Reaction of cyanide with glutathione peroxidase

Vol.

96, No.

October

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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Pages

16, 1980

1116-1122

REACTION OF CYANIDE WITH GLUTATHIONE PEROXIDASEl Richard Department Received

J. Kraus

and Howard

E. Ganther

of Nutritional Sciences, College of Agricultural Sciences, University of Wisconsin, Madison, WI

August

and Life 53706

18,198O

SUMMARY: An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 ti KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from These observations [14C]iodoacetate in equimolar ratio to enzyme selenium. suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and spontaneous oxidation of the selenol to a labile S-cyanoglutathione; oxidized form of GSH peroxidase leads to irreversible inactivation. INTRODUCTION: a tetrameric Although

GSH peroxidase enzyme containing

originally

enzyme was not

sensitivity

is

oxidized

correlated

enzyme

10 mM cyanide forms

shown (1).

We have with

under

Form C) is mild

The cyanide

susceptibility

supported by the College University of Wisconsin-Madison, Grant No. AM 14184.

2 Abbreviations:

moiety

covalently

of the

readily

inactivated

(pH 7.5,

25"),

whereas

other

or are

of Agricultural and by Public

less

thus

sensitive provides

1116

in the

to Se in a

derivative

by

oxidized

to cyanide a useful

and Life Sciences, Health Service

HEPES, N-2-hydroxyethylpiperazine-Ni2-ethanesulfonate; GSCN, S-cyanoglutathione.

0006-291X/80/191116-07$01.00/0 Capyrighr d I980 by Academic Press, inc. All rights of reproduction in any form reserved.

cyanide

and is

of GSH peroxidase

Stesearch

that

stable

unstable

form the

of a glutathione may be bound

and no heme.

whereas (2)

The glutathionyl

fairly

of the enzyme are either

discovered

is

an oxidized

by cyanide,

recently

Se) which

per mole

to cyanide,

the presence

conditions

EC 1.11.1.9)

of selenium

to be inactivated

form such as E-Se-SG.

(designated

oxidoreductase,

to be insensitive

(1 mole/g-atom

selenenylsulfide enzyme

4 g-atoms

thought

of the enzyme was later reduced

(GSH:H202

tool

(2).

Vol.

96, No.

for

3. 1980

differentiating of this

of the enzyme and its

from GSH by gel which (2).

of this

selenium

moiety.

that

if

GSH peroxidase

is

at 4',

undergoes

spontaneous

inactivation

to investigate

whether

to indirect

an oxidized

characterization

reduced

with

form of the

GSH and separated

enzyme

when diluted cyanide

inactivation

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and a better

in further

filtration

leading

RESEARCH

selenoenzyme,

may be helpful

We decided

the enzyme,

forms

BIOPHYSICAL

reaction

We have observed

at 25'

AND

oxidized

understanding

obtained

BIOCHEMICAL

(Form A) is

and incubated

might

also

reduce

of the enzyme by a similar

process. METHODS: Pure GSH peroxidase was isolated from ovine erythrocytes as previously described (2) and stored at 4" as a stock solution in buffer (50 n&i potassium phosphate , pH 7.2, containing 10% ethanol). [35S]GSH (1.3 mCi/mmol) was obtained from Schwarz-Mann and purified by thin layer electrophoresis at 10' on cellulose plates at pH 1.3 (formic acid:acetic acid:water, 100:150:750) and at pH 5.6 (pyridine:acetic acid:water, 20:5:2000). [14C]iodoacetate (12.6 mCi/mmol) was obtained from New England Nuclear. S-Cyanoglutathione was synthesized by reactin GSH with 2-nitro-5-thiocyanobenzoic acid (3). Procedures for assay of 18 C and 35s by liquid scintillation spectrometry, total Se by a fluorometric procedure, protein by a Lowry procedure, and GSH peroxidase by a coupled assay were described previously (2). For preparation of oxidized Form C of GSH peroxidase, a desired volume of the stock enzyme solution was treated with either unlabeled or [35S]GSH (SH/Se = 4) for 20 min at 25". After the enzyme was reduced (iodoacetatesensitive), the mixture was exhaustively dialyzed at 4" against buffer (10% ethanol in 50 mM KP04, pH 7.2) to give oxidized Form C. Sensitivity to iodoacetate and to cyanide was monitored as previously described (2) to verify the oxidation state of the enzyme. The molar S/Se ratio in the dialyzed enzyme prepared with [35S]GSH ranged between 0.9 and 1.18. RESULTS :

To test

initial

product

product

with

peroxidase of the

whether of the

but

enzyme labeled of iodoacetate

enzyme that

underwent

inactivated

rapidly

whereas iodoacetate

little

cyanide

iodoacetate, (4),

presence

a reduced

with

we decided

is known

inhibit

the

rapidly

oxidized

enzyme.

to minimize

at 15", autooxidation when treated

KCN alone).

thermal

to Form A (2). with

was lost

KCN plus in

When the

1117

with

reaction

be the

the reaction

with

reduced

Oxidized cyanide

inactivation

treated mixture

GSH

Form C in the of any

GSH peroxidase

iodoacetate

controls

might

to trap

to react

35S from GSH was treated

or no activity (or with

of GSH peroxidase

treatment,

which

does not

form

(Fig. with

was l), KCl plus

was chromatographed

Vol.

