Enthalpy changes for hemoglobin-ligand interactions: Revised calorimetric data

Vol. 59, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ENTHALPY CHANGES FOR HEMOGLOBIN-LIGAND INTERACTIONS:

REVISED CALORIMETRIC

DATA

H. T. Gaud, B. G. Barisas, and S. J. Department of Chemistry University of Colorado Boulder, Colorado 80302 Received

May,

1974;

accepted

June

12,

Gill

1974

SUMMARY Recently we reported values for the successive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin [Biochem. Biophys. Res. 555 (1974)l. Systematic errors in the data render comm., E, these results invalid. New data shows that cooperativity prevents resolution of successive heats of reaction and only average heats are currently accessible. At pH 7.4 and 6' we obtain: AH[&Hbt02+$Hb(02)4]=-13.2t0.4, AH[%,Hb(O2) +CO+%jHb(CO)4 +o~~=-‘i.o~o.~, AH[&HbtCO+$Hb(CO)4]=-17.7kO.4 kcal ? mole ligand. These data form a consistent thermodynamic cycle. INTRODUCTION In

the

course

of

of

stepwise

determination successive errors

ligands

in

describe

the

our

heats the

earlier

of heat

of

our

results.

values

required

of

state

on the

changes

for

reported

previous

the

(1).

report

corrected

compared

the

and O2 replacement

carbonylation

to establish

by our

functions,

current

independent

that

techniques of

binding

of

We wish

as well

also

calorimetric

systematic

errors

and to

We have

obtained

studies

we discovered

oxygenation

direct

enthalpy

our

equipment

hemoglobin of

enthalpy

previously

sources in

our

to hemoglobin,

measurements

improvements for

extending

reaction

to

as recent values summed by CO with reaction are,

as is

path.

METHODS Except

as noted

and calorimetric Experiments

Copyright All rights

data were

below,

wereobtained

performed

@ 1974 by Academic Press, of reproduction in any form

hemoglobin

Inc. reserved.

at

samples

as reported

6O in 1389

a sodium

were

prepared

previously maleate

buffer

(1,2). of

Vol.

59, No.

ionic

4, 1974

BIOCHEMICAL

strength

monitor

0.3.

Corp.;

volume,

reservoir

required of

thus

found

the

earliest

evaluating

that

the

the water

exerts

previously

due to

decrease

constant system

pressure volume

tion.

two

into

system

reaction

liquid

pressures,

it

by the

The second

of water

reduces gas volume

systematically dq/dz

from

high being

the it;

change values measured

more

liquid to

At

would for

these

in

the

6'= and normal of

0.12

baro-

kcal

per

correc-

When gas into

phase

to

the

calori-

increase

the The

its

equilibrium

value.

pressure

at

same time

of

indicate. the

in

which of

from

introduced

more moles

the

1390

the

gas are This

differential early

and

The reduction

serious.

the

hence,

inert

We now make this

is

system

both

of water

water

vapor

we

to maintain

an evolution

the

with

increases

gas uptake

is

evaporates

pressure

to

We can

effects

required

solution.

effect

saturated

water

the

amounts

gases,

The first

phase.

for

modifications,

proceeds.

the

allows

opening.

significant

volume

time

reaction.

account.

into

evaporation

binding

the

vapor

partial

than

of

condensation

incompletely meter,

these

results

the

stainless

volume

valve

of

as the

gas uptake

the

content

the

reaction

performance

the

of

valve,

the

forces

saturated metric

with

taken in

this

the

the

into

by a 2" x l/16"

of

vapor

gas to

a thermostatted

portions

on our

we had not the

into

Engin-

directly

from

calorimeter

associated

reactant

to

variable

(Validyne

injection

Upon opening

transients

reacting,

the

gas to diffuse

observe In

to

used

a sensitive

of

now by diffusion

a valve.

for

with

Admission

COMMUNICATIONS

formerly

transducer

by rapid

connected

through

decay

is

RESEARCH

manometer

replaced

DP45).

