Enzymic hydrolysis of intramolecular complexes for monitoring theophylline in homogeneous competitive protein-binding reactions

Vol. 103, No. 4,198l December

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31, 1981

ENZYMIC HYDROLYSIS OF INTRAMOLECULAR COMPLEXES FOR MONITORING THEOPHYLLINE IN HOMOGENEOUS COMPETITIVE PROTEIN-BINDING REACTIONS Thomas M. Li and John F. Burd Ames Division, Received

November

Miles Laboratories,

Inc. , Elkhart,

IN 46514, USA

2, 1981

SUMMARY A novel approach in the design of fluorogenic substrate-analyte conjugates that can be used in a substrate-labeled fluorescent immunoassay (SLFIA) is described. The new SLFIA uses an enzyme substrate molecule that contains a fluorophore component and a quencher component, separated by a chain containing a bond which can be hydrolyzed by an enzyme. The feasibility of using this approach, in the construction of a fluorophore-quencher-analyte conjugate for monitoring analytes in homogeneous competitive protein binding reactions was demonstrated by using flavin-N6-(6-aminohexyl-theophylline) adenine dinucleotide (FADTheophylline) as the intramolecularly quenched fluorogenic substrate. Hydrolysis of the FAD-theophylline by nucleotide pyrophosphatase yielding FMN and AMP-theophylline restores the fluorescence to the expected level of FMN. Antibody to theophylline, however, inhibits the enzymic hydrolysis, and this inhibition is relieved in competitive binding when theophylline is added.

INTRODUCTION Competitive protein-binding assays for a variety highly

sensitive,

tative

determination

methods allow specific and sensitive

of substances.

competitive

Radioimmunoassay (RIA)

protein-binding

technique for the quanti-

of analytes in biological fluids.

separation step to separate the antibody-bound is referred

is a

to as a heterogeneous method.

RIA requires a

and free fractions

and

The use of radioactive

isotopes requires a special permit and the useful lifetime of an assay kit is limited by the half-life

of radioisotopes.

developing homogeneous non-isotopic

Our laboratory

immunoassays (l-7)

has been

which do not

require a separation step or the use of radioisotopes. 0006-291X/81/241157-09$01.00/0 1157

Copyrighl 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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In this paper, we describe a new homogeneous competitive proteinbinding assay using fluorescence methods assay will be described first,

The general principle of the

followed by a specific example of an assay

for the determination of the concentration

of theophylline,

a widely used

drug for the treatment of asthma. PRINCIPLE OF THE ASSAY METHOD Our assay involves the use of an intramolecularly

quenched fluoro-

genie enzyme substrate molecule that contains a fluorescent

component

and a quencher component, separated by a chain containing a bond which can be hydrolyzed

by an enzyme.

The length of the chain will

be such that it will allow the quencher component of the molecule to contact the fluorescent

component of the molecule for very

quenching of fluorescence (8). can be in close proximity absorbed by the fluorescent

Alternatively,

efficient

the quencher component

with the fluorescent

component.

Energy

component can then be transmitted

to the

acceptor chromophore (the quencher) within 10 to 60 A according to the Forster

type

resonance transfer

For a competitive protein-binding covalently

of electronic

excitation

energy (9).

assay, the enzyme substrate can be

labeled with the analyte ligand on the quencher component of

the molecule.

Enzymic hydrolysis

relief of fluorescence quenching, longer covalently

of the labeled substrate brings about since the quencher component is no

linked to the fluorescent

quenching by direct intramolecular

component and fluorescence

contact or resonance energy transfer

is no longer possible. The general principle intramolecular

of our assay using enzymic hydrolysis

complexes is exemplified

shown in Figure 1 using theophylline enzymic reaction,

hydrolysis

by the following

scheme as

as the analyte of interest.

of the FAD-theophylline

of

In the

by nucleotide

pyrophosphatase yielding FMN and AMP-theophylline

restores the fluores-

cence to the expected level of FMN.

