Nitrogen mustard: An “in vitro” inhibitor of erythrocyte sickling

Vol.

48,

No.

BIOCHEMICAL

3, 1972

NITROGEN

MUSTARD:

AN

“IN

AND

VITRO”

BIOPHYSICAL

INHIBITOR

OF

Eugene F. Roth, Jr., Ronald L. Robert M. Bookchin and Arthur I. Department of Medicine. Division Montefiore Hospital and the Albert Einstein The Bronx, N.Y. 10461

Received &!/&WRY inhibited increasing hemoglobin gelation Preliminary erythrocytes intravenous

June

to low

oxygen

amino

acid

which

enables

cell

disease

the

near molecule

macromolecular

in addition

From

substitution

and

that

is characterized

tensions.

mechanism,

in the

ERMHROCYTE

SICKLING

Nagel Grayzel of Hematology College of Medicine

12,1972

sickling

it seems

COMMUNICATIONS

- Sickling of erythrocytes from patients with sickle cell anemia can be completely by exposure to nitrogen mustard (HN2). Reaction of hemoglobin (Hb) S with quantities of HN2 produces a progressive rise in the minimum concentration of required for gelation on deoxygenation. Quantities of HN2 sufficient to alter cause no observable change in oxygen affinity or in heme:heme interaction. studies of osmotic fragility and autohemolysis showed no abnormalities after were treated with HN2. Due to the known toxicity of HN2, no “in viva” therapy with this agent can be considered.

Sickle

the

RESEARCH

such

more

the the

molecular

one

to hydrophobic

protein-protein

pair

location

to inhibit

molecule.

This

preliminary

report

alkylating

agent,

nitrogen

mustard

that

sites

not

and

in sickling.

by altering

describes

sickling beta

chain

the

the in vitro

the

fully

cells

is the

(1).

sites,

is required (3),

result

sickling

binding are

electrostatic

interactions

to attempt

polymer

of binding

of the

of red

deoxygenated

of the

of the

bonds,

reasonable

when

deformation

of view,

residue

to polymerize as the

gross

point

N terminal

structure than

by the

upon

result

of a single

of hemoglobin Many

types

(Hb)

details of bonds

of the involved

understood.

Nevertheless,

and

likely

(2), possibly

it is very

hydrogen

Accordingly, surface

charges

bonds

that

are

blood,

anticoagulated

seem

of the

hemoglobin

inhibition

of sickling

or EDTA,

as indicated,

by an

(HN2).

Copyright 0 19 72 by Academic Press, Inc. All rights of reproduction in any form reserved.

with

either 612

citrate

involved

it would

METHODS Venous

exposure

was

S

Vol.

48,

No.

obtained

from

of Drabkin and

patients

(4) and

agar

gel

amount

all

(K and

cell

were

(5)

the

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

anemia.

Hemolysates

examined

by electrophoresis

presence

was

5%,

were

as the

by the

on starch

of Hb A or other

measured

prepared

variants,

gel

The

proportion

the

effect

K Laboratories, (PBS)

washed

three

of HN2

on sickling

Plainview,

N.Y.)

were

added

times

in PBS.

deoxygenated

After

volume glutaraldehyde

counting

300 Minimum

which

pH 7.35,

at 25OC.

The

Solutions

at pH 8.6

maximum

of hemoglobin

equilibria.

These

a Cary

were

model

two

of 95%

and

(2) on solutions with

various

of methemoglobin and were

after

reacted

performed

resistant

the

CO2

buffered

saline

or to red

cells

mixtures

were

or by the

addition

of cells

evaluated

for

discs)

of HN2

was

then

sickling

blood,

of HN2

of red results

a concentration

cells

at pH 7.0 using

HN2 prior

on deoxygenation

of Hb

S in 0.15

amounts

of HN2

M K for

was

determined

as described

above,

to determination

tonometers

spectrophotometer

from

patients

in progressive of at least

two

with

but were

of oxygen integral

optical

(6).

with

inhibition 0.4

by

or deformed.

RESULTS Incubation

fixed

experiments. with

at 25OC

blood

at 37oC,

gelation

in samples

gelling

whole

(biconcave for

amounts

phosphate

An aliquot

as normal

reaction

varying

N2-5%

metabisulfite.

described

M K phosphate

14 recording

hours

required

of hemoglobin 0.15

for

M Cacodylate-buffer

after

cells,

to either

concentration

before

against

Na

classified

proportion

spectrophotometrically

dialyzed

were

as previously

buffer,

of 1:l

incubation

a 0.1

hemoglobin

determined

phosphate

with

cells

ratio

of 2%

red

in isotonic

in an atmosphere

of a solution

in 2%

of intact dissolved

in a volume

by equilibration

of equal

amounts

method

denaturation.

solution

and

sickle

samples

at pH 6.4

To test

hours

with

of Hb F in hemolysates

to alkaline

was

BIOCHEMICAL

3, 1972

mg HN2

613

sickle

cell

of sickling per

anemia (Fig.

ml of blood

with 1).

increasing Using

is required

whole to inhibit

cuvettes

Vol.