previously

reaction

BIOPHYSICAL

pressure

Model

calorimeter,

tube

has been

differential

eering

mole

The bubble

gas uptake

reluctance

AND

stages

consumed

results heat of

as

in of

the

ligand

is

Vol. 59, No. 4,1974

BIOCHEMICAL

AND BIOPHYSICAL

I I

0

I 2

moles

ii*

RESEARCH COMMUNICATIONS

I 3

02/mole

4

tib

Figure 1; The differential heat dq/dii of oxygen binding to diphosphoglycerate-free adult human hemoglobin as a function of the average number ?i of ligands bound per mole of protein. Temperature 6’; pH 7.4. The smooth curve is drawn merely to show that the data can be adequately fitted to a straight line and is not meant to imply that dq/dii remains linear as ii + 0 or ii + ,a.

reaction.

It

previous

results.

gases

is

measured

the

of

replacement

by adding

calorimeter

so that

was fully the

the

evolution

pressure

water

invalidates

our

presaturation

of

change

before ligand ligand

that

been

binding

of

that

oxyhemoglobin.

oxygenated capacity

replacement

indicating and

O2 by CO were

CO to a solution

due to

oxygenated

careful

which

of hemoglobin-bound

excess

had immediately

zero

effect

problem.

The protein

The heat

this

Extremely

remedies Heats

primarily

the

displaced

in

the

was known. proceeded

hemoglobin

with initially

O2 was released

into

gas phase. RESULTS AND DISCUSSION Figure

average reaction inlet vapor.

1 shows a representative

number

of

ligands

iiboundper

Hb + 40 2 + HbK)2)4 and careful Within

presaturation our

current

plot

of

dq/dE

vs the

mole

of

protein

for

obtained

using

of

reactant

experimental

1391

the

improved

gas with

uncertainty,.we

the gas

water now

all

Vol.

59, No.

4, 1974

observe

BIOCHEMICAL

no systematic

figure

1).

Atha

conclusion

variation

and Ackers

on the

basis

Unfortunately the

stepwise

enthalpy This

factors.

On the

curve 6" than at

at

low

recent of

such

hand,

the

the

the

region

These

a standard (5)

AH~ which

might

a data

set

results

of

that

the

heats

data

of

This

permit

data

ligand

may be fit

present

the the is

of

the

to accompany of

in

shown

value

O2 binding

1392

reliability reliable

errors been

of

collec-

dq/dz Statistical

errors

resolution

in of

I.

It

by hemoglobin

the such

The

obtain.

Table

in

E s .2 to

02.

standard

in

the

So how then

from

cooperativity

are

data

We have

the

proportional

probable

kcal/mole

other

problematic

necessary. the

small. of

at

least

uniformly

0.5

is

lowest

a constant

about

degrees

a calculation

cooperativity

to

calculation

be expected

is

binding?

spaced

of

On the

ciq/dz

data

determine

at

shown by a

measurements

reduces

is

vs G

as pronounced is

Moreover

accuracy

of

fact

resol.ution

of

our

dq/dz

The consequence

that

rate

further

two

much lower

i? + 0 and s + 4.

dq/dG

deviation

when various such

of

(4).

precise

reaction.

our

in

of

the

temperature

measurements

greatest

of

resolve

successive

is

is

Consequently

37 values

techniques

our

the

of

successive

; ,I, 3.8. with

the

precision

about

where

evolution.

E + 4.

shape

is

a similar

combination

and Tyuma

gas solution

where

the

fitted

heat

for

precisely

ting

of

the

room

1,

to

of

cooperativity

at

Imai

reached

binding

revised

AHi requires

of

and end of

correction the

the

precision

beginning

is

z (contrast

confidently

cooperativity

end regions

rate

the

COMMUNICATIONS

studies.

able

cooperativity

among

in

does

of

with

recently

from

that

as it

study

for

the

the

undiminished

dq/dg

in

That

RESEARCH

calorimetric

arises

guarantees

careful

differences

to

changes

temperatures

have

flow

one hand,

25'.

dq/dE

no longer

situation

no longer

BIOPHYSICAL

in (3)

of

we are

ligands.

