Alkali denaturation of human hemoglobin: Possible role of disulfide formation

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMlJNlCATlONS

Vol. 20, No. 5, 1965

ALKALI DENATUEATION OF HUMAN HEMOGLOBIN: POSSIBLE ROLE OF DISULFIDE FOFXATION bY Elizabeth

A. Robinson from

The National

Institute

of Arthritis

and Metabolic

Diseases

of the National

Institutes

of Health,

U. S. Department

of Health, Bethesda,

ReceivedJuly15,

hemglobin,

the markedly

thirty

Education, Maryland

greater

differences

resistance

the sequences

resistance

between normal human hemoglobin,

of the latter

(Schroeder

0+r2,

to denaturation

of the S and y chains et al.,

have not yet been related

Adult

Service

and Welfare

and normal human fetal

amino acid positions

differences alkali

a+,,

Although

alkali.

Health

1965

One of the most striking adult

Public

1963),

to the great

is

by

differ

at over

these

difference

in

of the two hemoglobins.

hemoglobin

of y+

two of which

(Allison

and Cecil,

(lib A) has six are "reactive"

1958).

Fetal

two reactive

and two non-reactive

non-reactive

sulfhydryls

sulfhydryl and four

hemoglobin (Allison

groups of which

per molecule are "non-reactive"

(l-lb F) has four et al.,

1960).

two occupy the 104th position

sulfhydryls,

Of the

from the N-terminus

of each a chain

(Hb A and Hb F) and, in the case of Hb A, the others

occupy position

112 of each 8 chain.

sulfhydryls

occupy the 93rd position

y chains, respectively

In both Hb A and Hb F the reactive from the N-terminus of the S and

(Goldstein et al.,

573

1961).

Vol.. 2Q No. 5,1965

BlOCH~lCAl.

The data herein bonds plays

reported

a significant

suggests

role

hemoglobin.

It is proposed

denaturation

exhibited,

presence

AND &lOPHYSCAh RESEARCH COMMUNICATIONS

that

described

(Itano

hemoglobin

denaturation

resistance

aulfhydryl

samples were prepared and Singer,

1958).

of

to alkali

in part,

to the

per half-molecule.

reacted

coupling

with

with

HbCO A was reacted

carried

after

with

precipitation

of denatured

1929) at various continuous

recording

(Raurowitz

et al.,

of HbCO in alkali recorded

Results

protein

with

stages

reactions

and Snow (1962). by

(WH4)2S04 (Haurowitz,

(HbCO and Hb02) and secondly at a specific

1949).

prolonged

wavelength

complete W and visible

by (Hb02)

When rate of change of absorbance

was measured,

at various

acidic

by

of iodoacetamide

(1964);

of Cecil

et al.,

was measured in two ways,

of absorbance 1954).

(Irving

and Konigsberg

time intervals

was determined

molar excess

denaturation

A @lb02 A)

of HgC12 and pCMb

acid hydrolysis

out at pH 9 in the buffers

Rate of alkali

1958).

Bound mercury

a sixty-fold

to Guidotti

et al.,

molar excess

and Snow (1962).

dithizone

(INH 2) according

(Allen

from cord blood

A (RbCC A) and oxyhemoglobin

an eight-fold

to Cecil

from whole blood as previously

Rb P was purified

by column chromatography

according

also

the increased

by Rb F may be due, at least

Carbonmonaxyhemoglobin

were

of alkali

of disulfide

and Materials Hemoglobin

were

the formation

in the process

of only one non-reactive

M&hods

that

at 250 w

spectra

were

of the reaction.

and Discussion Figure

1 shows the results

of alkali

samples in 0.10 N Na0I-I as determined Untreated curves. sulfhydryls Hgf2/mole

HbCO A and HbCO A with Point

readings

were blocked

denaturation

by the precipitation

1.8 moles Hg'2/mole

of HbCO A- INH2 in which gave similar

Hb was more resistant

of HbCO

results.

to denaturation

574

technique.

Hb have identical

only the reactive HbCO A with than HbCO F.

6.1 moles Untreated

BIOCHEMCAL AND BIOPl-tYSICAli RESEARCH COMMUNICATIONS

Vol. 20, No. 5, 1965

TlUE IN YINIJTES

Fig.

1.

Rlkali

denaturation

reaction

mixture

of HbCC in 0.10 contained

and 1 ml HbCO (3-S%). pipetted

into

Zero

time

A A-pCMB

A/HgC12(1.8), untreated

blocked,

('NH4)2S04. (3.7),

0

FII

(Itano

The

1 ml was

mixed,

filtered,

measured

determined

A in presence

N NaOH, pH 13),

at 538 I+J.

by adding

A untreated A

A-INH2,

HbCC A,

17 hrs

at

of 1.4 M 2-mercaptoethanol et al.,

1964),

A-HgC12(6.1).

HbCO A in the presence resistance.

fraction

were

0

0

sulfhydryls

soluble

concentrations

intervals

50% (NH4)2S04,

neutral

(1.5

intermediate

of the

20° C.

N RaOH, 4 ml H20

At specified

to 33% saturated

pH 9, 0

were

5 ml 0.20

2 ml acidic

and absorbance

N R&H,

of 2lnercaptoethanol

If more than resistance to that were

two but to alkali

of untreated

blocked.

fewer

exhibited than

denaturation

six

intermediate

sulfhydryls was increased

Hb A and Hb A in which

The results

575

with

of Hb A

Hb02 samples,

all

but six

as determined

Vol. 20, No. 5, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(140

a30 0.20I-K

a06 0.04 -

0.03 -

Y

0.02 -

0.01

Fig. 2.

