Chemical modifications of SH groups of intraerythrocytic hemoglobin

Vol.

74,

No.

BIOCHEMICAL

4, 1977

CHEMICAL

MODlFlCATlONS

HEMOGLOB

OF

SH

GROUPS

OF

INTRAERYTHROCYTIC

I N

Antoni

ni

C.N.R.

Centre

mi stry

and

Rome,

,

loppol

E., of

o,

C.,

Molecular

Gi ardi

na,

Biology,

Chemistry,

ltal

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Faculty

B.,

Brunori

Institutes

of

Medici

, of

ne,

M.

Bioche-

University

of

y

January

lo,1977 ABSTRACT

Chemical

modifications

of

intraerythrocytic

hemo-

globi n have been obtai ned by reacti on of (3 93 sulfhydryl groups with di sulfides i .e. cystami ne and cysti ne-di methyl ester. The respiratory properties of the modified erythrocytes are simi I ar to those of the hemoglobi n reacted with the same reagents and the changes observed compounds. Although other properties modi fi ed 1, the resistance to hemolysis I ts The resu suggest the, possi bi I i ty simi ar ones) to modify red ccl ( or poses. l NTRODUCTI

Chemical used

to

obtai

ships.

This

hemogl ci

obi

date

I i ng

ns

means

if

for

exact

can

be

attempts cations

of of

“i

n,

modi

fied

sickle-ccl

I

i ntraerythrocyti

Copyright 0 1977 by Academic Press, Inc. AN rights of reproduction in any form reserved.

ns

of

n vivo”,

thi

provides

a c

both are impaired. pur-

the

HbS

thus

s

which 1647

al

to

el

I owed

(1).

target

proa

pathological

example

would

u-

i n control-

provide or

y

onwith

would

on

ati I

molecule

present based

rel

wi del

successful

of

an

are

been

chains

physiological

vi vo”; anemi

has

have

modifications

n

for

compounds

on

side

function

obi “in

protei

noacid

of

hemogl

of

changes

chemical

achieved

ng these therapeutical

particularly

ami the

usi for

Is

structure-functi

been

modulation

Agai

on on

several

of

are the same the erythrocytes is not vastly

ON

chemical

and

be

processes. which

of

specific

could

on

known

role

cati

has

structure

Ideally, tei

where

the

fi

i nformati

approach n

the

n

modi

of

of

a

protei

n

therapeutical chemical prevent

modi

fi

-

gel-

ISSN

0006-291X

Vol.

74, No.

ation

of

the

initiate

a

which

can

objective of

red

here,

achieved

of

obtai cells.

concern

the

modi

I able

ber

of

fi

ed

the

ni

was choi

previous obi

Materi

al

bank three

with times

n

results

obtained

on

of

SH

a

study

simple

cat

i oni

c

on

the

the

fi ne

compounds

hemogl

02

binding SH

promi

si ng,

and

and

the

cysti

ne-di

made

of

far,

of

des,

was

reaction

n

thi

on

01

with

the

properties

various

more

disul cystami

the

to

modifications

so

93

of

appeared

with

chemical

respiratory

(j

Of

aims

hemoglobi

The

that

work

i ntraerythrocytic

erythrocytes.

results

hemogl

of

i n the

were

of

present

changes

and

performed ce

The

ng

reacti

ones

reasons,

3, 4).

(2,

i n

reagents

avai

The

n

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

investigation

be

blood

the

here

protei

systematic

sulfhydryl of

BIOCHEMICAL

4, 1977

the

and obi

reported n

with

properties reagents for

a

work

reported

methyl

ester.

basi

reagents

num-

s

of

with

human

(5).

and

Methods

Fresh blood samples ACD as anticoagulant. with 1% NaCl and

mM pH = 7.4. Cystami ne and cysti and Fluka respectively, and then added to

were Red resuspended

83

obtai blood

ned

from a blood cells were washed in phosphate buffer

* ne-di the

methyl ester were dissolved ccl I suspensions

,

obtai ned from Merk i n the same buffer to give final ratio

di sul fi de/heme from 6 to 25. The hemoglobi the ccl I suspensions was general ly 1.5 mM. at 20°C the ccl Is were washed and resuspended buffer .

n concentration After i ncubati i n the desi

of on red

Membrane preparati ONS were performed accordi ng to Steck and Kant (6); determi nat i ons of membrane SH groups were carri ed out after sol ubi l i zati on with SDS usi ng El I man’s procedure (7). Protein determi nati on was carried out by the method of Goa (8), Titrati ons of suI,fhydryI groups i n hemogl obi n were performed by the method of Boyer (9). The rate of reduction of ferri hemogl obi n was measured usi ng the methods previ ousl y described (10, 11). Oxygen di ssoci at i on curves were determi ned spectrophotometrical ly (1); the hi gh turbidity of the materi al was compensated for using a scattering opaque glass in the reference beam. *

Abbrevi Cystami dodecyl

ati ons: ne; ACD, sulfate.

DMC, Aci

L-cysti ne-Di d-Citrate-Dextrose;

1648

methyl

-ester; SOS,

Sodi

CYS, urn

Vol.

