Organic phosphates and ligand binding in hemoglobin

Vol. 40, No. 6,197O

Quentin H. Gibson Department

of Biochemistry

Biology,

Wing Rail, Ithaca,

Supported by United States

and Molecular

Cornell

New York

University,

14850

Public Health Service GM 14276-06

Received August 3, 1970 Summary: Although several organic phosphates appear to react rapidly with hemoglobin, and to be nearly at equilibrium with the various intermediates during oxygen binding, pyridoxsl phosphate reacts quite slowly, allowing study of the properties of intermediates bound both to organic phosphate and to oxygen. The affinity for owgen and the n value are much decreased as compared with stripped hemoglobin. It has bzen calculated that about a third to a half of the heme-heme interaction energy is associated with binding of organic phosphates or other anions, in a linked reaction occurring only with tetrsmeric hemoglobin, which must therefore be considered as the smallest unit permitting a meaningful description of hemoglobin-ligand interactions. The discovery

and documentation

of organic phosphates plicated

attempts

by mass action

to describe

equations,

ences in opinion

is little

the kinetics

hemoglobin

kinetic

constants,

work such as that

simple interpretation phosphates considerably

and equilibria

of phosphates will

that

equilibrium the situation

that

equilibrium

be rapid as compared with the ligand slower.

In an attempt

to avoid this

1319

com-

reactions by differare

work is concerned,

only the interpretation

of Gibson (5), either

of ligand

So far as equilibrium

altering

requires

has greatly

has not been clarified

since binding

provided

(1,2)

and particularly

to which organic polyphosphates

(2,3,4).

on the form of the equations,

of salts,

by hemoglobin

the extent

difficulty,

upon the binding

binding

and the situation

concerning

bound by liganded there

on ligand

of the effect

have no effect to be placed

is indeed attained.

In

is more complex and between hemoglobin

reactions

studied,

and

or very

issue Gibson (5) used. a high

Vol. 40, No. 6,197O

concentration brim

BIOCHEMKXL

(O.lM) of inorganic

AND BIOWYSICAL

phosphate in the eqectation

with the hemoglobin nvuld be rapid.

a complete answer, since although may refer

to all

proportions

RESEARCH COMMUNICATIONS

equili-

This &es not, however, provide

the kinetic

species having a definite

of free and phosphate-bound

that

constants

in a k-step model

number of ligand molecules,

the

species are undetermined.

An approach to the problem has been made by examining the rate of reaction

of several

in functional

organic polyphosphates

behavior

as in the single

with ATP (Dr. E. Antonini,

that JYJC%W&. phosphate reacts of minutes rather

using the change

of the hemoglobin as an index of rektion.

most appear to react rapidly, acting

with hemoglobin,

personal quite

than milliseconds.

case of deoxyhemoglobin

communication),

slowly, Stripped

Although re-

it has been found

with a half time of the order hemoglobin prepared according

to Benesch, Benesch, and Yu (6) was allowed to react with oxygen in bis-!I& buf'fer

alone,

with oxygen in OJM phosphate buffer,

with oxygen in O.lM phos-

IOOr

60-

0

20

msec.

J.&GENDFOR FIGURE 1 Binding of oyygen in 0.1 M phosphate pR 7.0 nith (0) and without (0) 200 @i pyridoxal phosphate, hemoglobin concentration 110 a (before mixing), oxygen concentrations, upper pair of curves 250 *; middle pair 125 IJM; lower pair 63 fi; all before mixing. 2 cm path, 470 rnp, 20° Ordinate : percent saturation; abscissa : time.

12,2-Ms(hydroxymethyl)

- 2,21,2"-nitrilotriethanol. 1320

Vol.40,No.6,

1970

phate buffer hemoglobin

BIOCHEMICAL

containing

200 wpyridoxalphosphate,

with pyridoxal

preincubated

react with oxygen in phosphate indicate

that

hemoglobin

pyridoxsl binding

binding

of additional in fig.

The results

reaction

of equilibrium.

doxal phosphate

is followed

several

and partially

01

with

by slower

This point

is

minutes for the

The slower phases may represent

from fully

the

appears to be complex, being

complete in about one second, but requiring

attainment

only if

preincubated

of some seconds.

The slower phase itself

1 and 2

decreases the rate and extent

In the case of hemoglobin oxygen binding

allowed to

the reaction

substantially

oxygen with a half-time 2.

stripped

shown in Figs.

which influences

with it,

the rapid

andlastly,

phosphate was similarly

buffer.

phosphate,

reaction.

phosphate,

illustrated half

pyridoxal

is preincubated

of the ligand

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the loss of pyri-

oxygenated hemoglobin molecules.

It

TIME

-LFGENDFORFIGURE 2 Binding of oxygen by stripped hemoglobin in 0.05 M his*tris buffer pH 7.0. before mixing: upper Other conditions as for Fig. 1. Oxygen concentrations curve, 250 IJM; middle, 125 $4; lower, 63 $4. Time intervals between points: third group; 20 seconds. is possible functional pyridoxal

First

to draw from these results properties phosphate.

of hemoglobin

group 4 msec; middle group 200 msec;

sune approdmde

liganded

simultaneously

On the basis of the saturation 1321

conclusions

about the

to oxygen and to

reached about 80 msec.