! I 5

0

I I I IO I5 20 TIYE IN MINUTES

I 25

I 30

Increase in absorbance at 250 w of HbCOin 0.10 N NaOH at 25' C. Equal volumes of 0.1% HbCOin H20 and 0.20 N NaOHwere mixed, saturated with CO, and the increase in absorbance recorded with a Cary Model 14 until

Do was determined by mixing equal volumes of

change (b).

0.1% HbCOand phosphate buffer A, 0 FII,

there was no further

pH 6.0,

p = 0.2.

A

untreated

0 A-HgC12(5.5).

by either method, paralleled

these findings.

Figure 2 gives the results of absorbance changes of HbCCin 0.10 N NaCHat 250 w. w decreased.

Absorbance at 250 w increased as

that

at

538

The order of rates determined at 250 w paralleled

those determined at 538 w. Alkali

denaturation

changes in the visible

of hemoglobin is characterized

spectrum, Q.,

576

not only by

changes in the heme environment,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COhVvt~NlCATlONS

Vol. 20, No. 5, 1965

but

also

by changes

in

mechanism

is

discussed

on the basis

involved

of formation

It

case then

it

data

have

long

can be

the possible

sulfhydryls

in alkali.

the molecule

merely

by returning

to neutral

groups

be protected

could

namely,

hemoglobin

in 1956 that

to disulfides

either

bonds

If

this

is

the native PH.

by prior

of such reagents

of disulfide

one

one aspect

to be labile

to regain

effect

than

been known

be impossible

concentrations

However,

obtained,

would

could

more

role

bonds.

by Riggs

aerobically

phenomenon.

of the

sulfhydryls

was suggested

oxidized

in this

of disulfide

Protein

Unquestionably

solubility.

If

are indeed

the of

sulfhydryl

or by high

as mercaptoethanol,

on reassumption

alkali.

conformation the

reaction

in

the restrictive

of the native

conformation

be circumvented. Does the

disulfide

data

presented

formation?

interpreting

absorbance

phenylalanine, Winitz,

On the

hand,

unpublished

results).

significant

change

Robinson,

unpublished

even after

in spectrum

and the

namely,

(Greenstein

and

exhibit

pH is

over

a ten-fold

this

change

an

raised

from

increase

in

in spectrum

minutes

occurs

(Robinson,

with

alkali

(Benesch

and Benesch,

about

a one hundred-fold

There but

region,

tryptophan

sixty

in

saturated in

results).

in D250 initially,

is

However,

Cysteine,

decreases

w

and cysteine nor

of

be considered

in the 240-260

there

in alkali,

and is constant

absorbance

any evidence

at 250 ‘9-1 (D250 ) when the

immediately

increase

must

phenylalanine

other

D250 of tyrosine

2 offer

acids

tyrosine,

Neither

in absorbance

6 to 13.

amino

changes

tryptophan,

1961).

increase

Four

in Fig.

after

final

is longer

CO, likewise

exposure

spectrum

is

of the

twelve

shows a 1955;

to alkali

similar

to that

the of

cystine. Hermans in HbcO A, four

(1962) titrate

reported

that

abnormally

and that

577

tyrosyl

AD245 increased

residues with

time

Vol. 20, No. 5, 1965

BIOCHEMICAL

between

pH 11.4

and 12.3

Whether

this

also

is

been reported.

ionization

true

not

there

is a 2:1 ratio

ratio

in Hb F, it

to attribute

at pH 13 it

be time

of abnormal

was independent

of the tyrosyl

However,

would

AD245-250

but

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

this

of Hb F has not

due solely

other

hand,

to since

in Hb A and a 2.5:l

without

shown in Fig.

to assume that

A were

On the

to cysteine

not possible,

the curves

if

residues.

of tyrosine is

residues

at pH 12.3.

seems reasonable

dependent

tyrosyl

of time

further

2 solely

experimentation,

to changes

in cysteinyl

residues. If

one assumes

denaturation is

that

exhibited

disulfide

sulfhydryl rate

resistance

linkage

the non-reactive

sulfhydryls

is

of Hb A is

not

the pH of dissociation 1959;

Cottlieb,

linkages in the

process

non-reactive

the

sulfhydryls

denaturation,

the

do play

a significant

presence

of only

planned

as are

experiments

denatured

in

Detection

Hb A and Hb F will

above

role one

in Hb F may be significant.

Hb A and Hb F are

are blocked.

occurs

disulfide

apparatus

two sulfhydryls

the

reactive

interchain

silica

residues

that

and Vinograd,

special

cysteinyl

the

denaturation

in

of the

fact

when only

(Hasserodt If

the most

seem to be between

by the

that

half-molecules

blocked

and the non-reactive

chsnged

per half-molecule

studies

formation,

supported

fact

to alkali sulfhydryls

Hb A would

results).

of alkali sulfhydryl

location

alkali

into

non-reactive

Anaerobic untreated

and by the

unpublished

between

to disulfide

This

are blocked

to six

of the a chain

8 chain.

of denaturation

resistance

three

in untreated

sulfhydryl

of the

increased

by Hb A with

due to an increased

likely

the

samples

of disulfide

of treated

and

to determine

in which groups

more

than

in

be attempted.

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Allen., 80,

D. W., 1628

Schroeder,

W. A.,

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