A fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid

Avidin and biotin can be sensitively and accurately quantitated using the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS). In the presence of avidin, the fluorescence of 2,6-ANS is blue shifted with a large increase in quantum yield. Biotin binding causes complete displacement of the bound fluorophore with concomitant quenching of the fluorescence. The fluorometric monitoring of the displacement of 2,6-ANS can be used as a facile method of measuring the biotin-avidin interaction. 2,6-ANS displacement gives the same stoichiometry as the method using 4'-hydroxyazobenzene-2-carboxylic acid. Our initial studies of an affinity-purified avidin revealed that, of the four binding sites on the avidin tetramer, a mean of three remain available for biotin (or dye) binding; this finding highlights a caveat concerning the use of affinity-purified oligomeric-binding proteins with multiple sites. As compared with previous fluorescence methods, the use of 2,6-ANS gives high sensitivity without the necessity of preparing and purifying a covalent avidin conjugate. In addition, the present method;:is potentially more sensitive than those based on optical absorbance; uses a probe that has increased stability and a larger Stokes shift compared with fluorescein; is not subject to protein interference; and gives accurate results over a wide range of 2,6-ANS and avidin concentrations.     

Differential binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid to rhodanese catalytic intermediates

Studies have been performed to quantitate the binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid (2,8-ANS) to catalytic intermediates of the enzyme rhodanese: the sulfur-substituted form (ES) and the sulfur-free form (E). The molecular 2,8-ANS has not been extensively used for protein studies, and some characterization is presented to demonstrate its usefulness as a probe for apolar binding sites. The molecule 2,8-ANS binds to at least two classes of sites on rhodanese. One class (class 1) is present in the ES form and has a Kd of 1.7 mM. The E form of rhodanese appears to have a second class of sites (class 2) in addition to the class 1 sites. Two independent fluorometric methods of analyzing the class 2 binding of 2,8-ANS to the E form gave an average value for Kd congruent to 179 microM. These fluorometric titrations, together with a Job plot, clearly indicate that 2,8-ANS binds to more than one site on the E form of rhodanese. The apparent apolarity is slightly higher for class 2 sites than for the class 1 sites, but both give Z factors of greater than 85. The substrate thiosulfate is able to displace the probe that is bound to the class 2 sites on the E form of the enzyme. Further, 2,8-ANS is found to be a competitive inhibitor of the catalyzed reaction with an apparent Kd of 170 microM. Circular dichroism measurements detect no significant changes in the average conformation of rhodanese that can be ascribed to the presence of 2,8-ANS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis(8-anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 microM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 degrees C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.     

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.     

Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.     

Quantitative aspects of the development of a hydrophobic binding site on calmodulin by calcium binding

The interactive binding by calmodulin of Ca²⁺ and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been examined. In the presence of saturating levels of Ca²⁺, calmodulin develops one moderately strong binding site for 1,8-ANS, plus one or more weaker sites. The binding of 1,8-ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca²⁺ ions. Little further change occurs on binding additional Ca²⁺ ions.     

Alignment of anilinonaphthalene-sulfonate and related fluorescent probe molecules in squid axon membrane and in synthetic polymers

As a fluorescent probe for the squid axon membrane, the behavior of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) was found to be very different from that of its positional isomer, 2,6-ANS, or of the methylated derivative, 2,6-TNS. The degree of polarization of the fluorescent light contributing to a transient intensity reduction during nerve excitation was larger than about 0.7 for both 2,6-ANS and 2,6-TNS, while the corresponding value for 1,8-ANS in a squid axon was about 0.35.The physicochemical basis of this difference was investigated by measuring the fluorescence polarization of these probe molecules incorporated into poly(vinyl alcohol) sheets. In a stretched sheet of this synthetic polymer, 1,8-ANS showed poor alignment, while the 2,6-derivatives were highly oriented with their transition moments aligned approximately in the direction of stretching. Based on these findings, the experimental results obtained from squid axons were interpreted as an indication of the existence, at or near the membrane, of a longitudinally oriented macromolecular structure, bringing about a high degree of alignment of 2,6-ANS or 2,6-TNS molecules.It is clear that, as a probe for fluorescence polarization studies of macromolecular structures, 2,6-TNS is far superior to 1,8-ANS.     

