The role of water in deoxygenated hemoglobin solutions

Vol. 22, No. 6, 1966

The

BIOCHEMICAL

Role

of Water

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Deoxygenated

THOMAS

Hemoglobin L.

Solutions

FABRY

AND HASKELL

IBM

Watson

Laboratory,

Received

Columbia

February

no

the

is

tion

positions

The

globin

at

being

show

survey

for

decide

different

mechanisms

magnetic

fields

aqueous

solution of

magnetic

ions.

position

of

a water

is

divided to

York

This

paramagnetic

in

the

in

the

there

molecule

opinions

on

an

Since

effect

been in

ring, and

the

Haurowitz’ form

coordination other

position.

plausible

data

are

available

position.

A

very

used

macromolecules.

a nuclear

relaxation the

from

700

coordina-

literature

magnetic

spin-lattice

relaxation rates

relaxation

due

due

efficient,

a principal

comes has

ions

is ions

protons

the

of the

electrons

of the

deoxygenated

using

solution

relaxation.

paramagnetic

this

sixth

molecule

a ligand.

be

in

is

the

position

sixth

X-ray

in

porphyrin

dry,

matter

sum

by

no

there

question.

the

the

four the

may

this

aqueous

unpaired

water

up

Presently,

is

of

of the

that

ligand

coordination

taken

molecule

known

hemoglobin

part, of

fifth

a water

protons

of

the

that

out

the

nitrogens

investigate In

solvent

relaxation

New

well

of the

heme

usually

results.

technique.

of the

the

the to

is

is

there

indicates

resonance

by

pointed

whether

of

attached

these

identity In

evidence,

Lyster’

We

rate

up

position

there

explanation

the

middle

circumstantial

and

York,

is

deoxyhemoglobin.

the

is

coordination

George

New

of hemoglobin about

taken

residue

of hemoglobin

to

in

located

presented

University,

structure

evidence

position

iron

sixth

the

conclusive

coordination

REICH

16, 1966

Although still

A.

in

the

contribution

collisions

with

previously

to 3

The

to

the

to the case

of

to the the

para-

determine

the

solvent

protons

an

Vol. 22, No. 6, 1966

are

also

BIOCHEMICAL

relaxed

by water-water

According globin

are

carboxyhemoglobin

are

the iron

as ferrous

is present expect,

solutions relaxation

times

the paramagnetic

faster

than

wate:r

proton

should

we expect

male

carried

sample

then

to avoid

measuring

converted wet

also oxidizing

through

it also

oxygen

measured

this

effect

of hemoglobin.

These

measurements

Since

the relaxation

rate

with

done

sets

A typical

rate

of an

were

measured

The

measurements

hemoglobin with

a stopcock.

was

determined.

nitrogen,

saturated

through

the sample. solution

oxygen rate

deoxygenated

gas is para-

of the solution.

the relaxation

solution.

we

As a final

in a methemoglobin

It is

rate check

solution

due to we

by

ferricyanide.

of measurements

on three

prepara-

set of measurements

is shown

on Fig.

carried increases

interact.

blood

by hubbling

Since

by deducting

potassium

three

were

fitted

carboxyhemoglobin

in the hemoglobin

the hemoglobin

The

free

to the relaxation

the relaxation

We have tions

for

6

in deoxyhemoglobin

the solution.

contributes

to correct

the dissolved

into

could

times

in the concentration, rate

provided

drawn

solution

oxygen

The

has been

molecules

tube

of oxyhemoglobin

the relaxation

Further times

solution

(21-22°C).

that

methemoglobin

Mc/sec.

test

the

= 4.

for

in

provided

sixteen

the freshly

at 2.5

in a small

changes

with

molecules.

about

Relaxation

by bubbling

the hemoglobin

magnetic, possible

placed

times

of methemoglobin

temperature

rate

carbonmonoxide

the water

from

One

relaxation

forms

and the water

and Reich

deoxygenated

water

After

was

the relaxation

It was with

ml)

in hemoglobin

compared

protons

technique.

of Garwin

that

peff(Fett+)/peff(Fet+)

prepared

out at room

(1.5

At first,

was

by the Drabkin

on the apparatus

proton

in deoxyhemoglobin

ion in the heme

and

electrons.

