The a3 : Total heme a ratio in cytochrome aa3 of heart muscle

BIOCHEMICAL

vol. 18, No. 4, 1965

AND BIOPHYSICAL

: TUTALHEMEgFUTIO

THE %

RESEARCH COMMUNICATIONS

lNCYTOCDRO~jq,g~ UP HEARTMUSCIE

Walter H. Vauueste* Department

of Biochemistry,

ReceivedJanuary

studied

of Oregon klical

School,

Partlaud

13, 1965

The binding a3 content

University

of CO has proven to be useful

of purified

cytochrom

a Yonetani preparation

in the determination

2 oxidase.

(Yonetani,

of the

Gibson aud Greenwood (1963)

1960, 1961) and reported

that

out of every 3 berms p was able to combine with CO. Upon repetition the experiments

with Enzyme Institute

concluded that the CO-binding varied

preparations, greatly

frcm one preparation

to assume a 1 : 2 ratio

Morrison and Horie (1964) studied aration

(Rorie aud Morrism,

binding

hem occurred per total

gate the CO-binding capacity

of 3.8.

coutrwersy

A different

Results

of these experiments

Methods aud Materials, beef heart mscle

Keiliu-Hartree

mince serially

pH 7.4.

fuged,

washed with 0.1 M phosphate buffer

nation

which was virtually

* P'ulbright

grantee,

The final

approach to the

hems a ratio

'ke thought

of re-

was strictly

it useful

with a sensitive are reported particles

1:2.

to investi-

prepared mitochondrial

The homogenate freed of uuclei

bee below).

prep-

parti-

radiochemical

in this

conmuuication.

were prepared fram

washed with 0.1 M aud 0.02 M phosphate

buffer

a preparation

of their

(1964) who as a result

of both freshly

cles and cytochrorne 2 oxidase peparations technique.

oxldase.

1963) and fcmd that in the average 1 CO

suggested that the a:, : total

III view of the existing

to the

heme a ratio.

for the native

the CO-binding capacity

problem was made by Van Gelder aud Muijaers dox titrations

of

Gibson (1965) recently

other with extremes of 1 : 2.6 and 1 : 5 for the a3 : total The author was inclined

1

and cell debris

was centri-

pH 7.4 and recentrifuged

f?ee of myoglobiu or hemoglobin

to yield crmtsmi-

suspension medium was 0.25 M sucrose.

ou leave from the University 563

of Ghent, Belgium.

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL

Cytochrome E oxidase activity conditions

was assayed polsrographically.

of the assay matched those described

(1963, a and b) for obtaining chrome p was Sigma type V. m&m -1 as extinction

Its concentration

coefficient

Purified

for the difference

particles material

coefficient

instead

the reduced and oxidized

(Williams, same author, content

1964).

particles

relied

overestimates

by a radiochemical

in a nitrogen

dissolved)

method.

for a hemoglobin

standard run simultaneously.

of all

particles

Our experiments

gave the results

CO in chemical combination

under form with b&-k

of alkaline

of the gas phases

by reference

to the data

on the CO binding

shown in Table 1.

of Keilin-

The identification

with the % content requires 564

the

in counting rate was converted

of CO in chemical ccmbination

1 rtree

by the aotion

The radioactivity

counted and the difference

end Disa

The material

The CO from (a) (i.e.

was released

atmosphere.

by the

particles.

in micromolsrity

Results

to determine

under both (a) reduced and (b) oxidized

and (b) (i.e.

were

1963) was

more on a method described

a same pressure of a CUO, CO, N2 mixture.

so obtained

An extinction

the heme a content

however, with scme minor modifications,

study was equilibrated

ferricyanide

(1964).

in OD at 605 mp between

of a tspe cytochrome in mitochondrial

dissolved)

was analyzed as pyridine

(Van Gelder snd Slater,

apparently

We therefore

CO was determined

were success-

The hemochromogen procedure when applLed

also used for the same purpose. to Keilin-Hartree

(1962).

preparation.

for the difference preparation

from Dr. E. &rrison

of mitochondrfa

for this

1962).

