The peroxidation of molecular iodine to iodate by chloroperoxidase

Vol. 32, No. 5, 1968

BIOCHEMICAL

THE PEROXIDATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF MOLECULAR IODINE TO IODATE BY CHLOROPEROXIDASEl, *

John A. Thomas3

and Lowell

Biochemistry Division; Chemistry and Chemical University of Illinois;

Received

an apparent

catalyze

lack

electron

donor

oxides,

molecular

can serve

of substrate

varying

degrees

extends

this

chloride

ions

as well

as iodide

of molecular

chlorine,

halides

and hydrogen

report,

we present

of iodide

ion

is the

catalyzed

peroxide,

ion.

leuco

series

Hence,

as reported

appearing

dyes,

this

level

in the

literature

agent,

while

cytochrome

c, and Chloroper-

reactions.

from

bromide

the

et al --*

per-

oxidants

enzyme can catalyze

by Hager,

halogen

hydrogen

to include

and iodine

chloroperoxidase

with

or the

inorganic

oxidizing

in peroxidase

bromine,

that

substituted

as the

various

reactions

the oxidant

and various

in the halide

the molecular

report

acid

donors

list

evidence

beyond

first

donor

either

peroxide,

of success

as electron

oxidase

formation

hydrogen

ene-diols,

of oxidation

for

hypochlorous

amines,

can serve

number

specificity

Thus,

oxygen,

aromatic ion

a bewildering

molecule.

with

phenols,

This

Department of Engineering; Urbana, Illinois

July 25, 1968

Peroxidases

iodide

P. Hager

and the

respective (1967).

can catalyze

In this

the

to an oxidation

oxidation

state

of such a reaction

of +5. being

by a peroxidase.

IThis is the fifth paper of a series dealing with chloroperoxidase. ceding paper in this series is listed as reference (1).

The pre-

zThis investigation Foundation.

Science

was supported

by grant

3This author held a predoctoral fellowship Health at the time of the investigation.

770

GB 5442 from the from

the National

National Institutes

of

8lOCHEMlCAL

Vol. 32, No. 5, 1968

Methods

and Materials.

Chloroperoxidase

medium of Caldariomyces

fumago

(1966).

used

Enzyme samples indicating

(A403:Azao)s prepared

concentrations

sium

iodide

O.lM.

were

was added

a buffer

Experimental mechanism noted initial

of horseradish peroxidase donor

peroxide

is

thus

has reported

that

reaction

iodine

in this after

the addition

reaction During from

after

required the

both

course

the brown

presence

color

absorbance

at

solvated

incubation

for

the presence

for could

disappears

absorption are

twenty (0.75

electron serve

Katzin

(1953)

disappearance,

minutes.

However,

to a clear

771

peroxide

solution.

at of triiodide

A shows the were

present

was observed thirty

absorption

(Figure

the reaction

located

1, curve

No change

of hydrogen

oxidation under

peroxide

the

in

from solution

pH 2.8.

disappeared

donors

I

as an electron

peaks,

Figure

PM),

of the

characteristic

and 10 mM hydrogen buffer,

was

to complex

to some higher

iodine.

had completely

of iodine

iodine

two peaks

on the it

(analogous

typical

three

studies

the decomposition

of chloroperoxidase.

have

phosphate

of iodine

is

iodine

of chloroperoxidase

of iodine

of

of tyrosine,

accelerated

that

The latter

from

O.lM potassium

the presence

that

when 1 mM iodine

spectrum

Potas-

in a Cary 15 spectropho-

of chloroperoxidase

solutions

The 462 peak arises

in

(1963).

from the

of spectral

and be oxidized

in the

462 mu, 353 mP, and 287 mP.

together

(Baker).

concentration

iodination

appeared

established

conditions

obtained

course

Such behavior It

easily

spectrum

the

rapidly

complex

peroxidase).

peroxidative

ion.

