Identification of intermediate substrate free-radicals formed during peroxidatic oxidations, by electron paramagentic resonance spectroscopy

Vol. 1, No. 6

BIOCHEMICAL AND BIOYHYSICAL RESEARCH COMMUNICATIONS

IDENTIFICATION

OF

INTERMEDIATE

FORMED BY

DURING

ELECTRON

PARAMAGENTIC

I. Department

SUBSTRATE

PEROXIDATIC

of Biochemistry,

and

Received

We tions

of

field

flow

system

with

modulation

water

oxidase

(R.

kinetic

Z.

cifically

purified

a sharp

Peroxidatic

oxidation

asymmetric

line,

the

semiquinone

during

the

The be

measured

*

For

which,

rates by

we

was buffer

x

the

are

Alto,

Cal.

peroxidatic

oxida-

hydroquinone.

The

spectrometer, 0.5”

x

positioned

ob-

using

0.01”

attached

to avoid

dielectric

recrystallized and

of ascorbate

Japanese

dihydroxyfumaric

with

at pH a splitting

the

gave

peroxidatic

a five-line

hyperfine

pattern (Blois,

enzyme-catalyzed

free-radical

present

but

grateful

technique,

to

Dr.

4.8

100 to

a losses

turnip acid

produced

of

1.7

per-

were

spe-

F. 336

the

ban.

a free-radical

gauss,

g = 2.0043.

a free-radical

oxidation

of hydroquinone

of

and

0.9” and

doublet

while

autoxidation

Palo

x-band

of dihydroxyfumarate

with

Portland

autoxidation*.

oxidation by

cell

enzyme

Acetate

avoid

Peroxidatic characterized

V-4500

a sample

Our

2.7). to

School,

during

acid,

measurements C.

=

Medical

of free-radicals

a Varian

and

at 25O

Oregon

Associates,

dihydroxyfumaric

made

for

Mason

Piette

Varian

formation

acid,

were

of

SPECTROSCOPY

1959

observed

ascorbic

k. c.

to

4,

have

servations

due

Division,

December

H. S.

University

L. Instrument

FREE-RADICALS

OXIDATIONS,

RESONANCE

Yamazaki

Dec. 1959

with

of hydroquinone identical

with

a single. produced

that

obtained

1955).

steady

formation

were

state

free-radical

too

high

to

Vol. 1, No. 6

BIOCHEMICAL

concentration

from

function

of

AND

peroxidatic

substrate,

stant

flow

rate

were

consistent

BIOPHYSICAL

oxidation

enzyme,

(Table

and

1).

with

the

RESEARCH

These

COMMUNICATIONS

of ascorbic

hydrogen

acid

peroxide

steady

state

[AH~]

[Complex

Dec. 1959

was

measured

as

concentrations

free-radical

a

at con-

concentrations

expression: k3

112

II]

kd where

[AH*1

the

is

s

the

concentration

AH2

to

by

This

tions

and

Complex

by

concentration

acid, II,

relationship

proposed

1953),

state

of ascorbic

AH.

decay.

steady

k3

and

kd

appears

Saunders

Yamazaki

is is

to fit

(Saunders

(Yamazaki,

acid

in

rate

the

mechanism

Peroxidase,

M

for

constant

Mann,

for

oxidation

of

free-radical

of peroxidatic

1940),

George

oxida(George,

1

produced

acetate

constant

1957).

concentrations

ascorbic

rate

the

Table Free-radical

the

and

r.~21is

of free-radical,

buffer,

pH

H202,

M

during 4.8,

the

0.1

M,

Ascorbic

peroxidatic at 25O

Acid,

oxidation

of

G.

M

Free

-Radical,

M

1

x

IO8

2 x

10 -2

2

x

1o-2

1.7

x

10 -6

4

x

1o-8

2 x

10 -2

2

x

1o-2

3.6

x

10 -6

2 x

10 -2

2

x

1o-2

7.2

x

10 -6

1.6

x

lo -7

4

x

10 -8

2 x

1o-2

5 x

10 -3

1.1

4

x

10 -8

5 x

10 -3

2

1o-2

3.0

Free-radicals time,

in

these

experiments

out

more

our

have

thus

enzyme-catalyzed

studies

oxidations has

detailed

been

other

and

and

identified,

substrates.

for

of the

peroxidases

detected of

submitted

examination

to

been

x

A complete

publication.

systems

for

here

We

Blois,

M.

S. , P.,

J.

Chem.

Phys.,

Biochem.

Saunders,

B. C.

Yamazaki,

I.,

and Proc.

J., Mann, Internat.

2,

and

oxidases.

23,

1351

267

(1953).

P. J. G., Symp.

J.

(1955).

Chem.

Enzyme 337

Sot., Chem.,

769 224

the

first

account

REFERENCES

George,

10 -6

x

propose

described,

10 -6

x

(1940). (1958).

to to

of carry

extend