96, No.

3, 1980

BIOCHEMICAL

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Y

0,0

20 MINUTES

4c

Inhibition of cyanide-treated GSH peroxidase by iodoacetate. [ S]GSH-labeled oxidized Form C (Se = 15.4 PM) in 1.5 ml of 20 mM HKPES (PH 7.5) containing 10% ethanol and 1 mM iodoacetate was treated at 15' with 10 mM KCN (pH 7.5) (0) or 10 mM KC1 (0) (control). Aliquots were taken 20, and 40 min, diluted 1:40 in 20 mM immediately after mixing (zero time), and 10 pl aliquots assayed for GSH peroxidase HEPES (pH 7.5) plus 10% ethanol, activity.

%F:

KCI

6

1

0

2 FRACTION

I

Fig. 2: Release of 35S from [35S]GSH peroxidase (oxidized Form C) treated cyanide. The reaction mixture from Fig. 1 was chromatographed on Sephadex (1.5 x 17 cm), equilibrated and eluted with 50 OIM potassium phosphate (pH plus 10% ethanol at 4'. Flow rate was 20 ml/h, and 1 ml fractions were collected.

1118

with G-25 7.2)

Vol.

96, No.

3, 1980

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KCN+IA

ION

NUMBER

Fig. 3: Alkylation of cyanide-treated GSH perolddase by 114C]-labeled iodoacetate. Stock (14C]-iodoacetate was prepared by mixing 200 pl of 10 mM [14C]iodoacetate with 800 ~1 of 10 mM unlabeled iodoacetate. Oxidized Form C (15.8 PM Se) prepared with unlabeled GSH in 1.5 ml of 20 mM HEPES (pH 7.5) containing 10% ethanol was treated at 15' with 10 mM KCN (pH 7.5) or KC1 in the presence of 1 mM [14C]iodoacetate for 40 min. The reaction mixture was then chrometographed on Sephadex G-25 as described in Fig. 2.

on Sephadex whereas

35

G-25,

enzyme treated

and Se in a 1:l

in Fig.

control

I4 C in.

treated

ratio

with

completely

KC1 plus (Fig.

experiment

3, the

incorporated

with

molar

The trapping shown

S was nearly

iodoacetate

from

the

was eluted

enzyme,

with

35S

2).

was repeated

enzyme treated slightly

released

over

using

with

KCN plus

equimolar

KC1 in place

[14C]iodoacetate. labeled

ratio

with

of KCN incorporated

As

iodoacetate Se, whereas

only

a trace

the of

radioactivity. The low molecular (see

Fig.2)

subjected

weight

was concentrated to thin

layer

from

in the

area

GSH and showed

under

with

of GSSG); that

from

[35S]GSH

peroxidase

vacuum by rotoevaporation

chromatography

35S peak co-chromatographed remainder

35S released

at 40"

or to electrophoresis.

The major

GSCN and GSH at Rf = 0.37(with electrophoresis

35S was mainly

associated

1119

the

at pK 8.6 separated with

, then

GSCN (Fig.

GSCN 4).

Vol.

96, No. 3, 1980

BIOCHEMICAL

AND

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RESEARCH

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SF-

-

GSSG GSH

-

GSCN

-

ORIGIN

SSCN GSH 7) ‘2 Qm

;sSGN3IGIN -

05

04

from Form C of GSH peroxidase by cyanide. Fig. 4: Properties of 35S released Fractions 22-25 of (B-treated GSH peroxidase (see Fig. 2) were lyophilized, then chromatographed (lower panel) on cellulose plates in n-butanol:acetic acid:water (12:3:5) or electrophoresed (upper panel) in barbital buffer, The plates were scanned with a windowless pH 8.6, for 50 min at lo", 20 V/cm. Arrows show position of known compounds. proportional counter. Fig. 5: Spectrum of oxidized Form C of GSH peroxidase before and after treatment with cyanide. The sample cell (path length 1 cm) contained 2 ml of oxidized GSH peroxidase (Form C) in 50 mM potassium phosphate (pH 7.2) containing 10% ethanol, to which was added 0.985 ml of 61 mM HEPES (pH 7.5) containing 10% The reference cell contained ethanol (14.5 pM Se final concentration) The temperature was maintained at 15"; gas everything except enzyme. phase = air. The spectrum was recorded in an Aminco DW 2 spectrophotometer (1.0 nm band pass, 0.5 A full scale), then 15 pl of 0.2 M KCN (pH 7.5) was added to bring the cyanide concentration to 1 mM. The absorbance change was monitored at 249 nm (inset), and a final spectrum (solid line) was recorded A similar sample of enzyme treated with 1 m&l after A24g had stabilized. KC1 (control) showed no spectral changes.

