Vol.

167,

March

No.

30,

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1990

1146-1153

EVIDENCE FOR COOPERATIVE BINDING OF CHLORPROMAZINE WITH HEMOGLOBIN : EQUILIBRIUM DIALYSIS, FLUORESCENCE QUENCHING AND OXYGEN RELEASE STUDY Maitree

Bhattacharyya,

U. Chaudhuri

ob Biophybic4, Unive&kty oi, Cakuttc~,

Mokc&cul

Depuntment

Received

February

and R.K. Biology

Ca.kutta

700

009,

und

Poddar Geneticd, Indm

20, 1990

SUMMARY:Binding of chlorpromazine (CPZ) with human hemoglobin has been studied by equilibrium dialysis and fluorescence quenching. Results of when analysed by Hill plot revealed equilibrium dialysis experiment that the binding was positively cooperative with overall affinity constant K = 3.6 x 10’ M-’ . CPZ quenched the fluorescence of hemoglobin and the analysis of the quenching data by Stern-Volmer equation indicated two types of quenching process, namely, dynamic and static quenching. Dynamic quenching constant was measured from the decay of fluorescent life time of tryptophans of hemoglobin in presence of CPZ. Static quenching constant concerned with the ground state complex formation between CPZ and hemoglobin was found to be 5 x lo3 M-’ . Almost all the tryptophsns of hemoglobin were found to be accessible for CPZ to interact with. Oxygen was found to be released when CPZ was added to hemoglobin. Extent of release of oxygen depends on the D/P ratio of CPZ(D) : hemoglobin(P). 0 1990 Academic Press,

1°C.

Chlorpromazine of drug, is

(CPZ : molecular weight widely used as antidepressant

of psychosis (1). Investigation components of RBC cells is of this

drug

interaction (2.

3,

4).

on the

cell

itself.

317)) the tranquilliser

phenothiazine group in the treatment

the interaction of CPZ importance in understanding

M

Some of

of CPZ with the RBC membrane CPZ can penetrate through cell

the

studies

with the

concerned

protein have already walls and membranes

different effect of with

the

been done (5). Hemo-

globin is the major protein component of RBC Cell, and as an allosteric protein it plays an important role in the transport of oxygen from lungs to different tissues. In this perspective it is interesting and relevant to study the interaction of this drug with hemoglobin. We report here our results on the fluorescence quenching studies on hemoglobin in presence of CPZ. Affinit]. constant obtained from such study agrees well with that obtained by equilibrium dialysis of hemoglobin are found to be released from hemoglobin when

method, and almost all of the tryptophans accessible for CPZ interaction. Oxygen gets CPZ is added to it. The extent of release

0006-291x/90 $1.50 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

1146

Vol.

is

167,

No.

coupled

BIOCHEMICAL

3, 1990

with

the physiological

the

extent

importance

of

AND

CPZ binding

of this

MATERIALS

BIOPHYSICAL

RESEARCH

to hemoglobin.

drug

binding.

