

not appreciably

in aquocobalamin (George g&

as such a ligend

In presence

complex.

AND BIOPHYSICAL

of cyanide

dissociate

and factor

B ars raadily

1960).

The mercaptide

forming

a mercaptan-cobalamin

a stable

in aqueous

RESEARCH COMMUNICATIONS

cyano-complex

solution

and is,

dis-

anion

is formed, therafore,

RS-

which

does

relatively

inactive. The aquocobatimin-catalyzed flavins

in presence

of mercaptans

published

observations

synthesis

of methionine

(Aronovitch light vation vitamin

of DPNH and the reduction

are of interest

on the participation

observations

of enzymes by glutethione in the activation

of Dubnoff and vitamin

or protection

in view of the recently

and formaldehyde

Peel's

1961).

of

of 812, DPNH and FAD in the bio-

from homocysteine

and Grossowicr,

on the earlier

oxidation

findings

(Hatch

and our own may throw

and Barton

(1956)

B12, indicating

of protein

et al,19591

sulfhydryl

on the actia role

of the

groups.

References Amnovitch J. and Gmssowicz N., Bull.Aes.Counc.Israel, & 68 (1961). Brady R.O. and Barker H.A., Biochem.Biophys.Res.Comm., & 373 (1961). Bmwnstein A.M. and Abeles R.H., J.Biol.Cham., 236. 1199 (1960). Dubnoff J.W. and Barton E., Arch.Biochem.Biophys., 61. 99 (1956). Eggcrer H., Stadtman E.R., Overath P., Lynen F., Biochem.2.. 333. 1 (1960). George P., Irvine D.H., Glauser S.C., Ann,N.Y.Acad,Scien., 88. 393 (19601, Grunert R.R. and Phillips P.H., Arch.Biochcm., 2 217 (1951). Hatch F.T., Takeyams S., Cathou R.E., Larrabee A.R., Buchansn J.M., J.Am.Cham.Soc., & 6525 (1959). Helgeland K., Jonsen J., Laland S., Biochem.J., 81, 260 (1961). J.Biol.Chem., 2J& PC 264 (1962). Peel J.L., Pratt J.M. and Williams R.J.P., Biochim.Biophys.Acta, &, 191 (1961). 184, 313 (1950). Racker E., J.Biol.Chem.,

420