BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EVIDENCE FOR A QUATERNARY STRUCTURE CHANGE IN THE COOPERATIVE BINDING OF CYANIDE TO FERRIHEMOGLOBIN A Patrick

F.

Coleman*

Stauffer Laboratory for Physical Chemistry Stanford University, Stanford, California 94305 Received

December 21,1976

SUMMARY: At pH 6.5 in a 0.05 5 bis-Tris-0.1 M_Cl- buffer, tetra aquo ferrihemoglobin A (HbA+) binds CN- with a Hill coefficient of n = 1.4. The Hill coefficient increases slightly and the average CN' affinity decreases in the presence of excess spin labeled triphosphate (SLTP). This is probably the result of the finding that the SLTP exhibits a twofold higher affinity for HbA+ than for tetra cyano HbA+. Over the course of heme saturation with CN-, a certain fraction of the SLTP is specifically released. This shows linkage between organic phosphate binding and heme ligation. These findings bear a marked resemblance to the ligand binding phenomena in hemoglobin A (HbA) and provide good evidence that under these experimental conditions, HbA+ is undergoing a quaternary conformation change as the hemes become saturated. INTRODUCTION:

Ferrihemoglobin

in 1939 to bind

aside

coefficient

for

data

(n)

of Keilin ion

studies

were carried

state

with

the

that

out

address:

Abbreviations:

and the

He also

coefficient similar

(high

HbA + there

at pH 6.0.

1.76.

for

spin

to

is

a correlation

quaternary

1544

in binding)

et al. --

of

Stanford

HbA+, ferrihemoglobin A; HbA, hemoglobin spin labeled triphosphate.

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

change

between

structure

Department of Biochemistry, Stanford, California 94305.

These

upon ligand

Perutz

the

of hydro-

1.84.

the

low spin

More recently,

showed from

is

to correlate

(1)

The Hill

the binding

conditions

in an attempt

phenomenon. for

is

the Hill

to HbA" under

cooperative

cooperatively

saturation

of the heme groups

*Current

Copyright AN rights

(HS)

(N3-)

of the heme iron

have reported state

the

(2) that

sulfide

spin

ion

A (HbA+) was shown by Coryell

the

(3,4) spin

the protein.

University, A; SLTP,

ISSN

0006-291

X

Vol. 74, No. 4, 1977

It

is felt

ture

that

the high-spin

and the transition

some kind

of quaternary

There of

BIOCHEMICAL

aside

observe

however,

to HbA+.

that

ion binds

from high

spin

structure

change.

in careful

investigation

recent

n for

equilibration

time

associated

with

The results cooperatively saturation labeled findings

and with

a Hill

azide

azide

binding

binding

of the

following

CN-, there

triphosphate provide

is

at which

binding (7)

Banerjee process

of

1.5 at pH 6.0.

with with

they

the

their ligand

spectral

changes

are measured. study

demonstrate

a decrease

(SLTP) to HbA+.

good evidence

to involve

and Stryer

of the

varies

to aquo HbA+ at pH 6.5 and that with

struc-

likely

uncertainty

the wavelength

ligand

is

binding.

coefficient

further

a T-like

the cooperative

and Epstein

with

introduce

(9) that

about

(5,6)

to HbA+ with

with

to low spin

cooperativity

Anusiem and Beetlestone results

is associated

Anusiem -et al.

(8) report aside

iron

some controversy

no significant

--et al. find

is,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

It

over

that the

in the binding will

a ligand

CN- ion binds course

of a spin

be discussed induced

of heme

that

these

conformation

change in HbA+. MATERIALS AND METHODS: Hemoglobin A (HbA) was isolated from fresh adult human red blood cells. The cells were washed three times with isotonic saline and lysed by the addition of one packed cell volume of distilled water. The lysate was made 0.1 E in potassium phosphate, pH 7.0 and the solution was spun at 20,000 rpm for 30 minutes in a Beckman JA-20 rotor. The supernatant was decanted and spun again for 30 minutes. This procedure completely removes cell debris and ghosts. A threefold excess (over heme concentration) of K3Fe(CN)6 was added and the solution was allowed to stir for one hour at 4 “C to insure complete oxidation of the hemes. The ferrihemoglobin A (HbA+) solution was then passed through a G-25 (Fine) column equilibrated with 0.05 E bis Tris-0.1 M Cl-, pH 6.5, in order to remove the by-products of the oxidation reaction as well as to strip the HbA+ solution of all phosphates. The stripped aquo HbA+ was stored at 0 OC as a 7.0 x 10-4 M solution (tetramer concentration). HbA+ concentrations were determyned at 545 mu after complete conversion to the tetra cyano form. The extinction coefficient (~545) used was 43,50O/mole of tetramer. The SLTP solution (0.02 M) was prepared fresh from the solid. The synthesis of the SLTP has been described earlier (10,ll). The binding of CN- to aquo HbA+ was followed by measuring the fractional change in optical density at 545 mu and 630 npl using the

1545

Vol. 74, No. 4, 1977

Figure

1.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The increase in spectral amplitude from the SLTP as a function of added CN-. The first plateau occurs when heme saturation is complete. The second plateau indicates that all of the label is free in solution.