the measurements

effect

Hemoglobin

unpaired

in solution

3 From

a measurable

the paramagnetic

were

since time

measured.

four

with

the water

deoxyhemoglobin5

previously

concluded

of the diamagnetic

in contact

relax

relaxation

They

and methemoglobin

are

deoxyhemo-

oxyhemoglobin

decreased

solutions

ions

methemoglobin

adult

ion with

g reatly

in the

and Coryell’

paramagnetic,

of deoxyhemoglobin

collisions.

of Pauling

diamagnetic.

therefore,

RESEARCH COMMUNICATIONS

and water-protein

to the studies

and methemoglobin

would

AND BIOPHYSICAL

out at four

different

concentrations.

as a linear

function

of concentration,

701

1.

Vol. 22, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I.

0

OXYHEMOGLOBIN

0

OEOXYHEMOGLOBIN

A

CARBOXYHEMOGLOBIN

l

WATER

30

1.0

I.5

c x

Fig.

the dissolved

protein

differences

within

relaxation globin.

most

rate The

the other

1.

concentrated with

hemoglobin

concentration.

enhances

rate rates

are

observable

of methemoglobin

that

rate.

carboxyhemoglobin

solution

for hemoglobin.

the hydration

sphere

of the protein

of the solvent

water,

one expects

undergo the observed

702

rapid

But no the

and deoxyhemolarger

on the graph.

we observed Since

of

between

is so much

it is not indicated

methemoglobin 1.45/set

as

the relaxation

error

of oxyhemoglobin,

relaxation

compared

rate

experimental

relaxation

a function

Relaxation

itself

2.0

to+3

the water exchange relaxation

1 /Tl

than For

the

= 3.42/set,

molecule5 with rate

in

those to depend

Vol. 22, No. 6, 1966

on

a c.orrelation

including by

time,

the

studying

the

state

of the

motion

the

the

iron,

Tc

and

with iron.

the

large

increase

of hemoglobin

suggests

figuration

which

out

molecule,

in

be

the

relaxation

sphere

of the the

lines.

magnetic

This

protein

also

do not

coordination

exchange

upon change

the

implied

position

observed

to enter

the

Brownian

accompanying

molecules

these

of the

time.

time

an

verified of

along

rotational

sixth

relaxation

be

dependence

independent

with

is

can

work

spin

there

water

assumption

further

to

occupies

that

allows

protein

temperature

identified

hydration ligand

the

observed

electron

whatever The

the

This

carry

be

the

the

of

part. or

is

can

not in

heme

to

rate

protons

readily

characteristic

intend

relaxation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C’

frequency

We

time,

that

and

rate.

Since

7

globin either

relax,ation

the

BIOCHEMICAL

sixth

of

oxidation in

con-

co-ordination

position. Our sixth the of

results

coordination water the

is

water

position so

her

E:.

firmly

that is

in

either

bound

that

deoxyhemoglobin not

solution

occupied

no

exchange

Professor

J.

by is

water,

the or

possible

that

with

the

bulk

molecules. We

M.

suggest

Riepe

are for

indebted

to

helpful

discussions

and

H.

Wang

to Miss

C.

and E.

to

Miss

Volin

for

assistance. References

1.

F.

Haurowitz,

2.

P.

George

Acad.

p.

A.

Wishnia,

J.

Phys.

4.

L.

Pauling

5.

J.

A.

Nuclear

6.

R.

Biol.

R.

J.

of Sciences

1958) 3.

and

J.

L.

Chem. Lyster,

443 (1951).

193, Conference

- Natl.

Res.

on

Council,

Hemoglobin,

Washington,

(Natl. D. C.,

36. J.

Chem.

Chem.

65,

and

Pople,

C. W.

Magnetic Garwin

Phys. 837

D. G.

H.

871

Coryell,

Proc.

Schneider,

and

A.

(1960);

R.

Lumry,

et al

(1961).

Resonance, and

32,

McGraw Reich,

Phys.

703

Natl. H.

J.

AC.

Sci.

Bernstein,

22,

210

High

(1936). Resolution

Hill,

New

York,

1959.

Rev.

115,

1478

(1959).