Oscillatory oxidations of reduced pyridine nucleotide by peroxidase

Vol. 21, No. 6, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OSCILI&TORYOXIDATIOESOF REDUCED PYRIDINEEIJCLEOTIDE BY PEROXIDASE

I. Yamazaki, K. Yokota and R. Nakajima Biophysics Division,

Research Institute

Hokkaido University,

of Applied Electricity

Sapporo, Japan

Received November 15, 1965 It is widely reoognized that periodic eysteme.

The nature of the oscillator

the attention oscillations

of many physiologists

phenomenaare commonin biologioal

driving

such phenomenahas engaged

and biochemists.

of the reduced pyridine

nucleotide

Recently clear damped

level have been found in

Yeast cells (Ghosh and Chance, 1964; Chance, Eetabrook and Ghosh, 1964; Hommes,1964 a, 1964 b) and in aell-free

extracts

1964; Chance, Schoener and Elsaeeser, 1964). of oscillations glycolytio

depends upon the level

(Chance, Hess and Betz,

It was shown that the waveform

of metabolite intermediate

system (Chance, Schoener and Elsaesser, 1964).

describes the observation of periodic the presence of reduced pyridine

1965).

catalyzed

nucleotide under aerobic conditions. of reduced pyridine

by peroxidase has been reported (Yokofa and Yaaazaki,

Fig. 1 shows a slightly

cribes three charaoterisfio the periodic

This communication

reaotions catalyzed by peroxidase in

The detailed mechanismof the aerobic oxidation nucleotide

in the

reactions.

upon the concentration

modified mechanism. The previoua paper des-

phenomenaof the reaction, 1.

Irregular

which might cause

dependency of the oxidation

of reduced pyridine

nucleotide.

Bo appreoiable aerobic

oxidation

is observed at low levels of the reduced pyridine

oxidation

velooity

increases exponentially

582

velocity

nuoleotide.

with the aoncentrafion

The

of reduced

Vol. 21, No. 6, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PN- or Fe’,‘,

/-

Fig. 1. Possible mechanismof aerobic oxidation of the reduced pyridine nuoleotide. This mechanismalso suggests the possible paths of formation and decay of Compound111 during the reaction.

pyridine

nucleotide.

reaction.

2.

CompoundIII,

dcoumulation of peroxidase CompoundIII however, is not a typical

active

intermediate.

In the presence of excess peroxidase the rapid oxidation

of Radii only occurs

when most of the enzyme has been converted to CompoundIII. then, seemsto be an inactive intermediate, III

after

probably the perhydroxyl

CompoundIII,

of the enzyme trapping an active

radical.

3.

Disappearance of Compound

oxygen has been consumedin the presence of excess reduced pyridine

nucleotide. ferric

intermediate

during the

Under these conditions

and ferrous forms.

peroxidaee exists as a mixture of the

The ratio

between the ferric

the enzyme depends mainly on the pli and the concentration nucleotide

and ferroue forms of of reduced pyridine

(Yokota and Yamazaki, 1965).

Aa can be seen in Fig. 2, addition

of glucose-6-phosphate dehyrogenase

to a solution containing DADP, Mn2+, glucose-6-phosphate and peroxidase results in a transient of oxidation

reduction

and reduction’until

of DADP, folloved all

by a few cyolic

responses

of the oxygen is oonaumed. be the HAI?P

is reduced at a constant rate during the reaotion in Fig. 2, it can be eeen that a remarkably rapid oxidation

of BADIf suddenly ocoura at a certain

583

reac-

Vol. 21, No. 6, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 2. Kinetics of NADPreduction in the presence of glucose-6-phosphate dehydrogenase and peroxidase. 4 mMglucose-6-phosphate, 1 m?JNABS, 10 horse-radish peroxidase, 0.05 M acetate (pIi - 5.2), 25O. A ; 0.33 EM En!? , 20 ~lgglucose-6-phosphate dehydrogenase per 3 ml. B ; 1 mMEn2+, 2 118 glucose-6-phosphate dehydrogenase per 3 ml. In the presence of high dehydrogenase very sharp reduction of NABPcan be observed immediately after oxygen has disappeared. The crystalline peroridase was prepared from wild horse-radish and the ratio of E403 to E27E for this enzyme was 3.4.

tion time (one minute in the case, A) whiah corresponds to the termination CompoundIII

accumulation.