AND

METHODS

COMMUNICATIONS

This

signifies

CPZ (made in USSR) was obtained as a gift through Sun Pharmaceuticals, India. Absorption spectrum of CPZ solution shows two peaks-,, one, at 254 nm (E = 33,200 M-’ cm-’ ) and the other at 305 nm (c = 4000 M cm ). It has no significant absorption in the wavelength region 360 nm to 660 nm. Stock CPZ solution in 0.15 M NaCl was prepared immediately before each set of experiments. Hemoglobin was isolated from red blood cells obtained from human volunteers. Isolation of hemoglobin was done according to the method reported elsewhere (6, 7). Freshly prepared hemoglobin in 0.15 M PBS (0.15 M NaCl + 0.001 M sodium phosphate buffer, pH 6.8) was used throughout the experimen1 . Absorption spectrum of hemoglobin shows three peaks in the visible wavelength range at 415 nm, 541 nm, and 577 nm respectively, characteristic for oxyhemoglobin form (8). With isolated and purified hemoglobin, no significant absorbance at 630 nm characteristic for methemoglobin (9) was observed. Even after addition of CPZ, absorbance at 630 nm was not observed. Isolated hemoglobin when eluted through calibrated Sephadex G-100 column (26 cm x 1.2 cm) shows a single peak corresponding to tetrameric molecular weight of 66,800. Concentration of hemoglobin was determined from its absorbance at 415 nm (E = 125 mM-’ cm-’ , monomer basis). All absorption measurements in the LJV and visible wavelength region were done with Carl Zeiss PMQ II Spectrophotometer, using 1 cm path length quartz cuvette (3 ml capacity). Necessary volume corrections, as when required, were made in the spectral data reported. Fluorescence measurements were done in Hitachi F-4010 Spectrofluorometer using 1 cm path length quartz cuvette (3 ml capacity). Excitation slit width was 3 nm, and excitation of hemoglobin was at 285 nm, emission being observed at emission maximum at 331.8 nm. For quenching measuremerits, 3 ml of hemoglobin (concentration 1.98 x lo-” M. tetramer basis) was taken, and to it CPZ from a concentrated stock solution was added so that the volume increment was negligible. Blank solution of CPZ of different concentrations (used in this study) when excited at 285 nm showed negligible fluorescence at 331.8 nm. Increasing concentrations of CPZ showed no quenching. Excited state life-time measurements were done in 199 Fluorescence Spectrometer (Edinburg Instrument). Excitation pulse was given at 285 nm and the emission was observed at 331.8 nm. Nanomoles of oxygen release from hemoglobin was measured by Gilson 5/6 oxygraph machine fitted with a temperature controlled oxygraph cell of 2 ml capacity. Release of. oxygen was recorded on a calibrated oxygraph chart and the percent release of oxygen was calculated on the basis of untreated hemoglobin to be in 100 percent oxygenated form as assayed according to the method of Huang and Redfield (10). Temperature during the experiment was maintained at 27OC. pH of the hemoglobin solution did not change even after addition of CPZ. No oxygen was released when CPZ was added to the solvent (0.15 M PBS buffer, pH 6.8) alone. Untreated hemoglobin did not release any oxygen even after one hour. RESULTS

2 ml of hemoglobin taken in Visking

solution dialysis

AND

DISCUSSION

of concentration 1.25 x 10 bag (USA) was equilibrium 1147

-4

M (tetramer dialysed

basis) at

10°C

Vol.

167,

for

No.

BIOCHEMICAL

3, 1990

90 hours the

against

18 ml

method

already

ding

to

per

mole

free

CPZ

concentrations

using

the

Hill

of

tetrameric

of CPZ reported

(Cf). (12)

(K Cf)

of

binding

K

log

=

3.8

denoted

as

brium

of

x

diallxis

the

Fig.

above

(7,

11). r, obtained

affinity

sites,

lo3

where

measurement.

plot

in

the

moles of different

constant

K

was

accorCPZ bound values of determined

n H are

the

total

“H

and

the

1 = 0 gives -1 M corresponding

r/rmax,

concentrations the at

g and

Hill

coefficient

is usually represented rmax 19/l- 81 versus log Cf (Fig.

[ 6/l-8

COMMUNICATIONS

“H , where

cf + 0~. r + g. the plot of log through

of varying

was

Binding

RESEARCH

:

1 + (K Cf) number

BIOPHYSICAL

solution

hemoglobin

equation

r=g

AND

rmax Hill region

the

value to

=

of

6.8

the

was

1148

Since

the

intersection

log

Cf axis

the overall

affinity binding.

obtained

from

nH calculated

50% saturation

1 : Hill plot of log [O/l01 versus centration of free CPZ expressed brium dialysis method.

g and with

50% saturation

coefficient of

as 1)

respectively.

binding

of passing

constant Here,

above from (i.e.

at

8 is equili-

the

slope

8 = 0.5).