48,

No.

3, 1972

BIOCHEMICAL

0.0

AND

I 0.2 NITROGEN

Figure

BIOPHYSICAL

I 0.4

I 0.6 MUSTARD

RESEARCH

I

0.8

1 2 3

614

I 1.0

mg/ml

The effect of nitrogen mustard on erythrocyte metabisulfite in vitro. Solid circles: sickling circles: sickling of RBC’s in buffered saline. deformed ccl Is before deoxygenation.

1:

COMMUNICATIONS

sickling in whole Dashed

in 1% sodium blood. Empty line: level of

Vol. 48, No. 3, 1972

sickling; per

however

ml of cell

that

this

33%

of the

with

agent

increases bands

the

cathodal

hemoglobin

between

majority

progressive

these

in the

deoxygenation

(Fig.

ratio

as low

Methemoglobin than

anodal

minimum

as l:l,

10 per

cent

in the

“S”

position.

total

reoxygenated and

51,

after

gelling

within

diffuse

migration ratio

of HN2

for

of

of 20:1,

results

required

in a

gelling

on

at a mustard:heme

gelling

* 5 per

which

electrophoretic

a HN2:heme

is elevated

than

new

some

quantities

point

greater

hemoglobin,

are

of hemoglobin

gelling

at ratios

of samples

of the

increasing

shown

heterogeneity

of Hb S with

usual

have

portion.

There

with

minimum

while

2).

mg HN2

Approximately

electrophoretic

(Fig.

concentration

The

mustard

globin

even

of Hb S with

3).

show

as 0.1

erythrocytes.

isolated

position

as little

nitrogen

Nevertheless,

migrates

content

HN2

to the

bands.

of solutions rise

with

with

in intact

on the

of mustard

of hemoglobin

Reaction

molar

and

to globin

is recovered

concentration

is seen

l4 C-labelled

with

of Hb S incubated

with

inhibition

of binding

amount

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cells,

Studies

is capable

applied

both

washed

suspension.

Solutions

the

BIOCHEMICAL

is no longer

experiments cent

demonstrable.

were

of the

always

control

(no

sample. Measurements ratio

of 1:l

~50~12.5 of lO:l, curve

of oxygen

demonstrate mm Hg and

there with

normal

Figure

2:

n value

is a small

two

is near

normal

slopes;

of Hb S after

oxygen on Hill

increase one

equilibria

affinity plot

= 2.8).

in oxygen

component

and

affinity

has an n value

reaction

at a HN2:heme

heme-heme However, (p50=10 reduced

interaction with

molar (Fig.

a HN2:heme

mm Hg) to 1.4,

and while

4, ratio

a biphasic the

other

(n=2.37).

Electrophoresis hemoglobin anode is at as 1 treated 4) Same as

on starch gel pH 8.6 of nitrogen S hemolysates. The origin is at the the right. 1) Control:Hb A2 and Hb with HN2:heme 1:l. 3) Same as 2 2 but HN2:heme 1O:l. 5) Same as

615

mustard (HN -treated left, marked 6. The S. 2) Same hemolysate but HN :heme 5:l. 2 but H I4 2:heme 2O:l.

less HN2)

Vol.

48,

No.

BIOCHEMICAL

3, 1972

NO

GELLING 43 -

4

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

GELLING 4 1

2

230 MOLAR

1

RATIO

4I

6I

I

NITROGEN

Fig.

I

aI

10

I

MUSTARD:

HEME

Paz (mm Hqf

3.

Fig.

4.

Figure

3:

Minimum gelling concentrations of nitrogen mustard treated hemoglobin Sol id circles: minimum gel1 ing concentrations. Arrows: concentration at which experiment was stopped without gel formation.

S.

Figure

4:

Log-log plot of oxygen equilibria of hemoglobin S hemolysate (Hill plot). Ordinate: ratio of O2 saturated hemoglobin S to unsaturated portion 1 - S. Abscissa:p02 in mm. Hg. Open circles: native hemoglobin S n=2,87, ~50 = 12.5 mm. Hg. Solid circles: nitrogen mustard-treated hemoglobin S at molar ratio HN2:heme 1 :l. n=2.87 p50=12.5 mm. Hg. Triangles: HN2:heme 1O:l. n value for first phase = 1.4. n value for second phase = 2.37. ~50 =lO mm. Hg. Temperature 25OC., pH 7.04 potassium phosphate buffer 0.15 M.

DISCUSSION Previous that

the

sites

on proteins

of the ring would

studies

rapidly

formed

and

electrophoretic

of the

species

pH,

side (8).

to reduce with

of nitrogen

ethylene

neutral

of histidine.

be expected

reactions

mustard At

(7).

C terminal nitrogens

on the

the slower

immonium alkylation

chains

with

ion reacts occurs

of asportate

Esterification net

mustards

most and

migration

charge towards

616

readily

of the

of the the

readily

glutamate

by alkylation

negative

proteins

protein

anode

than

have

shown

with

several

at the

carboxyl

residues,

and

carboxyl

groups

molecule the

native

and

anionic groups at the

produce protein.