During

of iodine

in the peroxidase It

were

iodine

of Hosoya

measured

chloroperoxidase-catalyzed

reactions.

state.

grade

of 1.1

blank.

the presence

hydrogen

reagent

determined

were

and Hager

solutions

Iodine

to a final

was then

Spectra

and Results.

of the

that

ion.

by Morris

80%.

method

solutions

of iodine

350 rnp due to triiodide against

iodine

growth

index

with

by the

the

had a purity

of about water

determined

from

as described

experiments

distilled

to the

The concentration

tometer

in these

glass

was isolated

and purified

an enzyme purity

by saturating

Iodine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

seconds

peaks

1, curve

B).

due to This

and chloroperoxidase. mixture

When iodide

rapidly ion

changed was added

BIOCHEMICAL

Vol. 32, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Oxidation of Iodine by Chloroperoxidase. The oxidation of iodine by chloroperoxidase was followed spectrally. The initial reaction mixture contained 1 mM iodine, O.lM potassium phosphate buffer, pH 2.8, and 10 mM H202 in a total volume of 1 ml. The spectrum was taken in a Cary 15 spectrophotometer versus a buffer blank (curve A). Thirty seconds after the addition of chloroperoxidase (30 up) the spectrum was repeated (curve B).

to this

clear

of iodine

solution,

that

reformed

To quantitate was substituted Hager

--et al.

iodide

ion

this for

the

(1966)

that

a fixed

iodine

During

+ amount

quantitatively.

of iodide

ion the

iodine

depended

on the

reaction

more carefully,

iodine

were

incubation

solution.

iodine, +

Hz02

of iodide

ion

In separate incubated period,

with iodide

reformed original

It

chloroperoxidase

to form molecular 21-

Thus,

molecular

instantaneously. concentration a known

has been

catalyzes in the

12

2

+

amount

rapid

peroxidation

by of

equation. (1)

to generate

experiments,

different

was oxidized

ion

documented

OH-

chloroperoxidase

present.

of iodide

following

can be used

772

of iodine

previously

the

as shown

ion

The amount

a known

amount

initial

concentrations

and excess to the

hydrogen molecular

of

peroxide. species,

BIOCHEMICAL

Vol. 32, No. 5, 1968

which

then

minute for

incubation

the

tion

disappeared

through

under

formation

period

as it

the

iodide

ing

of triiodide

ion

shows

that

originally

immaterial initial

as long iodide

reaction

ion

mixture,

for

ion

3 molecules (Table

as it

the

decomposition

of iodine

was well

of the

amounts

excess

this

for

the

were

formed

period,

and the resultThe stoichi-

peroxide

ion was initially

peroxide

incubation

each molecule

(10 or more times

incuba-

hydrogen

of O.lM,

of hydrogen

iodide

of iodine

to be ample

Also,

After

formed

The amount

If

iodine.

ten

A

spectrophotometrically.

were

in excess

state.

1 was found

concentration

was measured

concentration). trivial

of molecular

to a final

1).

oxidation

shown in Table

of chloroperoxidase.

was added

present

to a higher

oxidation

activity

1 M potassium

ometry

conditions

and further

catalatic

formation

was oxidized

the

was sufficient

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of iodide used was

greater

than

absent

(see Table

from

1, lines

the the lc

and 2b).

Table

Reaction l.

1 umole

1.

Mixture

2. 10 umoles

KI

in the

Product

of

Equivalents of Iodine in Product (mpmoles)

Relative Equivalents of Iodine in Product per Original I-

30 273 269 6

3.0 2.7 2.7

3050 3120 230

3.1 3.1

H202 plus...

a. 1000 mnmoles b. Minus

Equivalents

Hz02 plus...

a. 10 mumoles KI b. 100 mumoles KI c. Minus

Number of Oxidizing Iodine Oxidation.

KI

KI

All reactions were carried out in 0.9 ml of solution, containing, in addition to the listed components, 100 umoles of potassium phosphate buffer, pH 2.8, After a 10 minute incubation period 100 umoles and 30 ug of chloroperoxidase. of potassium iodide (0.1 ml) was added. The amount of iodine present after the addition of potassium iodide was determined from the absorption at 350 mu (see Methods and Materials). For samples lb and 2a, dilutions of 1 to 10 and 1 to 100 respectively were made in O.lM potassium iodide, O.lM potassium phosphate buffer, pH 2.8, to adjust the absorbance values to a reasonable range.