C is the conlog c I where in ii Mf as measur Qd by equili-

Vol.

167,

No.

BIOCHEMICAL

3, 1990

AND

200

0

400

CONCENTRATION

2 : Stern-Volmer

Fig.

was

found

to

about

be

BIOPHYSICAL

600

OF

plot

RESEARCH

CPZ(Q)

of Fe/F

800

1000

IN PM

versus

3 .O indicating

COMMUNICATIONS

the

CPZ concentration

binding

mode

(Q].

to

be

positively

cooperative. Fluorescence

of hemoglobin

mainly- . Fig.

2 is

tration

following

and in

(0). F are

the

absence

to

lot)

be

of Fig.

modified tence

with of

an

observed

the

that

in

the

Fig.

type,

formation modification,

of

at

268

2 consists

called between and

the

is of

hemoglobin also from

(1

Ksv

is

and

the observed

solely

of

due

the

therefore

quite

likely

One

quenching and CPZ. equilibrium 1149

due

is as

to

evidenced

dialysis

F.

an

taken upward

quenching collisional

to

gets ratio

type

dynamic

is

involved

hemoglobin

: hemoglobin

types.

has

possibly

indicates

called

F.

that is

where

Stern-Volmer

process.

nm

two

CPZ concen-

the

linear

not

process

residues

of hemoglobin

is

CPZ

process static

+ KsvQ),

not

spectrum

It

= F

and

CPZ

increase CPZ.

versus

of tryptophans

is

other

absorption

of Fe/F

indicating

by

to tryptophan

collisional

plot axis

some

point and

F.

bimolecular This

to

gradual

:

intensities

Fe/F

collisional

bimolecular

other

UV

isosbestic

hemoglobin

between

to

due

plot

(13)

hemoglobin

due

285 nm is

CPZ respectively,

always. of

shows

of

towards

Quenching 3

equation

fluorescence

percent

fluorescence

at

Stern-Volmer

related

concaved

process.

the

presence

constant

curvature

simple

relative

and

quenching.

the

excited

gradually

(D/P).

complex that is

(13). Exisformation

the quenching concerned

quenching, ground from

measurements.

with and

state

the

complex spectral

above

Such

ground

Vol.

167,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0.6

\. :’ , I i

0.3

I-

r/P=0

\ 0.i

\

P 24 IO

250

260

270

280

WAVELENGTH

Fig.

state

and

complex

of

reduces

that

resembles

therefore

L(FO in

Q is

the

quenching the

effectively

hemoglobin) the

can

be

binding

the

amount

excited

affinity

(13,

of fluorophors 14).

constant

Static

(here quenching

between

hemoglobin

CPZ.

thing

ching

formation of

thus

We have plot

300

spectra of hemoglobin (1.2 x 10s5 M, tetramer 3 : UV absorption basis) in presence of CPZ. 2.5 ml of hemoglobin solution and 2.5 ml of solvent buffer (0.15 M PBS) were taken in sample and reference cuvette respectively. Equal volume of CPZ was added to both the cuvettes and absorbance of the sample cuvette at each addition of CPZ was noted with reference to the reference cuvette. CPZ : hemoglobin ratios (D/P) are mentioned on the figure. D : molar concentration of CPZ; P : molar concentration of hemoglobin.

try-ptophans constant

290

(nm)

the

analysed

-

F)/FI/Q

constant slope

of

(13)

(which

respectively. which

gives

0,

:

concentration

constant

quenching

versus

equation

molar

our

F.

of is

KSKD

using

incorporating = F

[l

dynamic

as affinity above

= 6.66 1150

KS and constant) plot

such

x

modified

+ (Ks+KD)Q

CPZ as quencher,

same

The

data

lo6

and

Stern-Volmer static

+ KSK,Q2], KD are and

the

dynamic

quenwhere static quen-

is found to be linear, -2 M , and the intercept on

Vol.

167,

the

No.