_

Vol.

48,

No.

BIOCHEMICAL

3, 1972

However,

conformational

charges

at the

molar

ratios

higher amount

changes

surface

and

of nitrogen

molar

ratios,

of HN2

is not

mobility

used

yet

in these

clear

on gelation

minimum

also

can

than

gelling

red

that

negative

anode.

At

predominate,

with

alkylation

at

the

largest

S unmodified

various

and

low

while even

hemoglobin

hemoglobin

that

quantities

change

of

in electrophoretic

from

increase

in the oxygen

by reaction

with

persons

(and

amounts red

However,

possibly

with

of HN2

are

red

that sufficient

cell

other

effect

cells. the

sickling

Elevation

concentration

of intact

quantities

smaller

tendency

sickle

inhibitory

100 ml,

much

the sickling with

intact

33 gm per findings

to reduce

a marked

of the

our

suffice

significant

in sickling

with

complete.

the

to have

of Hb S above

inhibited

of washed

neither

of HN2

elevations

to a level

trait.

Since

constituents

of the comparable

nitrogen

mustard

of whole

required

to inhibit

sickling

required

to inhibit

gelling

blood) in whole

it blood

cells.

amounts

of HN2

methemoglobin affinity.

Thus

formation gelation

in hemolysates

and

sickling

must

and

sickling

nor an appreciable be inhibited

by another

Hb S molecules.

It is important of nitrogen cell

new

However,

of the

appears

and

is nearly

larger

produce

mustard

correlates

proteins

It is noteworthy

ml red

cells,

cells

plasma

in suspensions

amounts

most

of the

the

species

observed.

between

solutions

may

by red

with

on the

leaves

concentration

point

surprising

effect

are

expose

towards

moving

species

correlation

be completely

exhibited

is not

fast

faster

of reaction

the

deoxygenation

reacts

migration

experiments

hemoglobin

within

erythrocytes

to that

and

gelling

of hemoglobin

minimum

and

of Hb S by nitrogen of the

when

accelerate

might

COMMUNICATIONS

to be determined.

Alkylation

even

slow

RESEARCH

by alkylation

to heme,

the extent

remains

of the

thereby

both

BIOPHYSICAL

produced

mustard

electrophoretically; HN2

AND

(whole

to consider mustard. blood)

the

effects

P re I iminary suspension

produce

617

on intact studies

red

show

no increase

cells

that

of treatment

with

large

treatment

with

1 mg HN2

in osmotic

fragility

or auto-

per

Vol. 48, No. 3, 1972

hemolysis.

The

Fourteen and

red

others

cell

group

observed

that

present

sickling

data

in properties

required

to inhibit

possibility

suggest

sickling

explored

of the that

oxygen

treated

has not

unchanged

vivo”red

been

examined.

exposure

after

survival

of Hb S by nitrogen of the The

its direct

after

properties

cell

yet

data

to HN2,

treatment

with

would

be

inhibit

gelling

cells.

equilibrium.

treatment

cells

its storage

polymerization

preclude

in our

retains

alkylation

the

red

qualitatively

However,‘Fn

with

of extra-corporeal

actively

blood

integrity

of the

were

bank (9).

by interfering

change

being

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on enzyme-deficient

antigens

of HN2

to determine The

the

of mustard

blood

amounts

required

and

effect

have

comparable

BIOCHEMICAL

deoxyhemoglobin,

very

use in patients or the

mustard

high

with

concentrations with

use of less toxic

li+tle

of mustard

sickle

cell

alkylating

anemia, agents

but are

laboratory.

ACKNOWLEDGEMENT - This work was supported by PHS grants AM15053, AM12597, AM05082 and AM13430. We thank Dr. Joseph Kochen for the red cell hemolysis studies and Dr. Parviz Lalezari for the blood group antigen determinations. Drs. Nagel, Bookchin and Grayzel are Career Scientists of the Health Research Council of the City of New York. Requests for reprints should be sent to: Dr. Eugene Roth, Jr., c/o Dr. Ronald L. Nagel, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, U.S.A. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Ingram, V., Nature. 180, 326, 1957. Bookchin, R.M. and RI Nagel, J. Mol. Biol. 60, 263, 1971. Bookchin, R.M. and R.L. Nagel, Clin. Res. 19,Tl, 1971. Drabkin, D.L., J. Biol. Chem. 164, 703, 19x. Smithies, 0. Biochem. J. 71, 58r1946. Nagel, R.L., Q.H. Gibsonnd T. Jenkins, J. Mol. Biol. 58, Ross, W.C.J., in Biological Alkylating Agents, Butterworths,‘ondon, Burnap, V.C.E., G.E. Francis, D. E. Richards and A. Wormall,

9.

Hartman,

504, 1957. F.W., G.H. Biol. and Med.,

643,

1971. 1962: Biochem. J.,

66,

Mangun, 70, 248,

N. Feeley 1949.

618

and

E. Jackson,

Proc.

Sot.

Exp.