The reaction may produoe hydrogen peroxide,

is again wed to keep the reaction is reached wherein the efficiency sufficient

for the reaotion

back to the ferrio diffioult pyridine nucleotide

until

to proceed.

reproducible

in Fig. 1 is no longer

A part of CompoundIII

reactions when using a oombination of

reductases have optimum activity

nucleotide most effeatively

seemsto go

slows down. It is very

reduotase and horse-radish peroxidase.

horse-radish peroxidase catalyzes

at a slightly

the aerobic oxidation

Most pyridine alkaline

at an acidic pE.

when oxygen is supplied continuously Addition

by bubbling air diluted

of NABEto an aerobic solution

results in a rapid appearance of CompoundIII !Phe rate of EABEoxidation

pSi, while

of reduced pyridine

More stable periodio reaotione oan be observed in the oxidation

into the solution.

which

a low level of NADPE

of the.ohain reaction

state and the EADRI oxidation

to get highly nucleotide

going further

of

of EABE

with nitrogen of peroxidase

as is illustrated

in Fig. 3.

increases suddenly when the formation of Compound

584

Vol. 21, No. 6, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INAOH

Pig. 3. Oscillation of oxygen concentration in the presence of NADR and peroxidaee when nitrogen-oxygen mixed gas (24 I 1) is bubbled into the reaction solution at constant rate (10 ml per minute). Reaction starts with the addition of NADH (final concentration = 0.8 mM). Increase in abaorbancy at 418 mclis due to the formation of Compound III (Yokota and Yamaraki, 1965) and recorded by a Hitachi recording epectrophotometer. Oxygen concentration uaa followed simultaneously by using a sputtered platinum electrode which use kindly aupplied by professor M. Mochizuki in this Institute. This type of oscillation stops when RADIi is consumed and repeats again on the further addition of NADH. Total reaction volume in the wide cell light path = 1 cm) is 6 ml. 10 W¶ horse-radish peroxidaae, pH 5.12), 250. 0.1 M acetate

III

is

as was reported

completed

rate of ooncomitant and the

oxygen

decomposition

consumption

concentration

lation

of Compound III

itself

until

this

almost

is

all

of oxygen of the

of Compound

(Yokota and Yamasaki, 1965).

previously

overoomea

solution

III.

As oxygen

again

observed

of the NADH is

decreases, is

supplied

oxidized.

provided

in the presence which

has a high

solve

this

the

of oertain

by the

continuously

It

the aocumu-

reaction

repeats

may be euggeeted, of the reaction

of NAM! remained oonstant,

as it

from

would

does for

be

example,

HADreductase eyeteme. The use of a peroxidaee for aerobic

activity

oxidation

of RADH at neutral

pH*e might

problem.

The formation mechanism

level

of the oxygen supply followed

and the oxdation

experiment, that a more sustained periodicity

expected

that

The

responsible

and deoay of Compound for

the periodioity

585

III

seems to be an essential

of the reaction

vhich

is

observed

Vol. 21, No. 6, 1965

in Fig. 3.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The formation of CompoundIII

peroxidase with the perhydroxyl the RADHoxidation. by the RADradical,

radical

is mainly due to the reaction

of

which causes the chain reaction

of

It ia probably also formed in the reduction of peroxidase the product of whioh oonverts to CompoundIII

when combined

with oxygen (Yamazaki and Yokota, 1965).

The sudden decay of CompoundIII

the presence of low oxygen concentrations

is the most interesting

periodic likely

reaction.

The mechanismof the reaction

that the reaction

diates,

CompoundIII,

step in the

is not clear but it

is due to the reduction of CompoundIII

such as the RAD radical

in

is most

by intewe-

and ferrous peroxidase (See Fig. 1).

thus, is thought to be a regulator

of RADHoxidation

by peroxi-

daee. It might be worth while to emphasize here that reduced pyridine tide is the only substrate which oauses the periodicity the well known peroxidase-oxidase triose reduotone and indoleacetate.

tion.

of the reaction among

substrates , such as dihydroxyfumarate, It is obvious that a number of experi-

ments remain to be done with the reduced pyridine system, particularly

nucleo-

nucleotide-peroxidase-oxygen

the demonstration of sustained periodicity

This may be achieved when the reaction

of the reac-

is carried out in the more

complete open system which is now under investigation. ACKNOWLEDGEMENTS The authors wish to thank Professor L. H. Piette, University of Rawaii, for his helpful comments. The expert technical assistance of Mr. S. Miura and Mr. T. Arai for making a platinum electrode is greatly acknowledged. This investigation has been supported in part by Research Grant ~~-06518 from the United State Public Health Service. REFEREXCES

Chance, B., Estabrook, B. W., and Ghosh, A., Froc. Ratl. Aced. Sci., Jl, 1244 (1964). Chance, B., Hess, B., and Retz, A., Bioohem. Biophys. Res. Commun.,&6, 182 (1964). Chance, B., Sahoener, B., and Elsaesser, S., Proc. Ratl. Acad. Sci., z, 337 (1964). Ghosh, A. and Chance, B., Biochem. Biophye. Bee. Commun.,& 174 (1964). Hommes,F. A., Arch. Bioohem. Biophya., 108, 36 (1964 a). Hommes,F. A., Arch. Bioohem. Biophye., 108, 500 (1964 b). Yamasaki, I. and Yokota, K., Biochem. Biophys. Res. Commun.,a, 249 0965) Yokota, K. and Yamazaki, I., Biochim. Biophys. Acta, u, 301 (1963). l

586