AND

is

clear

Vol. 59, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Standard Errors in Stepwise Enthalpy Table I: Expec.ted from Resolution of a Typical Data Set Various Deqrees of Cooperativity in Equilibrium Cooperativity Typical for Hemoqlobinb

Chanqes Assuminq Constants

Intrinsic Equilibrium Constants Equalc

Totally anticooperative

'AH~

(kcal/mole liqand)a

2.69

.43

.16

'AH2

(kcal/mole liqand)

7.81

.84

.16

'AH3

(kcal/mole liqand)

8.51

. 84

.16

'AH4

(kcal/mole liqand)

3.40

. 43

.16

d

aThe data set consists of 37 values of E spaced uniformly from i-i= .2 to ii = 3.8. The standard deviations of dq/dE about the fitted curves are taken as 0.5 kcal/mole liqand. bK 1=. 316 mm-l, K2 = .443 mm-l, K3 = .50 mm-l, K4 = 1.09 mm-l (8). cKl:K2:K3:K4 ::4:3/2:2/3:1/4. dKl>>K2>>K3>>K4.

renders Thus

the only

values

averaqe

are

three

successive

the

g = 3.8

miqht

dq/d& In

heats sistent reaction

of

number

(6).

are

the

next

paraqraph.

points

the

data

are

be collected

points

There

are,

beinq

pursued.

the

between

Finally,

data.

and these

in

collected

increased.

present

accessible

AHi and all

miqht

our

the

however,

end reqions. z = 0.2 standard

and error

be reduced.

earlier

studies

oxyqenation with

bindinq

to

of

from

of

be qreatly

miqht our

in

routes

a few more

Second,

in

heats

presented

possible

First,

AHi indeterminate

known

we observed

and carbonylation values

Because

of

for

the

differences

1393

heat

that

our

values

of

hemoqlobin

of

the

in

for

were

replacement

handlinq

techniques

the incon-

Vol.

59, No.

BIOCHEMICAL

4, 1974

Table II: glycerate-free

Heats

%Hb(aq)

of Adult

AND

BIOPHYSICAL

RESEARCH

Reaction of Gaseous Ligands Human Hemoglobin at 6O

COMMUNICATIONS

with

Diphospho-

AH

+02(g)+%Hb(O2)4(aq)

=-13.2t0.4a 02

~Hb(02)4(aq)+CO(g)+~Hb(CO)4(aq)+02(g)

AH0 ~Co

= -4.oko.1

%Hb(aq)

AHco(calc)

=-17.2tO.4

AHco Cobs J

=-17.7kO.4

+CO(g)+%Hb(CO)4(aq)

aHeats

the

of

reaction

oxygen

and carbon

presaturated the

in

with

errors

both

value

of

yields II.

kcal/mole

in

the

The heat

(6)

using

van't

Hoff

tent

with

most

recent

ligand.

were

these

neither

methods. 25O value

for

The heat of

-11.7

equally

rise

presaturation

accords sheep

nor

giving

calorimetric

replacement

obtained

thus

Complete

self-consistent of

totally

studies,

previously.

-5 kcal/mole

Tyuma (4) if -1 mole value

in

monoxide

water

discussed

gases Table

given

well

hemoglobin of

of

data

shown

with

the

by Roughton

oxygenation

2 0.6

to

reported

is

consis-

by

AC is assumed to have the same -100 cal deg -1 P observed for the carbonylation reaction (7). REFERENCES

1. 2. 3. 4. 5. 6. 7. 8.

Nell, L., Barisas, B. G. and Gill, S. J., Biochem. Biophys. Res. comm., z, 555 (1974). Rudolph, S. A., Boyle, S. O., Dresden, C. F. and Gill, S.J., Biochemistry, e, 1098 (1972). Atha, D. H. and Ackers, G. K., in preparation. Imai, K. and Tyuma, I., Biochem. Biophys. Res. Comm., z, 52 (1973). and Error Analysis for Berrington, P. R., Data Reduction the Physical Sciences, New York, Mzaw-Hill, p. 169. Gghton, F. J. W., J. Physiol., z, 359 (1954). Gaud, H. T., Gill, S. J., Nell, L. and Rudolph, S. A., in preparation. Tyuma, I., Shimizu, K. and Imai, K., Biochem. Biophys. Res. Comm., g, 423 (1971).

1394