(Ab) to theophylline

When antibody

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REACTION

nucleolidt pyrophosphatase

high fluorescence ANTIBODY

BINDING

REACTION

Ab

+

COMPETITIVE

BINDING

nucltotide pyrophosphalrse -

+

no hydrolysis,

no fiuorescmct

REACTIONS

+Ab

lr

+

4 hto

nucleotidt

Fluorescence

Proportional l0

x

IQ i

Theophyllint

Concentration.

hto :Ab

FIGURE

1.

Schematic illustration assay using enzymic

binds to FAD-theophylline,

the enzymic hydrolysis

in the competitive binding reactions, is relieved when theophylIine MATERIALS

A

of the principle of the theophylIine hydrolysis of intramolecular complex.

the inhibition

is inhibited.

Finally,

of enzymic hydrolysis

is added.

AND METHODS

Figure 2 shows the reaction leading to the synthesis of the FADtheophylline conjugate. The description of the synthesis of this compound has been published elsewhere (3). N6-(6-Aminohexyl)adenine dinucleotide (1) was synthesized according to Morris, et al. (3). The synthesis of 8- (3-carboxypropyl)theophylline Coupling of (2) was base% on the procedures by C . F . Cook, et al. (lo), tEe flavin N - (B-aminohexyl)adenine dinucleotide and 8- (3-carboxypropyl)theophyUine to form the FAD-theophylline (3) conjugate was effected by conversion of 8-(3-carboxypropyl)theophylline to 1,3-dimethyl-1,6,‘7,8tetrahydropyride[ 1,2e]-purine-2,4,9[3H]-trion~ by sublimation (lo), followed by its condensation with the flavin N -(6-aminohexyl)adenine dinucleotide in dimethylsulfoxide (3).

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CH -(CHOH)3-CH2-0-P-0-P-O-CH2 I * I

1

AND

I

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I

OH OH

(3) FIGURE 2.

Synthesis of flavin N6-(6-aminohexyl)adenine dinucleotidetheophyhine conjugate.

Theophylline-BSA immunogen was prepared by coupling the 8-(3carboxypropyl)theophylline to BSA and antibody to theophylline was raised in rabbits immunized with this immunogen as described earlier (4) * Fluorescence was determined on an Aminco-Bowman spectrophotofluorometer at room temperature. Snake venom nucleotide pyrophosphatase (EC 3.6.1.9) was obtained from Sigma Chemical Company and theophylline was from Matheson, Coleman and Bell. FMN concentration was determped _sfectrophotometrically using the extinction coefficient of 12,500 M cm at 445 nm (11). Enzyme specific activity is defined as pmole of FMN formed/min./mg enzyme. A linear calibration curve with relative fluorescence vs. nmole of FMN was first obtained. Hydrolysis of FAD and FAD-theophylline to FMN by the snake venom nucleotide pyrophosphatase was then followed by monitoring increases in fluorescence due to the appearance of FMN (excitation wavelength 445 nm, emission wavelength 525 nm) . Control experiments showed that under the conditions of the assay, the enzyme had no effect on FMN. RESULTS The present theophylline

assay is based on the observation that

adenine, adenosine or adenylic acid are strong quenchers of the fluores-

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cence of the isoalloxazine ring of flavine mononucleotide (FMN).

Addition

of adenine, adenosine or adenylic acid to a solution of FMN results in intermolecular (12).

The fluorescence of the isoalloxazine

dinucleotide very

quenching when the purine is at milhmolar concentratio;l

(FAD)

effective

is only 10%of that observed in FMN.

and is brought

about by intramolecular

isoalloxazine ring with the adenine moiety. hydrolysis

ring in flavin

adenine

Quenching is

contact of the

Nucleotide pyrophosphatase

of FAD into the component nucleotides restores the fluores-

cence to the level of FMN. Conjugation of theophylline

at the N6 position of the adenine ring

of FAD does not alter significantly

the fluorescence properties

of FAD.

The emission maximum is still at 525 nm and the FAD-theophylline very

low fluorescence intensity

has

similar to that of FAD.