Richardson and Hatefi

to the method of Williams

of I.2 mM%m'l

with 21.1

in OD at 550 9

a gift

Heme a in cytocbrome s: oxidase preparations hemochromogen according

Cyto-

sample (Van Gelder and Slater,

or was prepared by the procedure of Fowler,

used as starting

rates.

was determined

cytochrome 2 oxidase was either

However, Keilln-Hsrtree

The

by Smith and Camerino

maximal turnover

consistent

between a reduced and sn oxidized

fully

RESEARCH COMMUNICATIONS

the proof

Vol. 18, No. 4, 1965

BIOCHEMICAL

that no other CO binding Table 1.

CO binding

AND BIOPHYSICAL

pigment besides cytochrome a3 is present.

to Keilin-Hartree

Reme 4 from

$&type cytochrcmpe

pyridine hemochromogen method

frammodified Willisms procedure

62 pM (l)**

particles. Carbonmonoxide in chemical combination

56.3 v”’ (7)

29.6 p

p59.1p)

(52.9

+

RESEARCH COMMUNICATIONS

: total 2 eme B *

(4)

(27.9 p31.4

p)***

The first and second ratio were obtained with the results of the hemochromogen procedure and of Williams1 method far simultaneous estimation of the cytochromes, respeotively. The values given are the means of the number of observations indicated between brackets. lktremes.

** w*

Myoglobin is the most likely

conteminent

(Colpa-Boonstra

end Minnaert,

below indicates

both myoglobin

nm428.5

-lx5

globin

(or hemoglobin)

of this

ratio

1959).

of heart muscle preparations

The figure

of merit described

and hemoglobin contamination. - 422 in the CO difference

/ A rn428.5

heart mPlacle preperation

(Fig.

1) is very sensitive

which wffl

increase

is about 5.0 in purified

i

to traoes of myo-

appreciably.

5% of that

of the CO bound to the heart muscle preparation the ratio

5.4 with succinate indicates

to 5.9.

in a concentration

We found with this

and 5.9 with dithionite

a contamination

too small,

The value

cytochrome E oxidase.

that the presence of myoglobin

increases

The ratio

spectrum of the

calculated

%

lA.90

1:2.@

equal to of Table 1

preparaticw

as reducing

1tcanbe

ratios

of

agent, which

however, to seriously

&fect

our

: heme a ratio. On the basis of the identity

tion

and the 9 content,

peek to trough differences region

(590 - 605 7)

preperaticn.

between the CO in chemical ccmbina-

we calculated

extinction

coefficients

in both Soret (428.5 - 445 f)

of the CO differenoe

snd visible

spectrum of the heart nuscle

We found 136 mM%mel and 10.lm&cm~l, 565

for the

respectively.

BIOCHEMICAL

Vol. 18, No. 4, 1965

A OD km-')

AND BIOPHYSICAL

0.

-0.100

RESEARCH COMMUNICATIONS

_-.

I

I 400

I 450

I 500

ml-L

Fig.1. Carbon monoxide diffsrence spectra of the 20 x diluted heart puscle preparation and 0.001 mM -lobin. The full and dashed line6 represent the differences in abeorption between the CO derivative and the reduced form of cytochrome and myoglobin, respectively. The Gary ecattared transmission attac ha nt and high intensity light source were used if necessary to allow observations on turbid samples. The Keilin- Hartree particles were suspended in 0.1 M phosphate buffer pH 7.4. Beducti~ was acoompliehed byaddingl-2 mgdithionite to both oelJ.6. Abaseline correction was done as described by Chance (1953 a).

These values are higher than those reported obtained

from the photochemical

action

by Chance (1953 b) which were

spectrum and an aseumed

analog

with lactoperoxidase. Turnover number of cytochrome+ ino (1963 b)aa

02uptake

inpper

has beendefined

by Smith and Camer-

second x4/~c,ytochramb

3.

Ueing

the concentration

of 3 obtained frumthe CO binding egerirmsnts, we found cytochr~ p concentration cwed to this TN to be 475 set -1 at infinite

330 - 390 isec-l reported

by Smith et al.

difference

extinction

is that their

630 difference -paration)

(1963 b)

se~pltl to overestimate

more, we used a slightly lower concentration

(23 mMW1umolfa

coefficient

in the reduced mdnus oxidized

for cytochrome 2. 566

the 605 -

speatnxn of the heart ~lscle

the a3 content

higher extinction

The reason for this

by about 10%

coefficient,

resulting

Furtherin a

Vol. 18, No. 4, 1965

’ TN

BIOCHEMICAL

Isec)

