BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

December 29,1978

TITRATION

CURVES OF INTERACTING

HEMOGLOBIN BY ISOELECTRIC PIER GIORGIO RIGHETTI',

ODepartment

GERARD GACON", ELISABETTA and JEAN-CLAUDE KAPLAN"

of Biochemistry,

Via

Celoria

"Institut

de Pathologie

et U.129,

24,

CYTOCHROME b5 AND

FOCUSING-ELECTROPHOREESIS

DANIELE LOSTANLEN"

GIANAZZA',

1575-1581

University

2, Milan0

20133,

Moldculaire,

rue du Faubourg

of Milano, Italy

Groupes

INSERM U.15

Saint-Jacques,

75014 PARIS

France Received

November

14, 1978

SUMMARY: A strong interaction between cytochrome demonstrated by titration curves in isoelectric The pH of maximum interaction is in the pH range predominant role of Lys of met hemoglobin in the acids of cytochrome b5. The stoichiometry of the (cytochrome b5: hemoglobin subunit) with similar and 8 chains.

b5 and hemoglobin has been focusing - electrophoresis. 8.0-8.3, which suggests a binding to acidic amino complex appears to be 1:l binding affinities for a

INTRODUCTION:

idea

By further

(l),

we have recently

rial

representation

resis

parallel

(such possible

gel.

curves

in the mutant

that

revealed

phenotype

as hemoglobins to correlate

(2).

which

curve

and its

pH 3-10

Moreover,

1575

were

run

bed of in

of the respective

had been

any given similar

electropho-

in a flat

mutants

acid

mobility

ABBREVIATIONS: met Hb = met hemoglobin; isoelectric focusing; UV = ultraviolet.

a picto-

the shape

amino

of very

the electrophoretic

to obtain

by performing

genetic

within

or cytochromes)

et al.

preformed

gradients,

charged

of Rosengren

is possible

pH-gradient

When a protein stationary

it

titration

to an Ampholine

in these

pa-mobility

an original

demonstrated of a protein

perpendicular

polyacrylamide

developing

family

size

of proteins

and shape,

at any given

Cyt b5 = cytochrome

substituted

b5;

it

was

pH with IEF =

0006-291X/78/0854-1575$01.00/0 Copyright 0 1978 by Academic Press, Inc. AN rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

the number

of protons

bound

frictional

resistance

of the proteins

within

a given

technique

family

the bound

their

complexes

species

the

We have

isolation

that

duction

(4).

red-blood keeps

cell,

range

phosphates,

as well

to see

ones,

in fact

able

and to iso-

such as inositol their

half-life,

as the stoichiometry

on the use of this

metHb and soluble did

of

the kinetics

(5),

oxidized

the half

de-

of metHb reis

reduced

that

in the

soluble

Cyt b5 is

b5 reductase

of the splitting

to determine

was recently

interaction

and Passon

of NADH-Cyt

it

species.

in the course

of this

In turns,

technique

protein

Cyt b5 since

interact

significance

any metHb formed.

possible

unliganded

of interacting

two proteins

form

in order

and to measure

data

as shown by Hultquist

by a soluble

was indeed

organic

we report

The biological

By following it

We were

the present

(3).

article

these

reducing

reduced

studies.

the

constant

recently,

of proteins

of the complex

as a model

More

from

and characterization

chosen

monstrated

(2).

since

is essentially

be resolved

with

complex

In the present

the gel

hexasulphate,

of stability

protein-ligand

in

states

properties

and inositol

the pH range

for

could

of hemoglobin

hexaphosphate

the

to liganded

physico-chemical

late

by the macromolecule,

of macromolecules

was extended

whether

or released

RESEARCH COMMUNICATIONS

Cyt b5

continuously

(5). of the Cyt b5-metHb

life

of the complex

complex,

and the pH

of maximum stability.

MATERIALS AND METHODS: MetHb was prepared from pure HbA by ferricyanide oxidation. Homogeneous soluble Cyt b5 (t-b5) was obtained from trypsin-treated rat liver microsomes according to Omura and Takesue (6). The Cyt bg was in fully oxidized state as checked by spectral analysis. The two freshly prepared proteins were desalted and mixed in a 1:l molar ratio (Cyt bg:Hb subunit) just prior to electrophoresis. The two-dimensional IEF-electrophoresis technique (Z), pH measurements (7) and gel staining (8) were performed as previously described. The spectrophotometric studies were carried out on a Beckman Acta III spectrophotometer. RESULTS AND DISCUSSION: and Cyt b5,

respectively,

temperature,

gel

in this

there

case

Figs. run

strength is

1A and C report

the titration

singly

under

and ionic

no perturbation

in a gel strength

conditions.