Michaelis constants and Vmax values calculated from double reciprocal plots (Table 1) show that the Km value of FAD-theophylline higher than that of FAD.

With FAD-theophylline,

is ten times

the Vmax is decreased

to * 40% of that for FAD. Figure

3 shows the effect

enzymic hydrolysis

of antiserum to theophylline

of FAD-theophylline.

The fluorescence decreases as

a function

of increasing volume of antiserum.

hydrolysis

is specific because normal rabbit

corresponding

decrease in fluorescence.

antiserum to theophylline hydrolysis

on the

This inhibition

of enzymic

serum does not cause a

In another control experiment,

is found to exhibit

no inhibition

of enzymic

of FAD.

Figure 4 shows a typical

dose-response curve.

is present in the reaction mixture the enzymic hydrolysis

When theophylline

in increasing amount, inhibition

is progressively

relieved and a standard curve

with increase in fluorescence as a function of theophylline is obtained.

1161

of

concentration

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TABLE KINETIC

CONSTANTS

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1

OF FAD-THEOPHYLLINE Km $5

Substrate

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AND FAD

Vmax Pmole/min ./mg

FAD-theophylline

5

0.04

FAD

0.5

0.1

Calculated from plots of reciprocal velocities against the reciprocals The assay contained 2 mM Mg and 50 mM of substrate concentrations. N ,N-bis(2-hydroxyethyl)glycine buffer, pH 8.5 in a total volume of 2 ml. T = 23%.

DISCUSSION Our ment

of

immunochemistry homogeneous

determination protein-binding

laboratory

has been

non-isotopic

of analytes assays

0

fluorescent

in biological monitored

by

interested

immunoassays

fluids.

We have

enzymic

hydrolysis

1

1

I

2

4

6

in the

1

6

develop-

for

developed

,

10

Effect of rabbit antiserum to theophylline on the enzymic hydrolysis of FAD-theophylline. Varying levels of antiserum to theophylhne (0) or normal rabbit serum (A) was added to 2.0 ml Bicine buffer, pH 8.5 containing 50 nM FAD-theophylline. At timed intervals, 0.16 units of nucleotide pyrophosphatase was added and mixed. The fluorescence intensity was measured 20 minutes after the addition of enzyme. The fluorescence of cuvettes containing all components except enzyme was subtracted from the appropriate reaction cuvettes.

1162

competitive

of fluorogenic

ul antiserum

FIGURE 3.

the

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I

I 0

FIGURE

4.

substrates analyte

AND

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1

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L

100 200 ng 01 Theophylline

I

4

300 400 per ml (in cuveite)

500

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Effect of theophylline in competitive-binding reactions. Reaction mixtures contained 2.0 ml Bicine buffer, pH 8.5, 8 t.11of antiserum to theophylline, 0.16 unit of snake venom nucleotide pyrophosphatase and theophylline at the indicated levels. At 20 second intervals, 100 t.11of 1 .O pmolar FAD-theophylline was added to the cuvettes Fluorescence was measured 20 and the contents mixed. minutes after the FAD-theophylline addition.

covalently conjugate

coupled should

the free fluorophore

to specific

analytes

,

After

be non-fluorescent.

The

intact

substrate-

enzymic hydrolysis,

should be highly fluorescent.

The use of fluorogenic

substrates in the assay of enzyme systems

is the subject of several review advantages of using fluorogenic

articles

(13-14).

In general,

substrates are great sensitivity

the and the

requirement of minute quantities of enzyme. Two approaches have been described in the design of fluorogenic substrate-analyte

conjugates.

In the first

use of an analyte-fluorophore coupled directly Hydrolysis

cent product.

conjugate (1))

to a dye (umbelliferone)

of the non-fluorescent

approach which involves the an analyte (biotin)

through

is

an ester bond.

ester with an esterase yields a fluores-

The bond of enzymic cleavage is immediately adjacent to

the fluorophore . of the fluorophore

The advantage of this approach includes the conversion from a fluorescent

when the analyte is coupled covalently

1163

form to a non-fluorescent

form

at a specific site on the fluorophore.

