The binding of n-butyl isocyanide to human hemoglobin

Vol. 48, No. 3, 1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

THE BINDING OF n-BUTYL ISOCYANIDE TO HUMAN HEMOGLOBIN* W. H. Hue&is**

and M. A. Raftery***

Division of Chemistry and Chemical Engineering., Institute of Technology, Pasadena, California Received

June

$ California 91109.

28,1972 Abstract

The binding of n-butyl isocyanide to hemoglobin has been investigated magnetic resonance spectroscopy. The I0 F-nmr spectrum of by lo F-nuclear hemoglobin trifluoroacetonylated at cysteine j393 exhibits chemical shift changes Comparison of these changes to the on binding of ligands to the 6 chains. fractional change in the visible spectrum, shows that in the presence of diphosphoglyceric acid initial ligands bind preferentially to Q! chains. In the absence of DPG, ligation of /3 chains increases linearly with overall fractional ligation, indicating that binding to cy and /3 chains is random under these conditions. The classical p chains as equivalent (5) evidence

models

for ligand

binding

sites (l-3).

has been presented

being bound in a preferential

binding

occurs is of interest process,

to hemoglobin

Recently,

which indicates

ligands

cooperative

binding

order.

since analysis

kinetic

the (Y and

(4) and structural

that the subunits

Knowledge

in understanding

treated

are nonequivalent,

of the sequence

the molecular

in which

basis of the

of cause and effect in the known structural

changes is then possible. Extensive

studies

Gibson and co-workers

on the subunit (4) for several

binding ligands,

isocyanide

(BIG). * The experimental

interpreted

to show that BIC binds preferentially

concentrations. *

** *** $

A series

of studies

results

order have been conducted most notably

for n-butyl

of these workers

has been

to p chains at low ligand

were conducted

Supported by USPHS Grant GM 16424 National Science Foundation Predoctoral Fellow National Institutes of Health Career Development Contribution Number 4488.

in this laboratory

Awardee

on the

1972

*Abbreviations used are: BIC, n-butylisocyanide; nmr, nucle&pagnetic resonance; DPG, diphosphoglyceric acid; Hb, hemoglobin; Hb , trifluoroacetonylated hemoglobin. Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

678

by

Vol. 48, No. 3, 1972

BIOCHEMICAL

carbon monoxide the visible

binding

spectrum)

the nmr spectrum

of a fluorine conflicted

so concurrent

were undertaken of different

comparing

to the ligation

of these experiments BIC binding,

process,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

label attached

studies

(observed

concurrently

to the B chains)

with the conclusions

(8).

in

from

The results

of Gibson on the order

of BIC association

whether differences

of

with Hb

exist in the order of binding

ligands. MATERIALS

Hemoglobin

-- Human

AND METHOD8

hemoglobin

titrated

blood.

The packed erythrocytes

sodium

chloride

solution,

stroma

were removed

gel filtration buffer

ligation

of /I chains (observed

nmr-visible

to determine

the overall

through

was prepared

were washed three times

and lysed with distilled

by centrifugation a BioGel

P-2

from freshly

with 0.9%

water and toluene.

and the supernatant

column

drawn

The

was desalted

(2.5 x 45 cm) equilibrated

by

with a

containing

0.05 -M bistris and 0.1 -M NaCl at pH 7.0. Hemoglobin solutions were stored at 4”’ and used within four days of isolation. Trifluoroacetonylated

hemoglobin

was prepared

Reagents

as described

-- Diphosphoglyceric

the pentacyclohexylammonium with Dowex 50-X8. research,

top.

-- Visible

stages of ligand This combination

Deoxygenated

and converted

a small

septum,

and aliquots

was a product

was a product

and nmr spectra

binding

were recorded

Fourier

transform

capability.

as

were obtained

Chem-

Chemical

Co.

concurrently

at

with a Gilford

into the argon-filled

with a microsyringe. Model

using a Varian The temperature

679

Glass Co.

The opening was covered

of BIC were injected

spectra

of Peninsular

of Aldrich

were introduced

access hole in the side.

