Spin assay as a general method for studying plasma protein binding. Bilirubin — Albumin binding

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SPIN ASSAY AS A GENERAL METHOD FOR STUDYING PLASMA PROTEIN BINDING. BILIRUBIN

D.J.

- ALBUMIN BINDING

Wood and J.C.

Hsia

Department of Pharmacology Faculty of Medicine University of Toronto Toronto, Canada M5S lA8

Received

April

22,1977

Summary. The binding of a spin label to pre-existing binding sites of a protein can be monitored by electron spin resonance spectroscopy and can be interpreted in terms of a spin assay for any ligand which shares these sites. As an example of this procedure, a method is presented which evaluates the reserve capacity of the bilirubin high affinity binding site of human serum albumin for additional bilirubin binding. Hyperbilirubinemia in pre-mature or low-birth-weight term infants can result in severe neurological damage. The maturation of this technique will provide a needed clinical assay in the management of jaundiced newborn infants. Hyperbilirubinemia can result

in

and in severe the

cases,

(1,2).

to the

site

% 10 a

(4);

capacity for

the

high

There

Copyright All rights

and hold,

uptake

into

site.

This

is is

site

site

is

and the with

and thus

the

binding

reserve

generally

termed

the

produced

by

blood albumin

one high

(HSA)

affinity

sites

(Ka s 10 5 litres/

taken

as a measure

much greater

secondary

bilirubin

the tissues

retardation,

circulating

to involve

due to its

tissues

1) is

human serum

of secondary

bilirubin

affinity

serum,

neonates

and motor

in the

thought

affinity

to the body

detoxification

affinity

capacity

for

in the

of the high

loss

detoxified

and number

of the high

available

is

or premature

(see Figure

to HSA is

litres/mole)

compared

bilirubin

Bilirubin

protein

of bilirubin

of HSA to bind,

The saturation

for

predominant

capacity

bilirubin,

being

death

of haem and normally

The binding

mole)

low-birth-weight

damage such as hearing

(3).

(Ka

in

neurological

catabolism

by binding

(jaundice)

sites

results the

reserve

affinity

(5).

in bilirubin

critical

capacity

of the

parameter

of the bilirubin bilirubin

binding

(RBBC). have been a number

0 I977 by Academic Press, Inc. of reproduction in an\’ form reserved.

of methods

proposed

to measure

the bilirubin

863 ISSM 0006-291X

Vol. 76, No. 3, 1977

available

for

affinity

bilirubin

BIOCHEMICAL

binding

to the tissues binding

index'(13-17).

experimentally

In addition,

optical

amount

in simulated

due to the

species. the

Also,

observed

Because

the

reserve

capacity

a spin affinity

probe

binding

are

application,

is

Figure free

separating are

distinct

calibration itself,

a detectable read,

The spin

1) so that

the

free

that

probe

the

label

from bound

and their free

can be easily with

standards

the

of the augment

spectral

understood

using

to measure

electron

technique.

spin

We propose

capacity

that

of the high

quantifiable. esr

contains

The spectra

to be

a method

method

can be tailor-made

can be determined

peak height

site

to the

compound

label.

that

here

the

of

can variably

and poorly

sense

is paramagnetic.

concentrations

straightforward,

of determining for

a nitroxyl

The amount without of the

moiety of label

which

physically free

determined (22,23).

a particular

and bound through The esr

labels a

instrument

requiring

concentration

sample-out"

label

samples

suggested

be easily

concentration

state

labeling

advantages

to macromolecule

of the is

of unique

only

the

and yet

the

to quantitate

to the binding

affinity

will

of serum.

tend

we present

which

volumes

required

complicated

spin

(4,7,18-20)

measurements

the

been

high

and the

site

requiring

or bound

a very

can be designed

parameters.

in

difficulties,

(esr)

a number

spectrum

has recently are

binding

These

species

of the bilirubin

bilirubin There

It

of these

spectroscopy label

other

of bilirubin

(21).

studies.

an "RBBC

criticized

large

usually

by the high

evaluate

or to measure

of the

from

which

require are

present

binding

absorbances.

area

resonance

is

insensitivity

spectra

characteristics

(see

which

contained

have been

and/or

measurements

RESEARCH COMMUNICATIONS

not

methods

procedures

complex

density

(i.e.