3, 1990

BIOCHEMICAL

axis

ordinate

gives

solutions obtained.

of the quadratic F’irst solution -1 1.35 x lo3 M . To decide dynamic

which

KD from presence decay case

of

straight

due

static

constant,

above

where

. The

The

plot

To (=2.23 absence the

solutions

estimated

for

actually dynamic

K two D’ = 0 were one is

corresponds quenching

to

constant

decay of tryptophans of hemoglobin in of CPZ. Excited state life-time will

quenching

(13, of

To/T

14). versus

It

will Q was

be unaffected found

to

in be

a

nanosecond) and T are life times of tryptoand presence of CPZ as quencher (4) res-

in

of

COMMUNICATIONS

plot gives an estimate of dynamic -1 quenching constant KD = 1.54 x lo3 M according to the equation : ~~ = M-l ) closel> of KD (= 1.35 x lo3 (1 + K,Ql 'r. Hence the second solution constant while the first solution corresponds to the dynamic quenching of K D (= 5 X lo3 M-l) refers to static quenching (KS) and it agrees to constant obtained by equilibrium dialysis a good extent with the affinity measurement.

slope

two

we have

to dynamic

of hemoglobin

pectively

the

quenching.

line,

phans

RESEARCH

equation : Ki - 6300 KD + 6.66 x lo6 of KD is 5 x lo3 M-1 , and the second

excited state life time of different concentrations

only

BIOPHYSICAL

KS + KD = 6300 M-1 . When solved

one of

quenching

AND

(K = 3.8 x lo3

above

M-l).

TIME IN MINUTES

of-4the release of oxygen from 2 ml of hemoglobin (0.45 x 10 M. t e t ramer basis) solution in 0.15 M PBS. pH 6.8. when CPZ at different final concentration was added at different time (arrow marked).

Fig. 4 : Gilson

5/6 oxygraph chart record

1151

Vol.

167,

No.

3, 1990

We

have

are

accessible

further

aof

for

to

be

to

be

molecule for

accessible

number

of

4 is

fixed

concentration

about from D/P

gives

an

the

are

6

plot

It

= 0.9

almost

(rmax)

is

in

the

)

measured

Fo/dF

the l/Q

tryptois

which

fa

tryptophans

be

=

fraction

versus

found

measures of hemo-

tetramer)

indicate

to

:

of

hemoglobin to

hemoglobin

accessible

from

all

relevant

was

for

of Fo/ @

tryptophans

interaction.

(13

fraction

constant

l/f, that

of

equation

accessible

The

intercept

(there sites

is

COMMUNICATIONS

tryptophans

the

quenching CPZ.

indicates

a representative

increasing

very.

fa

of

the

using

the

clearly

binding

when diate

and

K is

CPZ

all

RESEARCH

that

6.8

are

the

from

total

equilibrium

method.

diallxis

Fig.

and This

BIOPHYSICAL

whether

concentration

linear

AND

interaction,

AF = Fo-F

the

1.1.

globin

CPZ

hemoglobin,

Q is

and

investigated

where

:K++G plans

BIOCHEMICAL

concentration

after fast

of

of

then

gets

min

hemoglobin ratio

release

of of

of

oxygen

CPZ

particular

addition. :

increases

Fig.

at

added

x

low4

and

min

addition

With

increasing

30

20

O/p

extent

with

different

the

of

oxygen

time

to

the

release

saturation

of release of

CPZ) D/P

increase

of at

oxygen

different percent

the

40

RATIO

(measured for 15 min 5 : Percent release of oxygen from hemoglobin after CPZ addition) as a function of D/P ratio. Percent binding saturation ( 0 1 of hemoglobin with CPZ was measured from equilibrium dialysis experiment. D/P-patio is varied by keeping the CPZ concentration (D) (4 x 10 M) fixed and varying the hemoglobin concentration (P) accordingly. 1152

is

within

ratio, of

a

Imme-

basis).

CPZ.

reaches

after

parallel

10

of

the

release

M tetramer

finally

5 shows

15 in

of the

concentration down

for

record

was

hemoglobin(P).