IQ F-nmr

Calbiochem

using an nmr tube with a cuvette fused to the

solutions

were determined

from

to the acid by being stirred

cell was made to order by Wilmad

hemoglobin

through

absorbances

isocyanide

(6).

acid was obtained

Bromotrifluoroacetone

n-Butyl

Inc. Methods

various

salt,

previously

tube

with a rubber Visible

240 spectrophotometer.

XL-100

spectrometer

with fluorine

of the nmr probe was 27”; the

Vol. 48, No. 3,1972

air temperature

BIOCHEMICAL

in the Gilford

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sample

compartment

ments were made using a Radiometer of nmr peaks were determined BIC Binding in 5 ml of bistris/NaCl

Copenhagen

by gravimetric

Experiments

(pH 6.75,

fold excess of DPG was introduced solution

in a tonometer,

and transferred

a syringe.

had reached

a constant

contained

Magnitude!

175 mg of HbTFA

0.1 -M NaCl). A fivesolution of the same PH.

by repeated

to the argon-filled

washing with nitrogen

sample

was determined

was recorded.

tube and the tube was rotated

26 pH meter.

integration.

as a concentrated

of the solution

after each nmr spectrum

pH measure-

0.05 M bistris,

was deoxygenated

Absorbance

Model

-- Nmr solutions

buffer

The hemoglobin

was 26-2’7”.

Aliquots

manually

tube by means of

at 534 nm before

and

of BIC! were injected

to permit

mixing.

value after each addition,

into the

When the absorbance

the nmr spectrum

was

recorded. RESULTS The I9 F-nmr of a single

absorption

of the molecule

(HbTFA-CO), appearing

Liganded

acid,

within

exhibits

in addition

to a small

More

liganded

both the normal intermediate

(HbTFA-Oz),

on the ligand

state

carboxyhemoglobin

and HbTFA-BIC

The HbTFA-de02 hemoglobin

resonances

resonance

appears

Partially

liganded

peaks.

deoxy- and liganded resonance

consists

absorb about 480 cps upfield

(HbTFA-IHCN)

the same 20 cps range.

hemoglobin

the liganded

derivatives

with oxyhemoglobin

of the various

hemoglobin

shift changes depending

HbTFA

cyanmethemoglobin

50-70 cps upfield

labeling

of trifluoroacetonylated

whose chemical

(6).

of trifluoroacetic

on ligand

spectrum

AND DISCUSSION

hemoglobin

which appears

peaks,

10 cps upfield

of

peak in BIC spectra. specifically,

binding

reflect

the changes observed the structure

group is covalently

magnetic

environment

position

of the P-chain

attached

and ligation

in the nmr spectrum of the /3 chains.

to the /3 chains at cysteine

893.

of HbTFA The lg F The

of this group is influenced

primarily

carboxy terminal

(7), which changes on ligand

680

segment

by the equilibrium

BIOCHEMICAL

Vol. 48, No. 3, 1972

binding,

Experiments

with the ligand

at chains are locked in a liganded independently,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o2IIICN,

state hybrid

structure

showed that the chemical

and the p chains can be deoxygenated shift of the fluorine

of the ligand

state of the /3 chains (8).

Therefore,

magnitudes

of the nmr peaks as ligand

concentration

binding

all subunits

equally

be compared

in Figure

ligand

is increased

reflects

the overall (Y,),

and intermediate

reflect

ligand

fractional

determined

peaks.

the ligation

state of

ligation

(Y) could

from the combined

magni-

As is shown in the binding

curves

1 and in the plots of Y versus Y

(Figure 2), in DPG bound hemoglobin P until the visible spectrum change

in the nmr spectrum

that 15% of the subunits

were liganded.

curve) this lag in Yp disappeared.