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In the second approach (2,4), attached to a fluorophore (therapeutic

drug or specific protein).

is an effective

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an enzyme substrate (galactose) is

(umbelliferone)

the analyte is covalently

RESEARCH

that is coupled with the analyte Before and after enzymic cleavage,

attached to the fluorophore.

fluorescence quencher,

When an analyte

the fluorescence of the fluorophore

may be quenched if (a) the chain separating the analyte and the fluorophore is long and flexible enough so that the fluorophore interact

with each other intramolecularly,

spectral characteristics

and the analyte can

or alternatively

of the fluorophore

and the analyte and their

spatial arrangement permit resonance energy transfer fluorophore

if (b) the

from the excited

to the analyte.

The new approach described herein utilizes a fluorophore-quencheranalyte conjugate. fluorophore

It offers two distinct advantages over the analyte-

conjugate or the substrate-fluorophore-analyte 1.

Since the fluorescence of the fluorophore cularly

conjugate. is intramole-

quenched, the bond of enzymic cleavage can be

far removed from the fluorophore analyte to the fluorophore

and coupling of the

does not have to be restricted

at a specific site on the fluorophore which will affect its fluorescence properties. 2.

After

enzymic cleavage, the fluorophore is no longer

covalently

attached to the quencher or the analyte.

Thus, full expression of the fluorescence properties of the fluorophore is permitted after enzymic hydrolysis.

ACKNOWLEDGEMENT We are indebted to Dr.

Steve Thompson and Dr. Robert Carrico

for valuable discussion and to Dr. Dr.

Robert T. Buckler

theophylline

Robert Carrico,

and Dr. Jim Albarella

conjugate.

1164

Dr. David Morris,

for providing

the FAD-

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REFERENCES

(1)

Burd, J. F., Carrico, R. J., Fetter, M. C., Buckler, R. T., Johnson, R. D., Boguslaski, R. C. and Christner, J. E. (1977) Analytical Biochemistry 77, 56-67.

(2)

Burd, J. F., Wong, R. C. , Feeney, J. E., Carrico, R. J. and Boguslaski, R. C. (1977) Clinical Chemistry 23, 1402-1408.

(3)

Morris, D. L., Ellis, P. B., Carrico, R. J., Yeager, F. M., Schroeder, H. R., Albarella, J. P., and Boguslaski, R. C. (1981), Anal. Chem., 53, 658-665.

(4)

Li, T. M., Benovic, J. L., Buckler, (1981), Clin. Chem. 3, 22-26.

(5)

Boguslaski, R. C., Carrico, R. J., U. S. Patent No. 4,134,792.

(6)

Greenquist, A. C., Walter, B. and Li, T. M. (1981), CIin. Chem., 21, 1614-1617.

(7)

Li, T. M., Benovic, in press.

(8)

Weber, G. (1966) in Flavins and Flavoproteins pp. 15-21, Elsevier, Amsterdam.

(9)

Foster, T. (1951) Fluoreszenz organischer Verbindungen, Vandenhoeck u . Rupprecht, Gottingen.

R. T. and Burd, and Christner,

J. L. and Burd,

J. F.

J. E. (1979))

J. F. (1981), Anal. Biochem., (Slater,

E. C. ed. )

(10)

Cook, C. E. , Twine, M. E. , Myers, M. and Amerson, E. (1976) Res. Commun. Chem. Path. Pharmacol. 13, 497-505.

(11)

Whitby,

(12)

Erlanger, B. F., Borek, F., Beiser, S. M., and Lieberman, S. (1957). J. Biol. Chem. 228, 713-727.

(13)

Kanaoka, Y. (1977) Angew. Chem. Int.

(14)

Coleman, P. L., Latham, H. G. and Shaw, E. N. (1976) Methods in Enzymology XLV (L. Lorand, ed. ) pp. 12-26, Academic Press, New York.

L. G. (1953) Biochem. J. 3,

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