0.008

-

0.006

-

0.004

-

AND BIOPHYSICAL

I 0.04

0’

I 0.08

I 0.12

I

[cytochrome

RESEARCH COMMUNICATIONS

QJM-‘1

c]

Fig. 2. Plot of reciprocal of cytochrome g oxidase activity of KeilinHartree particles eqzeeased as turnmer number of + versus recipoaal of cytochrome p concentrlrtion. Oxygen uptake was measured with the GME oxyThe medium was O.O!.'M phosphate buffer pH 7.0, 50 mM ascorbate, 0.1 mM EDNA. Cytochroms E conaentration was varied between 7.1 and 35.8 JIM. Particles were treated with deozycholate before assay (Smith and &merino, 1963 b). graph

l

Table 2.

00 binding

capacity

of cytochr~

~oxidase Carbon

Preparation

frcm~idiE?fkm

ACEI reai8a

heIilOChrORlOg6j~

modified Fovler etal.

77.lpM

(2)**

Morrison

75.7 p 72.7 p

(2 (2

*, **,

d-,

monoxide

in chemical combination

76.7 pM (21

31.7yM

(8)

(27.4 p-36.9 3O.lpM (2) 30.7 p (2)

J+ : total heme& * k2.43,

k2.42

jnM)1:2.51 lr2.37

cf. Table 1.

The CObindinguas arations

preparations.

(Table 2).

also studied The relevant

ence spectrum were calculated a8 anexactmeasure

cnpurifiedcytochromepoxidase

extinction

again taking

qoefficients

prep-

ia the CO differ-

the CO in chemical dombination

of &3 (Table 3).

Table 3. Ektinction coefficients chrome ~oxidaseprepsraticns. Preparation

Modified Fouler et al. wl=riscm

for the CO difference

peak (428.5) (445) in(&~~jica

148 u9 567

to traqh

spectrum of cyto-

peak (5901 to trcush (60!5-608) in y$!i!~q* 10.1 9.7

Vol. 18, No. 4, 1965

The ratios

BIOCHEMICAL

now deviate

AND BIOPHYSICAL

consistently

ment with Gibson's result.

from 1 : 2 but there is general agree-

This may indicate

CO has been lost during the preparation. clusive,

hovever,

since two different

Acknowledaementsc couragement during this genercms gift

of purified

RESEARCH COMMUNICATIONS

that

some capacity

for binding

Our evidence is not completely methods of heme analysis

con-

were used.

I wish to thank Dr. H. S. Mason for his advice snd enstudy.

Thanks are also due to Dr. 14. Morrison

for his

cytocbrome .Q oxidase. This study was supported by grants from the American

Cancer Society

and the United States Public Health Service,

to whcanv8 are

indebted. Referencea Chance, B., J. Biol. Chem., 2Q2, 383 (1953 a). Chance, B., 5. Biol. Chem., 2Q& 407 (1953 b). Colpa-Boonstra, J. P. and Minnaert, K., Biochim. Biophys. Acta, 3, 527 (1959. Fovler, L. R., Richardson, S. K., and Hatefi, Y., Biochim. Hiophys. Acta, a, 170 (1962). Gibson, Q. and Greenwood, C., Biochem. J., & 541 (1963). Gibson, Q., in International Symposium on Oxidases and Related Redox Systems, edited by T. King. H. S. Mason, and M. Morrison, John Wiley snd Sons, N.Y. (1965) in press. Horie, S. and Morrison, M., J. Biol. Chem., & 1855 (1963). Morrison, PI. and Horie, S., Information Exchange Grou #l, 172 (1964). Smith, L. and Csmerino, P. W., Biochemistry, & 1.428 P1963 a). Smith, L. and &merino, P. W., Biochemistry, 2, I.432 (1963 b). Van Gelder, B. F. and Slater, E. C., Biochim. Biophys. Acta, 58. 593 (1962). Van Gelder, B. F. and kijsers, A. O., Biochim. Biophys. Acta, &+, 405 (1964). Williams, J. N., Arch. Biochem. Biophys., 537 (1964). Yonetani, T., J. Biol. Chem., 845 (1960 9 . Yonetani T. J. Biol. Chem., 923 ’ 1680 (1961). E.’ C ., Biochim. Biophys. Acta, p, 663 (1963). Van Geld&, 6. F., and Slat&r,

568