74, No.

4, 1977

BIOCHEMICAL

a

AND

BlOPHYSlCAL

RESEARCH

COMMUNICATIONS

0 0

1

2

3 Time

4

24

(hrs)

0 Time

Fi gure

1:

human erythrocytes incubated cystine-dimethylester (LJ). tion with su lfhydryl groups. Phosphate buffer 83 mM pH tration in cell suspensions heme

6.25

=

12.5

fO>,

(hrs)

with cystamine (a) and Time course of the reacConditions: T = 2U°C; = 7.4; hemoglobin concenN 1.5 mM; disulfide/

25

(W,

(ml.

RESULTS 1)

Time

course

of

erythrocytes

with Fi g.

SH

with

(fig.

2),

ne

reacti di sul

1 shows

cystami

throcytes

the

and

are

i ncubated

The

reaction

but

it

occurs

the cysti

on fi

of

the

HbA

and

of

des. time

course

of

react

ne-dimethylester, with

the

with at

human

free a

i on when

of human

(3

93 ery-

reagents. hemoglobin

substantial

1649

is rate

al ways also

with

faster ery-

Vol.

74, No.

Figure

4, 1977

2:

BIOCHEMICAL

Free

AND

SH/hemoglobin

BIOPHYSICAL

tetramer

RESEARCH

as

a

COMMUNICATIONS

function

of

disul-

fi de/heme ratio after 2 hrs of incubation with cystami ne (d) and 1 hr with cysti ne-di methylester (b). Red ccl I suspensions: fu I I symbols; hemoglobin solutions: open symbols. Conditions: T = 23’C; Phosphate buffer 83 mF1 pti = 7.4.; hemoglobin concentration in ccl I suspensions N 1.5 rnk!.

throcytes. with tic

Particul

arl

cysti

ne-dimethylester

hemoglobi

n

at

y

si

gni

fi

which a

higher

rate

cant

are reacts

than

1650

the with

cystami

results i ntraerythrocyne.

obtai

ned

Vol.

74,

No.

BIOCHEMICAL

4, 1977

AND

BIOPHYSICAL

60

Figure

Oxygen after

3:

Bohr complete

after

(0)

80

IibA and human wi th cystami

the cells;

Bohr (0)

0

cystami ne-treated methy I ester-treated

on

equi

2

of

I i bri

SH

urn

93

HbA

and

of

ne-

untreated

erythrocytes, hemoglobi erythrocytes. 83 mt/l and

of

with

erythrocytes ne and cysti

effect for cystamine-treated

methyl ester-treated Phosphate buffer

the

reacti

for ion

react

i ne-di

cysti ne-di conditions:

in

effect

represents and red

hemoglobin, ( q ) cyst

Changes

COMMUNICATIONS

70 PH

dimethylester. Ful I I ine hemoglobin

2)

RESEARCH

n,

( w )

Other 2G’C.

T =

erythrocytes

disulfides.

c9 O2 tes

after

cysti

equi

complete

those

neutral

but

the

O2

ester

red

are with ccl

HbA

range;

the

O2

equi

urn

of

the

same

as

those

is

di sul no

different

fi

des

affi

results reacted of the from

Hb

effect

that

with reacted

1651

with

the

confi

rm

i ncreased

i n

decreased,

essenti (i)

al the

ne-dimethyl-

with

cystamine; our

hemoglobins.

Iy changes

cysti

behavi of

or

ne

is

i s

functional that

ne

i s

curve show

erythrocy-

cystami nity

Bohr urn

human

3.

with

the

HbA

on cystami

Fi g,

O2

I i bri

the

I ibri

i n

the

pH

and

reacted

(5):

equi

both Is

on

addition

HbA either

shown

acid of

In

i n the

i )

results

and

unchanged.

on with

reported

shape

data

are

al ready

the

urn

reaction

ne-dimethylester The

(i

I i bri

of

reacted

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Decrease of free t i on with cysti of Sy/tetramer Cystine-dimethylester/heme PH

I

membrane ne-di methyl of i ntraerythroci

sulfhydryl ester.

groups duri ng i ncubaFor comparison the ratio ti c hemogl obi n i s reported. 6.25 Phosphate buffer 83 mM

=

7.4.

=

Treated

Untreated red cells

ccl

red

Treated ccl Is

Is after 2 hrs

4

red after hrs

c SH/Tetramer

nmol

3)

es

i )

SH/mg

Changes to

the

di ysi

ne

During

the

equi

I i bri for

c

sol

by

experiments

both

i s

also

of

si

free to

SH the

for

the

no

hemol

the

gni

fi

exposure

after

reaction ysi

sul

fhydryl

y

and

s

for

either red

osmotic

cant1

decrease

93

f

data of

i n was

Is

treated

cases

not

found

efficiency

i n erythrocytes reacted

ccl

fragi

for ccl

Is

I ity

was i n

i ncreased.

groups

titrable

i I I ustrated

5

red

ne-dimethylester

ons

was

The

urn

number

preparations of

of

required

sulfhydryl The

i i i )

time

i n

uti

Membrane

number

properties

cysti

Moreover

hypotoni i i )

other

50

sulfides.

or

observed.