If

inactivation

described,

the initial

from the there

subsequent

should

selenolate enzyme).

step steps

chromaphore, Treatment in a rapid

within

2 minutes

of

enzyme

at pH 7.5 occurred

of reduction

might

of oxidation

be a spectral

result

the

by cyanide

change E-Se-

and inactivation.

corresponding

(assuming

is

(Fig.

treated

5), with

without

with

absorption

loss

Upon

the reduced

mechanism separated reduction

to the appearance

this

in ultraviolet

indirect

be experimentally

of Form C of GSH peroxidase increase

by the

of the

form of the

1 mM KCN at 15' that

was complete

of enzyme activity.

An aliquot

1 rnM iodoacetate

1120

was

87%

inhibited,

showing

did

Vol.

96, No.

3, 1980

BIOCHEMICAL

AND

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RESEARCH

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C-LJJ MINUTES

Fig. 6: Inactivation of cyanide-treated GSH peroxidase. Aliquots of the enzyme which had been treated with 1 mY KCN at 15" (see Fig. 5) and the control sample treated with 1 mM KC1 were diluted 1:40 with the buffer from the reference cell, incubated at 25', and assayed at zero time and subsequent intervals for GSH peroxidase activity. The KCN-treated enzyme was also tested for inhibition by 1 mM iodoacetate (87% inhibited at zero time); KCl-treated enzyme was not inhibited by iodoacetate.

reduction

had taken

Upon dilution

place.

cyanide-treated

enzyme was inactivated,

retained

all

nearly

DISCUSSION: cleaved

There

of its

with

E-Se-

Se as in

papain

reaction

However, inactivation

ways that

would

by

be repaired

E-SeCN + GS---WE-Seit

appears

that

of GSH peroxidase

a control

2).

without

be explained

KC1

the thiocyanate

removing

can be

if

inactivation cyanide

of SeCN- (5),

the preincubation

treatment

1121

with

1);

The irreversible

B-elimination

with

the

a Se-S bond in E-Se-SG

+ GSCN (reaction

cyanide

treated

+ GS- (reaction

during

as occurs

at 25",

6).

at pH 7.5 could

1, followed

of enzyme activity, (6):

(Fig.

+ GSCN (reaction

by cyanide

E-SeCN derivative to assay

activity

E-SeSG + CN-jE-SeCN

E-SeSG + CN-of GSH peroxidase

whereas

are two possible

by cyanide:

and incubation

with

reacted unless

GSH prior

derivative

3).

at neutral selenium,

pH can cause by an indirect

of

the

Vol.

96, No.

3, 1980

mechanism which

involving

then

incubation at 15',

it

rapidly,

8lOCHEMlCAL

to an unstable

at 25".

By following

was possible

form.

than

group.

grounds

by cyanide,

because

The observation

that

place A number

peroxidase mechanism

are for

a selenocyanate further acid initial

cause

reaction (E-SeOH), reaction

cyanide

did

with

an ionized

the cleavage

be (RSe-)

of unsymmetrical

released

further

the enzyme

selenol

has a lower

is

very

The

E-SeSG would

[35S]GSCN

occur

of enzyme activity.

of cyanide

the selenol

the

by 1 mM KCN

indicating

mechanisms

pK than

the

thiol

upon treatment

evidence

for

possible.

Although

the

inactivation

that

does not

derivative

cyanide

present

that

the

with

by oxidation

inactivation

work

involve

of the enzyme,

of cyanide

with

during

induced

with

(reaction

of the

cleavage

2.

of different

formed

inactivated changes

loss

COMMUNICATIONS

to iodoacetate,

with

enzyme is

by reaction

is

to liberate

by analogy

[35S]glutathione-labeled takes

not

RESEARCH

form of the enzyme

a reaction

The reaction

the thiol,

that

the spectral

did

on thermodynamic

disulfides

form

enzyme was sensitive

was in a reduced

BIOPHYSICAL

of a reduced

to show that

reaction

cyanide-treated

rather

formation

oxidizes

and this

expected

rapid

AND

provides

the

such a form

an oxidation

product

of the reduced

direct could

of GSH a reasonable formation arise

of

by the

such as a selenenic

selenium

generated

in the

cyanide. REFERENCES

1. 2. 3. 4. 5. 6.

Prohaska, J.R., Oh, S.H., Hoekstra, W.G., and Ganther, H.E. (1977) Biochem. Biophys. Res. Comm. 74, 64-71. Kraus, R.J., Prohaska, J.R., and Ganther, H.E. Biochim. Biophys. Acta (in press). Keen, J.H., Habig, W.H., and Jakoby, W.B. (1976) J. Biol. Chem. 20, 6183-6188. Flohe, L., and Gunzler, W.A. (1974) In: Glutathione (Flohe, L., Benohr, H., Sies, H., Waller, H., and Wendel, A., eds.) pp. 132-145, Academic Press, N.Y. Ganther, H.E., Prohaska, J.R., Oh, S.H., and Hoekstra, W.G. (1977) Fed. Proc. 36, 1094. Lin, W.S., Armstrong, D.A., and Gaucher, G.M. (1975) Can. J. Biochem. 53, 298-307.

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2)