Cary 15 spectrophotometer. Isosbestic points were routinely observed in the spectra. The hemes were titrated by the addition of small volumes of a 0.1 M stock solution of NaCN. The total added volume was usually about-l% of the sample volume. It was found that a 20 minute equilibration time was sufficient to insure no further change in spectrum after each CN- addition. Cyanide uptake curves were measured both in the absence of phosphate and in the presence of equimolar SLTP and a tenfold molar excess of SLTP. The HbA+ concentration for the cyanide uptake curves was 1.25 x 10-5 M in tetramers. The low concentration of hemoglobin was necessary for accurate determination of the CN- levels due to the very high affinity of CN’ for HbA+. The SLTP binding studies were performed as described earlier (12). The change in the signal from the free SLTP was monitored as a function of added CN-. The spectra were recorded on a Varian E-4 spectrometer equipped for use with a quartz flatcell. The fractional saturation of the hemes with CN- was determined as before. The HbA+ concentrations for these experiments was 2.5 x 10-4 M. Due to the low affinity of the SLTP for HbA+, relatively high conceiitrations of both species were needed in order to observe significant binding. The nonspecifically bound SLTP was released by the addition of solid NaCN to the sample in excess of 0.1 M CN-. The increase in pH ($9.0) and the concomitant increase in ionic strength served to liberate all of the bound SLTP. RESULTS: cl-, is

From the

pH 6.5 at 17 OC, it twofold

sociation tetra

greater

for

constant

(KD)

cyano

HbA+ KU is

SLTP binding

studies

has been found aquo HbA+ than for 4.3

for

aquo HbA+ is + 0.2

x

that

the

affinity

HbA+(CN)4. 2.1

10B4 g.

1546

in 0.05 g bis

Tris-0.1 of the

M SLTP

The SLTP dis-

f 0.2 x 10v4 M and for

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.6

/ 0 l

F 0.4 1

.

1’ 1’

,,/’

1

of,,,‘---; I I t 0.2

04

0.6

08

1.0

7

Figure

2.

The fractional as a function

release (F) of the specifically of heme saturation (y).

20

Figure

3.

Saturation Tris-0.1 M 1.25 x 10-5 SLTP.

released

follow

upon ligation

the release

shown in Figure

of

1.

have become saturated

80

100

curves of HbA+ with CN' at pH 6.5 in 0.05 M bisCl', 17 OC. The concentration of HbA+ is M 0, stripped HbA+;W, 1.25 x 10-a M - in tetramers.

At pH 6.5 much of the not

40 60 CN-x106

bound SLTP

the

There

SLTP is

bound nonspecifically,

of the hemes with SLTP as a function is a plateau

in the

and the nonspecifically

1547

CN-. of

i.e., It

is possible

added CN-.

curve

it

This

is to is

where the hemes

bound label

is

released

Vol. 74, No. 4, 1977

BIOCHEMICAL

- 5.2

Figure

4.

with

fractional

pH and ionic

release

the'fractional

-42

-4.0

(y)

as more CN' is

added.

If

the

bound label

(F) is

plotted

against

a non-linear

curve

results.

See

2.

ation

([CN]50)

the presence

curves

for

of HbA+ with

in both

the

conditions.

slightly,

absence

are

titration

increases

DISCUSSION: Coryell

very

The results

(1) and Banerjee

of a strong

HbA+ in the range cooperative

a tenfold

but

significantly of

stripped

of

et al. --

of pH 6.5. binding

excess

(8) with

experimental CN- at pH 6.5

SLTP the Hill

respect

CN-, provide

1548

co-

4. the to

case cyanide data,

M. -

interactions

See Figure

The SLTP binding of

of

of SLTP

3.7 x 10'5

work corroborate

- in this

heme ligand

these

satur-

same in

excess

to

HbA+ with

to 1.5. this

molar

the

cooperative

SLTP under

of a tenfold slightly

of CN- at half

With

of of

absence and presence

3.1 x 10-5 M and is

indicative

and presence

In the presence

efficient

the

SLTP.

coefficients

CN- in the

The concentration

HbA+ is

of equimolar

For the

n = 1.4.

binding

3.

stripped

[CN]50 was shifted The Hill

with

-4.6 -44 log CN-

strength

heme saturation

of SLTP are seen in Figure

of

-4.6

of specifically

The saturation

the

-5.0

The Hill plots for the data from Figure 3. In the absence of SLTP n = 1.4; in the presence of 1.25 x 10-4 M SLTP, n = 1.5

increasing

Figure

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

findings

the

cooperative

ion

- to aquo

in conjunction

good evidence

that

Vol. 74, No. 4, 1977

there

is

a quaternary

binding. which

BIOCHEMICAL

structural

The existence is

shifted

phosphate)

and which

From this

and organic

related,

although

study

The findings

for

states

there

binding

which for

IHP,

spin

hexa-

of the heme groups

(3,4). observations

which

and SLTP binding

to HbA.