107

n

0

s

oxygen

cystami

protei

i n the

the

Hemol

0.86

2

which

investigated.

in

shown the

groups i n

paral

hemoglobin. i n

Table

obi The

1652

with

the

This

n n

membrane

finding

is

I.

methemoglobi hemogl

lel

i n

SH results,

reduci

groups

ng were reported

systems ful

ly in

Vol.

74, No.

4, 1977

BIOCHEMICAL

AND

TABLE Efficiency

of

the

ferri

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I I

hemogl

obi

n

reduci

ng

systems

measured

in the presence of glucose or I actate as substrate throcytes treated with cysti ne-di methyl ester. Human red ccl Is containing 957 o met Hb were i ncubated in 0.05 Tris HCI buffer pH 7.4 plus 0.1 M NaCl in of methylene blue and substrate as indicated. Treated

red

ccl

Is

% HbO2

ene

bl

ue,

50 PM

60’

HbO

2 90’

20

20

mM

18

14

Glucose,

10

mM

11

14

Methylene

blue

and

Lactate

16

20

Methylene

blue

and

GI ucose

17

18

Untreated

red

ccl

Is

ene

bl

ue,

% Hb02

50,+4M

% Hb02 60’

after

90’

14

15

Lactate,

20

mM

17

18

Glucose,

10

mM

12

15

Methylene

blue

and

Lactate

34

48

Methylene

blue

and

GI ucose

46

74

Table

Ii

l ene-bl

, ue

sence

of

D I SCUSS

clear1

y

observed I actate

show i n

or

that

the

untreated

glucose

catalytic

effect

erythrocytes is

of

methy-

whether

abolished

i n the

i n pre-

reacted

ccl

I ON The

blood on

cells of

results with i ntraerythrocyti

presented

above

disulfides

a c

hemogl

1653

show

specific obi

37°C presence

after

Lactate,

Methyl

ficati

at

15

after

red

ery-

the

%

after

Methyl

for

that

by

chemical n can

be

treating modi-

achieved.

is.

Vol. 74, No. 4, 1977

The

modi

mixed ci

f i cat

BIOCHEMICAL

i on,

which

disulfide

ated

ccl

with

Is

I ar

number

after

of

of

SH with

the

treated

cells

of

the

reacted

erythrocytes

with

reinjected with

into cysti

prel

imi

nary than

this

compound Is

not

the

i ndi

cystami

ne.

This

simi

I ar

ish

sickling

cate

therapeutical Along

of

measure

that

such are

resistance

to The

present

under the

I i fe

The

is

sulfide

study)

s

the

Is

obtained

di

under

hemolysi

ccl

this

ones

ed

of

interest

bi

fi

ga-

results

possi

modi

i nvesti

particular

the

as

viability

of

opens

the n.

membranes,

viva”.

I i nes

effect red

to

asso-

si much

I ity

less

of

usi

to

modi

i n

progress

rice

ng

fy

red

purposes. these

the

at

are

is

hemoglobi

impaired.

i s

“in

data

the

a

of

erythrocytes,

vastly

blood

93,

P

free

the

aimed

(or

for

establ

is

ne-dimethylester

toxic

ccl

sulfides,

of

properties

the on

di

i on

position

with

groups

experiments

in

format

respiratory

properties

treatment

on

the observed

free

i n the

group

those

of

ti

SH

in

to

other

consists

the

changes

simi

Although the

at

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

cells

experiments

are

treatment

with

containing

disulfides

to

on

the

HbS.

REFERENCES 1.

Antonini, their shi

2.

E.,

ng

Lubi

n,

T.B.,

Brunori,

reactions Company,

M.,

with Ii Amsterdam

B.H.,

Pena, L.

Paeker,

V.,

(1975),

“Hemoglobin

gands” - The Meuzer, Proc.

and

1971

North

Nether1

myoglobi Ho1

n

I and

Publ

E.,

Bradley, U.S.A.

in i -

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W.C., Nat.

Bymann, Acad. Sci.

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Zak, mack,

72, 4.

5.

S.J., M.,

Peller, Kri vit,

G.R., Finkel, K. (1975), Proc.

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Tukey, Nat.

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MC CorU.S.A.

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Wyman,

J.

Acad.

D.P., Sci

4153-4156.

Cerami,

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and

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68,

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Manning,

Taylor, J. Biol.

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ni., A., 241-248.

(1971),

Brunori

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,

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(1966),

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Steck,

7.

Ellman,

8.

Goa,

9.

Boyer,

P.D.

Rossi

Fanel

Ii,

A.,

Chim.

Acta

2,

476-481.

10.

11.

4, 1977

BIOCHEMICAL

T.L.,

Kant,

(1953),

J.

loppol o, Antoni L .,

(1954),

C., ni

,

Arch.

Stand.

J. J. Antonini,

BIOPHYSICAL

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J.A.

(1959)

G.L.

AND

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I+Iethods

Biochem.

Chem. E.,

Ani coni, G., Currel I, E. (1974) VOX Sangui

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