773

BIOCHEMICAL

Vol. 32, No. 5, 1968

The stoichiometry way to iodate shows

ion,

each iodide

tion

reaction

ion

is

via

I-

3

originally

+

ion

+

the

3 H,O

incubation

period

iodine

+

6 H+

3 molecules

+

of iodine

mixture

and used in a calorimetric

iodate

ion.

The complete

nitrate sample developed white

volume

mixtures

were

without

the precipitates 0.5 N HCl. After that the

originated other

by Charlot ion

washed

the purple

+

3 Hz0

buffer,

the presence

pH 2.8, for

of iodide,

and 15 ug

10 minutes.

chloroperoxidase,

iodide

0.1 ml of O.lM (Ag I)

hydrogen

silver

developed

peroxide.

complete

iodide

was isolated

1 mM potassium

period,

In the

each (3)

it

for

precipitates

lacking

for

reduction

be formed.

product,

either

Yellow

was tested

To each solution

shaking,

oxida-

in the.

No precipitate

reaction

mixture,

a

developed.

by thiocyanate were

Thus,

test

incubation

iodide.

precipitate

as described ion

subsequent

in the

3).

was incubated

lacking

enzyme and the sample

Each of the precipitates

of iodate

of

eventually

reaction

phosphate

the

to each mixture.

in the sample

ion,

For

one molecule

results

containing

of 3 ml, run

After

peroxide.

crystalline

iodate

mixture,

O.lM potassium

in a total

lacking

of the

reaction

peroxide,

was added

identity

would

a reaction

or hydrogen

2

(2)

31,

from

ion,

the

period.

and the

(equation

the

incubation

all Equation

mixture,

+

confirm

Similar

oxidized

incubation

to iodine

To further

chloroperoxidase

the

incubation

103-

after

5 I-

present,

10 mM hydrogen

being

equations.

during

of iodide

to form molecular

103ion

place

in the

oxidation

ion

the following

takes

H202

of iodide ion

iodide

species.

+

iodate

with

present

the

of the molecular

of the

that

originally

formed

The addition

consistent

as can be seen from

the overall

iodate

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(1954). in acid

thoroughly

in the complete

This solution

with

was added color

calorimetrically

of iodine reaction

precipitates.

774

water,

for

test

is

the presence

based

on the reduction

to form molecular and then

of

taken

All

iodine. up in

1 ml of

1 ml of CC14 and 0.1 ml of 1% KCNS. developed mixture.

only

from

No color

the precipitate

developed

from

BIOCHEMICAL

Vol. 32, No. 5, 1968

Summary and Conclusions. of molecular that

the reaction

number is

iodine

of oxidizing

the first

Chloroperoxidase

to a higher product

oxidation

is

iodate

equivalents

in

demonstration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

can catalyze

state.

ion.

A calorimetric

Stoichiometric

the product

of the enzymatic

the peroxidation

agree oxidation

test

indicates

measurements with

this

on the

conclusion.

of a molecular

This

halogen

species. It cance not

is for

grow

rather the

that

mold from which

in the presence

the halogenating iodinating

doubtful

system

equivalents

this

reaction

chloroperoxidase

of iodide

ion.

of the thyroid

has any physiological is

However, gland

with

obtained,

since

such a reaction a storage

system

signifithis might for

mold will provide the

of iodine.

References Brown, F. S., and Hager, L. P., J. Am. Chem. Sot., 89, 719 (1967). Inorganic Analysis”, JohnTiley and Sons, Inc., Charlot, G., “Qualitative New York, 1954, p. 597. Hager, L. P., Morris, D. R., Brown, F. S., and Eberwein, H., J. Biol. Chem., 241, 1769 (1966). Hager3. P., Thomas, J. A., and Brown, F. S., Abstract, Am. Chem. Sot. Meeting, Chicago, 1967. Hosoya, T., J. Biochem. (Japan), 53, 381 (1963). Katzin, L. I., J. Chem. Phys., 2-1, 490 (1953). Morris, D. R., and Hager, L. P., J. Biol. Chem., 241, 1763 (1966).

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