(6-12)

and also

of these

of bilirubin

any dyes used complex

site)

A number

and many are

the

AND BIOPHYSICAL

range

no day-to-day adjustments, lo-25 ul samples, -7 -6 down to 10 - 10 Molar and a 'sample-in,

procedure. MATERIALS

AND METHODS

Fraction V human serum albumin was obtained from the Sigma Chemical Co., St. Louis, MO., U.S.A. Bilirubin was also obtained from Sigma and further purified by the method of Fog (24). All procedures involving bilirubin were

864

BIOCHEMICAL

Vol. 76, No. 3, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

COOH

HOOC

BILIRUBIN

HOOC-CCH-C-COOH

TOPS

Figure

1

Structural formula oxyl&piperidylidene

of bilirubin succinic

and 2,2,6,6, acid (TOPS)

- tetramethyl-l-

A spin labeled probe, 2,2,6,6-tetramethylcarried-out in reduced light. l-oxyl-4-piperidylidene succinic acid (TOPS , see Figure 1) was synthesized by the method of Rozantsev (25). The experimental samples (100 ul) were prepared by the combination of aqueous stock solutions of TOPS, HSA and bilirubin disodium salt with a 50 ul Hamilton syringe. After vortex mixing, 25 ul of the sample was transfered to a 25 ul Micro et disposable pipette (Becton, which was used for the esr Dickinson and Company, Parsippany, N.J., T&-F The esr spectra of these samples consists of a sharp first-derivative sample. triplet due to the TOPS which is not protein bound, superimposed on a number of broad resonances near the baseline due to the TOPS which is bound to the The concentration of the TOPS which is free in solution is protein (22). proportional to the height of the high-field peak of the triplet and the proportionality can be determined through the use of standards of TOPS in 100 mM phosphate buffer. Since the total amount of added TOPS is known, the concentration of TOPS that is bound to HSA can easily be determined. The esr spectra were recorded on a Varian E6 X-band esr spectrometer at 20 t 1'C. The use of the thin capillary disposable pipettes and a variable temperature Dewar insert (J.F. Scanlon Co.) permits the recording of an entire series of samples without the need to change instrument conditions. The estimated error in these measurements under these conditions is less than 1%. Including pipetting and the other facets of sample preparation, the estimated accuracy of these measurement is 2 3%. RESULTS AND DISCUSSION TOPS was chosen similarity it

is

specific

with readily binding

for

this

bilirubin. water

soluble.

of the label

study

because

since

Furthermore, This

of the

considerably

to HSA.

865

dicarboxylic

TOPS is reduces

a small

acid polar

secondary

structural molecule,

and non-

BIOCHEMICAL

Vol. 76, No. 3, 1977

0

AND BIOPHYSICAL

10

5

2

The binding of TOPS and phosphate buffer at pH added TOPS, [TOPS] is bound [TOPSIP, for*Aodel equivalents (bilirubin of bilirubin (A ).

The proposal

that

2.

These

(not

shown)

are

with

an association

Figure

primarily

is

the shared

consistent constant

total

protein. with

Figure

constants

of bilirubin

interpretation

one,

binding

is

illustrates addition

and TOPS, this between

bilirubin

curve

of bilirubin

(bilirubin

given

displaced.

in Figure

the

the

The addition

to HSA) results This

in all

suggests

that

866

but not

label

site(s)

differing

affinity

expected.

and TOPS for

3.

of the

of bilirubin

same sites

of TOPS to HSA of one to one (27)) is more clearly

displacement

For TOPS

TOPS binding

Considering is

is

one.

binding

equivalents

inhibition

in

to albumin

binding

of around

the

given

TOPS binds

a partition

of 2.3

data

of the binding

and this

that

of TOPS to HSA.

(26) that

litres/mole

there

by the

analysis

TOPS to HSA ratios

as the

The competition

of TOPS being

the

2 also

bilirubin

inhibits

a solution

than

confirmed

a Scatchard

of 2, lo3

much greater

completely

(for

with

for

to HSA is

with

)

albumin is model sera (5 x 10 -4 HSA in 100 mM of 7.4). Total molar concentration of plotted against the molar concentration of sera with no bilirubin ( n ), with 1.1 to HSA) of bilirubin (W) and 2.3 equivalents

TOPS binds along

to one site

to HSA ratios with

data

20

15 [ M x lo4

[ TOPS],

Figure

RESEARCH COMMUNICATIONS

on HSA seen in the

of % 1.4 equivalents a four only

are

percent the

background

same sites

BIOCHEMICAL

Vol. 76, No. 3, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

.

[ Bilirubin],/[HSA]

Figure

3

Displacem nt of TOPS from HSA by bilirubin. The HSA concentration . 100 mM phosphate 4.7 x lo- EM in buffer at a pH of 7.4. The TOPS concentration is 5 x 10-4M. The square brackets denote molar concentrations.

shared

butthe

site.