of the classical

1576

curves

identical It

of metHb

voltage,

time,

can be seen that

sigmoidal

shape

of

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-+

C

9

IEF

+ t

7

5

Figure 1. Titration curves of metHb (A), Cyt b5 (C) and of a mixture of 150 ug each of metHb and Cyt b5 (B). The small arrows in A, B and C indicate the sample application trough. The gel position where the sample crosses the application pocket represents the zero-mobility plane of each macromolecule, i.e. its isoelectric plane. The gel contains 6% acrylamide, 2% Ampholine pH 3.5-10, 0.2% Ampholine pH 6-8, 5 mM Asp, 5 mM Glu and 5 mM Lys. Running conditions: LKB Multiphor 2117 chamber run at 13 W (600 V at equilibrium), with an LKB constant wattage power supply, for 90 min at 4"C, in the first dimension (IEF). Second dimension: 15 min run at 600 V (constant voltage), 4'C. Staining with a colloidal dispersion of Coomassie Brilliant Blue G-250 in 12% CC13COOH, as in ref. 8. The splitting of the upper curve in B (as in C) is due to inherent heterogeneity of the Cyt b5 preparation. The horizontal line between pH 4 and 7 represents part of the mixture of metHb and Cyt b5 precipitated in the application pocket, possibly due to further aggregation and concomitant sedimentation of the complex in this pH range. The bidirectional arrows and + and - symbols represent the direction and polarity of isoelectric focusing (IEF) and electrophoresis (El.). each the

titration Cyt b5 curve

ves as an anion),

curve. is

On the contrary, strongly

while

deviated

when running above its

the same happens

1577

for

the mixture

p1 (i.e.

metHb,

but

(Fig.

lB),

where

Cyt b5 beha-

especially

below

BIOCHEMICAL

Vol. 85, No. 4, 1978

its

pl

(i.e.

where

metHb behaves

as the formation though

within

can enter

the

mum distance of

of a strong the

complex

gel

only

the complex

with amino

acids

between pH range

ved.This tion) the

of Lys residues In fact

be accounted

of a repulsive Cyt b5 molecule.

This

is

between

Cyt b5 and Cyt c. That

strated

by running

(PI

5),

well

No interaction exhibited

known

for

its

was apparent

in

between

dity, is

observed added

creased. this

the

unperturbed

upon mixing

to a solution Since

the

can probably

due to the formation

is

titration

two proteins be ascribed

(4). again

each

Moreover,

1578

invol-

deprotona-

has been demonacidic

protein

of ligands. each protein

has already formation

in Fig.

been of a com-

increase

absorbance

complex.

of

interaction

in turbi-

2, when metHb

at 700 nm is markedly

light

on

shown).

by the

As shown

to an increased

of an intermolecular

(not

the

confirmed

minimal

on the

aspecific

other

obser-

located

in the mixture

the absorbance exhibit

is

the predictions

(10)

curve

the two species.

of Cyt b5,

possibly with

a 6-8

is

a multitude

since

with

over

of interaction

a rather

of binding

spectroscopy

two proteins

is not

case,

constant

interaction

at pH 8 (via

serum albumin,

this

at pH 10 as well

of maximum stability

of Ng et al.

ability

consistent to acidic

of the ionic

residues

the binding

they

and Glu and Asp large-

agreement

metHb and Cyt b5 do interact

shown by W difference plex

data

as

the mini-

is

split

suppression

by His

metHb and bovine

a completely

That

by the

and the experimental

readily

type

in excellent

(9)

Thus,

behavior

be roughly

pH range

generated

Salemme

ea.

should

an additional

for

force

is

the strength

a narrow that

This

deprotonated

However,

that

so that

of metHb in the binding

complex

groups

appears

the pH of maximum stability

8.0-8.3).

be partly

and carboxyl

pH 8 indicates could

the

Lys would

and the fact

ved around

pH range

respectively.

E-amino

of the complex.

phenomenon

It

the other,

represents

role

where

ly protonated,

two curves case,

of Cyt b5.

as at pH 3.5,

disaggregation

this

the two species.

neutralizes

this

a predominant

RESEARCH COMMUNICATIONS

We interpret

between

each protein

the

(in

as a cation).

complex

upon

between

AND BIOPHYSICAL

scattering

in this

in-

region,

phenomenon,

On the one hand,

as jud-

BIOCHEMICAL

Vol. 85, No. 4, 1978

OD

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7Wnm

I

OA-

0.2 -

I..

10

5

15

.)