In the absence of DPG (lower

Thus it appeared

BIC bound to (Y and p chains randomly, binding

to CYchains at low ligand

These findings

are similar

in preparation).

the disparity

In the presence

in affinities

preferential

concentrations.

as ligands

to a! chains at low ligand pressures.

that in the absence

while DPG produced

to the results

using oxygen and carbon monoxide script

changes in the relative

of hemoglobin

to the p chain ligation

no change appeared indicated

spectrum

and independently,

tude of the liganded

of DPG,

probe is a function

to the p chains. Since the visible

binding

2, in which the

of equivalent

(Hue&is

of DPG,

and Raftery,

all ligands

Depending

on the ligand

of the two chains was altered

experiments manu-

bound preferentially and pH studied,

or eliminated

in the

absence of DPG. These results in the native protein, on allosteric

from modified

hemoglobin

since introduction

parameters.

The Hill

with 2.7 for native hemoglobin;

are relevant

of the fluorine

coefficient

moiety has little effect of Hb TFA is 2.5, compared

the oxygen affinity

(6), and the Bohr effect and DPG effect are normal the fluorine

label caused the observed

since an increase

in /?-chain affinity

to ligand binding

depression

is very slightly (9).

681

Thus it is unlikely

of the /3 chain ligand

would be the likely

cation.

increased

effect of serious

that

affinity, modifi-

BIOCHEMICAL

Vol. 48, No. 3,1972

+ n - butyl

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

isocyanide

1.00 HbTFA+n-butyl pli 6.75

mcyanide

. wfth DPG

75

73 .50

.25

0.25

I

log [butyl

isocyonide]

Fig.

Figure

I --

X IO6

Fig.

1.

n-Butyl visible

isocyanide

binding

curves observed

and nmr spectra.

-o-,

Y; -o-,

Yp.

I

.50 Y

.25

.75

I

2.

concurrently

in

A. with DPG,

B. with-

out DPG.

Figure

II --

Comparison

of Y with Ys for n-butyl

l , with DPG; o, without

These experiments a chains of hemoglobin DPG is present.

affinity

binding

to HbTFA.

DPG.

show that in the early stages of ligand binding, exhibit

In agreement

to all chains randomly

isocyanide

higher

ligand

affinity

with the results

if DPG is absent.

chains which give rise to an isosbestic

than the /3 chains provided

of Gibson et al.

However,

the

it appears

(4), BIC binds that the high

point at low ligand

pressures

in

Vol. 48, No. 3, 1972

BIOCHEMICAL

stopped flow studies conclusion

of DPG-bound

is consistent

hemoglobin

their

low affinity

effect on ligand exhibits

which binds to deoxy

conformation

affinity

decreased

This

( 10) must be a-chains.

with the known effect of DPG,

p chains and stabilizes also have an indirect

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(11).

DPG binding

of the (Y chains,

complexed

hemoglobin

overall

affinity.

surprising

to find that the /3 chains are affected to a greater

must

since DPG However,

it is not

degree.

References 1. Adair,

G. S., J. Biol.

2. Monod,

J.,

3. Koshland,

Chem.,

J. Wyman, D. E.,

63, 529 (1925).

and J. P. Changeux,

G. Nemethy,

J. S., and Q.H.

and D. Filmer,

Biochemistry,

2, 365 (1966).

Perutz,

M.,

6.

Huestis,

W.H.,

7.

Huestis, (1972).

W. H. , and M. A. Raftery,

Proc.

Natl.

Acad.

Sci. U.S. ) 69, 0000

8.

Hue&is,

W.H.,

Proc.

Natl.

Acad.

Sci. U.S.,

9.

Lee,

10.

Olson,

11.

Arnone,

and M.A.

A.,

Nature,

Raftery,

Raftery,

Biochemistry,

Raftery,

unpublished

Gibson,

J. Biol.

J. S., and Q.H. E,

5241 (1971).

726 (1970).

and M.A.

and M.A.

E,

88 (1965).

5.

T.T.,

Chem.,

( l2,

Olson,

E,

J. Biol.

Biol.

4.

Nature,

Gibson,

J. Mol.

146 (1972).

683

11, 1648 (1972).

results

Chem.,

E,

(1972). 1713 (1972).

in press (19’