Extrapolations

intersect

primary

site

of the

linear

at a bilirubin

can be interpreted to its

shared

high site.

binding

of TOPS to HSA is is

affinity

site

(free

that

the

first

four

high

sensing

and since

the

molecules

investigation

for

at the

an RBBC assay

present

curve This

equivalent

affinity

capacity

behaviour of bilirubin

of TOPS from binding

background

binding,

binding

the

site.

of the bilirubin

we can accurately

of TOPS, TOPS and molecules

probe

one.

exclusion

percent

binding

displacement

added the

to the bilirubin TOPS is

bilirubin

of approximately

of the

for

affinity of the

and consequently,

except

on albumin

or bound)

suitable this

Hence,

apparent

portions

binding

site

to this

It

the high

to HSA ratio

as the

affinity

is

follow

of similar of newborn

the binding

structure sera.

high

will

state be

We are involved

time.

ACKNOWLEDGEMENTS Financial is Dr. T.E.

support

gratefully

acknowledged.

G.W. Chance Eagles

preparation

from the

for

of the Hospital many helpful

Ontario

We would for

Ministry also

Sick

discussions

like

of Health to express

Children, and to Dr.

of TOPS. 867

is

Toronto,

(Grant our

gratitude

Canada

R. Schwenk

#PR-524)

for

and Dr. the

to

in

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. lg. 20. 21. 22. 23. 24. 25. 26. 27.

Cowger, M.L. (1971) Biochem. Med., 2, 1-1.6. Amanullah, A. (1976) Am. J. of Diseases of Children, z, 1274-1280. Odell, G.B., Storey, G.N.B., and Rosenberg, L.A. (1970) J. of Pediatr., 76, 12-21. Jacobsen, J., and Wennberg, R.P. (1974) Clin. Chem,,z, 78X-789. Wosilait, W.D. (1974) Life Sci., I&, 2189-2198. Jirsova, V., Jirsa, M., Heringova, A., Koldovsky, O., and Weichova, J. (1967) Biol. Neonatorum, 11, 204-208. Chan, G., Schiff, D., and Stern, L. (1971) Clin. Biochem., &, 208-214. Kapitulnik, J., Vdaes, T., Kaufman, N.A., and Blondheim, S.H. (1974) Arch. Dis. Childhood, 49, 886-894. Chunga, F., and Lardinois, R. (1971) Acta Paedit. Stand., 60, 27-32. Pays, M., and Beljean, M. (1975) Clin. Chim. Acta, z, 121-128. Athanassidis, S., Chopra, D.R., Fisher, M.A., and McKenna, J. (1974) J. Lab. Clin. Meth., 83, 968-976. Bratlid, D. (1972) Stand. J. Clin. Lab. Invest., 3, 91-97. Odell, G.B., Cohen, S.N., and Kelly, P.C. (1969) J. Pediatr., &, 214-230. McClusky, S.B., Storey, G.N.B., Brown, G.K., More, D.G., and O'Sullivan, W.J. (1975) Clin. Chem., 21, 1638-1643. Lee, K-S., Gartner, L.M., and Zarafu, I. (1975) J. Pediatr., 86, 280-285 Bjerrum, O.J. (1968) Stand. J. Clin. Lab. Invest., 22, 41-48. Porter, E.G., and Waters, W.J. (1966) J. Lab. Clin. Med., 67, 660-668. Lucey, J.F., Valaes, T., and Doxiadis, S.A. (1967) Pediatrics, 2, 867-883. Krasner, J., and Chignell, C.F. (1975) J. Pediatr., 5, lOO8-1009. Levine, R.L. (1976) Clin. Chem., 22, 560-561. Lee, K-S., and Gartner, L.M. (1976) Pediatr. Res., lo, 782-788. Hsia, J.C., Wong, L.T.L., and Kalow, W. (1973) J. Immunol. Meth., 3, 17-24. Copeland, E.S., and DeBaare, L. (1976) Biophys. J., 16, 1245-1255. Fog, J. (1964) Stand. J. Clin. Lab. Invest., l&, 49-5. Rozantsev, E.G. (1970) Free Nitroxyl Radicals, pp. 219-220, Plenum Press, London. Scatchard, G. (1949) Ann. N.Y. Acad. Sci., 2, 660-672. A similar result is obtained for solutions of TOPS to HSA of two to one. This collaborates the interpretation of the one to one displacement data and indicates that an exact stoichiometric ratio of TOPS and HSA is not required for the displacement phenomenon.

868