BIOCHEMICAL

Vol. 40, No. 6,197O

after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mixing with oxygen it would seem that half-saturation

50 rJMoxygen as compared with about 3 $4 for stripped conditions.

suggests that the greatest

hemoglobin is in the binding and rapidly

is strongly

hemoglobin,

from the behavior

but relatively

phosphate hemoglobin.

It follows

on oqgen

binding

mixed with omgen + diphosphoglycerate,

suggesting

brate quite rapidly

equivalent

is substantially

experiments,

CO-hemoglobin or following

in the rate and extent of are able to equili-

and pyridoxal

under equilibrium free from

organic

phosphate can

conditions

(7),

phosphates in solutions

addition

of dithionite

to oxyhemoglobin,

combine to a small extent with Hb4X , and substantially 2 Hb4, where X is the ligand. may be expressed symbolically Kphos 1 rn4

%

of about 300/

but on removal of ligand by flash photolysis

will

'Ihe situation

hemoglobin was

as compared with the rate of oxygen binding

be regarded as functionally

used for kinetic

weakly bound by Hb4(02)2,

that hemoglobin and diphosphoglycerate

If diphosphoglycerate

hemoglobin

phosphate

and hemoglobin + diphosphoglycerate

sec. in these experiments.

liganded

slowly and loosely

in which stripped

was mixed with oxygen showed only small differences binding,

of stripped

that pyridoxal

bound by Hb4 and Hb4(02) but is relatively

Analogous experiments

at kinetic

of the second ligand molecule which is firmly

bound by stripped

bound by pyridoxal

difference

hemoglobin under similar An attempt

The value of E is reduced to about 2.

analysis

is reached with

w

HbP 4

+x

1322

phosphate

with HbkX and

by the equations:

of

Vol. 40, No. t&l970

BIOCHEMICALAND BlOPHYSlCALRESEARCHCOMMUNlCATlONS

where P represents Since all P-free

a molecule of orgfLnic phosphate and X is a ligandmolecule.

P-species have lower affinities

species,

and because the predmiuant

Rb4P, while the pre dominant ligended cooperativity phate.

of ligand binding

The estimates

globin yield energy.

result

underscores of its

fuuctional

increase

effect

containing

(fast)

this

in any serious

of the scheme given above is in treatin@;

upon the &oxygenation

constant

that

as the concentration

of diphosphoglycerate

arises because the transition

species occurs earlier

described

of oxyhemoglobin

there is a progressive of diphosphoglycerate

but that there was no obvious stoichiometric

This effect

inter-

is bound (2),

and Mizuksmi (8) who have recently

being observed with ratios

as 4o:l.

half the total

hemo@;lobin as a tetramer

They found, and we have confirmed,

in the velocity

was raised,

or nearly

hem-

properties.

of 2*3-diphosphoglycerate

by dithionite.

and unstripped

As only one phosphate molecule per tetmer

of Salhany, Eliot

the effect

with the loss of orgm3.c phos-

of about 1.5 kcal,

the need to treat

is

species is Rb4X4, a part of the observed

is associated

An example of the application the results

species in deoxyhmobin

of Gibson (5) for 5 in stripped

a contribution

action

discussion

for oxygen than the corresponding

relation,

an increasing

to hemoglobin as great

Finn P-free

in the chain of reactions

(slow) to Pas the

1.0

0.5 A

0.2 5 .I.25

b 0

40

80

Calculated time course of deoqqenation of hemoglobin in the presence of dithionite in bis'tris buffer pR 7, 20°, vith ~aryinl: am0unts 0f 2,3.diphosCurve B, 200 ~diphosphoglyCurve A, no diphospho@ycerate; phoglycerate. cerate; Curve C, 400 w diphosphoglycerate. 1323

BIOCHEMICAL

Vol. 40, No. 6, 1970

concentration

of diphosphoglycerate

of differential family

equations

of curves presented

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is raised.

with rate constants

Solving the appropriate taken from (5) provides

in Fig. 3, which is strikingly

similar

set the

to Fig. 1

of Salhany --et al.. Although

the scheme used in this example attempts

10 species of intermediates

in the reaction,

cx- and S-chains have not been considered,

to take account of

the known differences and it cannot,

therefore,

between be re-

garded as a complete model.

REFERENCES 1, :: 4.

2: 7. 8.

Chanutin, A. and Curnish, R. R., Arch. Biochem. Biophys. 121, g6 (1967). Benesch, R. and Benesch, R. E., Nature Land. 221, 618 (lgm. Luque, J., Diederich, D. and Grisolia, S., Bisem. Biophys. Res. Commuu. 3, 1019 (1969). @=by, L., Gerber, G. and de Verdier, C.-H., European J. Biochem. I-C, UO (1969). Gibson, Q. I-I., J. Biol. Chem. 245, 3285 (1970). Benesch, R., Benesch, R. E., Proc. Nat. Acad. Sci. z, 526 (1968). Remthal, R., Benesch, R. E., Benesch, R. and Bray, B. A., Fed, Proc. 3, 732 (1970).

Salhany, J. M., Eliot, R. S. and Mizukami, H., Biochem. Biophys. Res. Qmml. 2, 1052 0970).

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