0

Fig.

chart (0.45

slowed

(measured CPZ(D)

of

hemoglobin

addition and

lo-15

oxygraph

extent

Vol.

167,

of the

No.

BIOCHEMICAL

3, 1990

saturation

binding

release

of

( 8 ) of

oxygen

is

AND

BIOPHYSICAL

hemoglobin

closely

RESEARCH

with

coupled

with

COMMUNICATIONS

CPZ.

This

the

extent

indicates of

that

binding

of

the drug. In

summary in

globin

possible

our

results

give

clear

positive co-operative binding sites. Results

evidences

that

CPZ

binds

with

hemo-

mode and tryptophans are in or near the on oxygen release render the physiological

importance of CPZ binding to hemoglobin understanding the effect of this drug

and might on metabolic

be

of help processes

in further involving

hemoglobin.. ACKNOWLEDGMENTS Authors providing due

to

are grateful to Department of Atomic Energy, Govt. of India for one of us (M. B. ) a Junior Research Fellowship. Thanks are Department, Jadavpur Dr. S. Bera and Sri D. Sen, Chemistry

University, and

to

Physics, Thanks

for Dr.

P. K.

making

available

Sen Gupta

Calcutta, for providing are also due to Prof. A.

of Chemical

Biology

for

their

life-time

and Sm.

decay

M. Sarkar,

measurement

Saha Institute

the Fluorescence measurement Bhaduri and Dr. M. Roy of Indian

cooperation

in oxygen

release

facilities, of Nuclear facility. Institute

measurement.

REFBRENCI3S 1.

May and Baker Ltd. (Dagenham, England) (1971) The Red Book of M and B Medical Products, 9th Edition, pp. 67-75. L. Maher. P., and Singer, S.J. (1984) Biochemistry 23, 232-240. 3. Salesse, R., Garnier, J., Letterrier, F., Daveloose, D., and Viret. J. (1962) Biochemistry 21, 1581-1586. 4. Yamaguchi, T., Watanabe, S., and Kimoto, E. (1985) Biochim. Biophys. Acta 820, 157-164. 3. De Mol, N.J., Interaction 52, and Busker, R. W. (1984) Chem. Biol, 79-92. 6. Riggs, A. (1981) In Methods in Enzymology (E. Anatonini, L. Rossi-8ernardi. and E. Chiancoxe, Eds. ). Vol. 76. pp. 5-29, Academic Press. (1989) 7. Bhattacharyya, M., Chaudhuri, U., and Poddar, R.K. Biochemistry International 19, 1363-1372. 8. Di Iorio. E.E. (1981) In Methods in Enzymology (E. Anatonini, L. Rossi-Bernardi, and E. Chiancoxe, Eds, ). Vol. 76, pp. 68-72, Academic Press, Y. In Best and Taylor’s Physiological Basis of Medical Practices (1985) 11th edition (J.B. West, Ed.). Chapter 24, pp. 390-408. Williams and

Wilkins.

and Redfield, A.G. (1976) J. Biol.Chem. 251, 7114-7119. 10 Huang. T.H., 11 Das. G.C., Das Gupta, S., and Das Gupta. N.N. (1974) Biochim. Biophys. Acta 353, 274-278. 12. Hill, A.V. (1910) J. Physiol. 40, 4-10. (1983) In Principles of Fluorescence Spectroscopy. 13. Lakowicz. J.R. Chapter 9, pp. 260-270. Plenum Press, N.Y. (1978) In Fundamentals of Photochemistry. 14. Rohatgi-Mukherjee , K, K. Section 6. pp. 165-210, Wiley Eastern Ltd.

1153

Evidence for cooperative binding of chlorpromazine with hemoglobin: equilibrium dialysis, fluorescence quenching and oxygen release study.

Binding of chlorpromazine (CPZ) with human hemoglobin has been studied by equilibrium dialysis and fluorescence quenching. Results of equilibrium dial...
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