20

(HbLro’ M

Figure 2. Turbidimetric study of the metHb-Cyt bg interaction. A Beckmann a 100 uM soActa III spectrophotometer was used, zeroed at 700 nm against lution of Cyt bg. To 200 ul of this Cyt bg solution, as well as to the blank (dist. water), 2 ul of 1 mM metHb were added at each point of the turbidimetric curve, up to the 10th point, after which 4 ul increments were added. No corrections were made for the sample dilution, since the of Cyt bg is negligible. 7oonm absorbance

ged from

the maximum of the turbidimetric

stoichiometry

of 2:l

and above

this

ratio.

the stoichiometry is

(Cyt b5:Hb

On the other

by the fact

(Fig.

presence

1B).

Moreover,

of an excess

the titration

curve

on of a tight

complex

ble

to assume that

titration this

1:l

complex

is

have a

and seems to be redissolved

below

from

when the

to be 1:l curve

two samples

when the titration

be observed.

requires

a 1:l

This

obtained

each Cyt b5 molecule the physiological

again

stoichiometry.

complex

one is

1579

titration

binds highly

experiments,

(Cyt b5:Hb of free are

subunit).

Thus,

in a 1:l

were

no clear implies

This

Cyt b5 and free

loaded

experiments

of one of the two proteins, could

the complex

the

no titration

in the stable

experiments,

hand,

would

appears

that

metHb can be seen in the gel ratio

subunit)

of the complex

demonstrated

curve,

run

molar

in the

perturbation that it

of

the formatiseems reasona-

in the conditions

of the

to each Hb subunit. probable,

since

That in

the

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3

Figure 3. Kinetics of the titration curve of the mixture metHb-Cyt b5. The numbers 3 to 15 represent the minutes after the start of the electrophoreprior tic run at which pictures were taken. The same gel was photographed to staining up to the fifth frame. The sixth frame represents the same gel as the fifth, but after staining with Coomassie Blue G-250. All other conditions and symbols as in Fig. 1.

red that

cell

the concentration

of metHb (cu. Fig.

2~10~~

of the two proteins.

Fig.

1. Here,

this

M) under

3 shows the kinetic

ture

better

of Cyt b5 is very

appreciated

from

behavior

This

the pH range

physiological

is

(8x10 -7 M) as compared

in general

deviation

region.

1580

to

conditions.

of the titration

of maximum interaction

the strong

low

agreement

curves with

(pH 8.0-8.3) of both

titration

of the mix-

the data

of

can be even curves

in

BIOCHEMICAL

Vol. 85, No. 4, 1978

As the present of interacting ral

technique

appears

macromolecules,

to be very

we should

like

promising

for

to stress

the

the

study

following

gene-

aspects:

1) by running sible

pH-mobility

to determine

stability

curves

the nature

of the complex

2) equilibrium se complexes.

species,

run

if during

the

since is

half

even

life, will

within

in most cases favorable

of a few minutes,

in a mixture,

species

groups, all

in the most

of the order

to equilibrium transient

protein

of the interacting

focusing

In fact, complexes

of interacting

and its

isoelectric

fe of these

apart

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

will

it

is

the pH range a single fail

the

have ample

of

experiment;

to detect

cases,

pos-

the-

the half

li-

interacting time

to split

state.

ACKNOWLEDGEMENTS: Supported in part by INSERM (grants CL 37 76 69) and by Consiglio Nazionale delle Ricerche (CNR, 01471.04). P.G.R. thanks INSERM for a visiting professorship tut de Pathologie Moleculaire (Paris). We are grateful to Krishnamoorthy and H. Wajcmann for helpful criticism and

78 5 149 3 and ATP Roma, grant CT 77 to the InstiDrs. D. Labie,R. discussion.

REFERENCES 1.

Rosengren, A., Bjellqvist, B. and Gasparic, V. (1977) in Electrofocu-z sing and Isotachophoresis (Radola, B.J. and Graesslin, D., eds.) de Gruyter, Berlin, pp. 165-171. 2. Righetti, P.G., Krishnamoorthy, R., Gianazza, E. and Labie, D. (1978) J. Chromatogr., in press. 3. Krishnamoorthy, R., Bianchi Bosisio, A., Labie, D. and Righetti, P.G. (1978) FEBS Letters 94, 319-323. 4. Gacon, G., Leroux, A., Lostanlen, D., Labie, D. and Kaplan, J.C. (1978) 12th FEBS Meeting, Dresden, Abstr. No. 826. 5. Hultquist, D.E. and Passon, P.G. (1971) Nature New Biol. 229, 252-254. 6. Omura,T. and Takesue, S. (1970) J. Biochem. (Tokyo) 67, 249-257. P.G. and Drysdale, J.W. (1974) J. Chromatogr. 98, 271-321. 7. Righetti, P.G. and Chillemi, F. (1978) J. Chromatogr. 157, 243-251. 8. Righetti, 102, 563-568. 9. Salemme, F.R. (1976) J. Mol. Biol. 10. Ng, S., Smith, M.B., Smith, H.T. and Millet, F. (1977) Biochemistry 16, 4975-4978.

1581