It

to HbA+ and that

are occuring

in

the existence

HbA+ are the

show

can be argued

processes

support

in HbA+

inositol

state

are several

CN- binding

cyanide

of at

least

following:

higher

affinity

for

aquo HbA+

cyano HbA+.

fraction

conditions)

to the

SLTP has a twofold

tetra

(2) A certain

(e.g.,

by no means identical,

(1) At pH 6.5 the

accompanies equilibrium

and coworkers

phosphate

two conformational

than

coupled

between

oxygen

cases.

phosphates

by Perutz

marked similarities

both

is

current

change that

of a conformational

by organic

has been suggested

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

of

the total

specifically

bound SLTP (30-40% under

released

over

the course

these

of heme

saturation. (3) The fractional

release

of heme saturation label

is not

a shift site. but

would

lead

in the

(5) The binding

The major

indicates

but more likely

gives

rise

to a lower

would not

rule

out

this

affinity

that by binding

possibility,

to an ambiguous interpretation.) of a large

molar

[CN]50 to a higher

of SLTP there concentration

of CN- to HbAt at pH 6.5 is

and the Hill

This

by CN- binding

which

response

bound SLTP as a function

non-linear.

directly

in conformation (A linear

the specifically

is distinctly

released

(4) In the presence shift

of

coefficient

emphasis

of this

increases work is

1549

is

a measurable

of liqand.

significantly

cooperative

slightly

in excess

to point

out the

SLTP.

similarities

Vol. 74, No. 4, 1977

in the

ligation

oxygen. that the

BIOCHEMICAL

of

None of

havior

with

In the

HbA but

case of

as an allosteric binding

cooperative

CN' binding.

structural

overall

heme groups

to give

the

that,

rise

is

to the

the hemoglobin

still

the

loss

occurs

cooperative

of

favor

is

certainly

integrity

The

between

certain

and allosteric

the

become

conditions properties

of

molecule.

Research

N00014-75-C-0869

Agency

which

to the process.

under

work was sponsored

from the

and to

when the heme groups

ACKNOWLEDGEMENTS: This under

Foundation

be-

affinity

conditions

enough structure

basic

in its

lower

are crucial

changes

qualitatively

in cooperativity

which

which

dramatic

the differences

experimental

despite

of HbA with

in many ways,

are due to its

modifications is

ligation

show the

are,

The decrease

and the protein

there

oxidized,

they

SLTP binding,

under

conclusion

and the

quantities

effector

nonspecific

due to

cyanide

the measurable

are observed same.

HbA+ with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

contract

Grant

no. BMS 75-02381

facilities

through

the Center

for

work also

by the Advanced

Materials

Office

of Naval

and by the National This

AOl.

made available

by the

Research

Science

benefited

Research

Projects

at Stanford

University

REFERENCES 1.

Coryell,

2.

Keilin,

D.

3.

Peruts, (1974).

M. F., Fersht, Biochemistry

4.

Perutz, M. F., Heidner, E. J., Ladner, J. E., Biochemistry Ho, C. and Slade, E. F. (1974).

5.

Anusiem, A. C., Beetlestone, J. Chem. Sot. (A), 106-113.

J.

G. and Irvine,

D. H.

(1966).

6.

Anusiem, A. C., Beetlestone, J. Chem. Sot. (A), 960-968.

J.

G. and Irvine,

D. H.

(1968).

7.

Epstein,

C. D.

(1933).

H. F.

J.

(1939). Proc.

Phys. Royal

Chem. 43, Sot.

A. R., Simon, 13, 2174-2186.

and Stryer,

L.

(1968).

1550

841-851.

(London)

Ser.

B 113,

S. R. and Roberts,

J.

Mol.

393-404.

C. K. G.

Bettlestone, J. 13, 2187-2200.

Biol.

32,

G.,

113-120.

Vol. 74, No. 4, 1977

BIOCHEMICAL

Henry,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8.

Banerjee, R., 32, 173-177.

9.

Anusiem, 403-414.

A. C. and Beetlestone,

10.

Coleman,

P. F.

11.

Coleman,

P. F.,

12.

Ogata, R. T. and McConnell, H. M. (1971). Symp. Quant. Biol. 36, 325-336.

(1974).

Y. and Cassoly,

Ph.D.

Biopolymers,

J.

R. G.

Thesis, submitted

1551

(1973).

(1976). Stanford for

Eur.

J.

Biopolymers

Biochem. 15,

University.

publication. Cold Spring

Harbor