Tetracyanonickelate probes the active site of sulfur-free rhodanese

Tetracyanonickelate (Ni(CN)4(2-)) was used as a probe for the active site of sulfur-free rhodanese (E) in physical and kinetic studies. Ni(CN)4(2-) quenches the intrinsic fluorescence as well as the fluorescence of enzyme-bound 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS), an inhibitor that is competitive with respect to thiosulfate. A facile binding method based on centrifugation was developed to study Ni(CN)4(2-) binding to E. Binding studies performed using either of the electrophoretic variants A and B, fractionated by DE52 column chromatography, showed one high affinity Ni(CN)4(2-)-binding site in each species and additional weak sites on the more electropositive form A. The high affinity Ni(CN)4(2-) binding was corroborated by ultrafiltration binding (Kd = 3.95 +/- 0.35 microM), titration of intrinsic fluorescence (Kd = 1.8 +/- 0.11 microM), and displacement of enzyme-bound 2,8-ANS (Kd = 1.9 +/- 1.1 microM). A nonlinear least squares analysis of kinetic data collected under conditions used for the binding studies gave a Ni(CN)4(2-) inhibition constant of 21 microM. It is concluded that Ni(CN)4(2-) binds to sulfur-free rhodanese in solution near the active site as has been shown in x-ray crystal studies (Lijk, L. J., Kalk, K. H., Brandenburger, N. P., and Hol, W. G. J. (1983) Biochemistry 22, 2952-2957). In keeping with recent suggestions that the conformational state of the enzyme is dynamically determined, the discrepancy between Ni(CN)4(2-) affinity as determined by physical methods and that by kinetic methods suggests that Ni(CN)4(2-) may be able to distinguish the conformation of the working enzyme from those of the idle forms.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

A comparative analysis of extrinsic fluorescence in nerve membranes and lipid bilayers

Changes in extrinsic fluorescence intensity, associated with step changes in membrane potential, have been studied in intracellularly or extracellularly stained squid axons, and in lipid bilayers, using six different aminonaphthalene dyes: 1,8-TNS; 2,6-TNS; 1,8-MANS; 2,6-MANS; 2,6-ANS and NPN. In all preparations the optical signals were found to be roughly proportional to the voltage applied. All signals had a very fast initial component, which was followed in some case by a slower change in the same direction. The slow component was observed only in intracellularly stained axons, and not for all chromophores studied. 1,8-TNS, 1,8-MANS and 2,6-MANS yielded the largest fluorescence signals in all preparations. The sign of these signals was independent of the type of membrane studied. However, the fluorescence changes of 2,6-MANS were opposite to those of 1,8-TNS and 1,8 MANS. Staining of both sides of the axolemma with 1,8-MANS or 2,6-MANS showed that these dyes yield larger signals when applied to the extracellular face. The changes in fluorescence light intensity of 2,6-TNS, 2,6-ANS and NPN were smaller and their sign depended on the membrane preparation studied. The comparison of the extrinsic fluorescence signals from the nerve membrane and the phosphatidylcholine bilayer suggests strong similarities between the basic structures of the two systems. The variety of observed signals cannot be easily interpreted in terms of changes in membrane structure. A possible alternative interpretation in terms of electrically induced displacements, rotations and changes in partition coefficient of bound chromophores, is discussed.Abbreviations 1,8-TNS 1-toluidinonaphthalene-8-sulfonate, and similarly, 2,6-TNS - 1,8-MANS 1-N-methylanilinonaphthalene-8-sulfonate, and similarly, 2,6-MANS - 1,8-ANS 1-anilinonaphthalene-8-sulfonate, and similarly, 2,6-ANS - NPN N-phenyl-1-naphthylamine     

Location of a binding site for 1-anilinonaphthalene-8-sulfonate on calmodulin

The quenching by radiationless energy transfer of the ultraviolet fluorescence of Tyr-99 and Tyr-138 by bound 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been employed to determine the separation of a hydrophobic binding site of 1,8-ANS from each of the tyrosines. The results suggest that the dominant binding site is located in the N-terminal region of domain III.     

Bis(1,8-anilinonaphthalenesulfonate). A novel and potent inhibitor of microtubule assembly

Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Dietary cholesterol caused modification in the structure and function of rat hepatic microsomes, studied by fluorescent probes

A 4% cholesterol diet fed to rats for four weeks was found to increase the phospholipid and cholesterol contents and the activities of drug metabolizing enzymes in rat liver microsomes.Microsomes from rats on a high cholesterol diet were able to enhance the fluorescence of membrane bound 1-anilinonaphthalene 8-sulphonate (1,8-ANS) and ethidium bromide more than microsomes from rats on a standard diet.In the case of 1,8-ANS, the enhanced fluorescence was found to be due to the increased affinity of the molecules for microsomes. In the case of ethidium bromide the fluorescence increased partly because of the larger amount of binding sites and partly because of the enhanced quantum yield of the molecules.P-nitrophenol was found to compete with 1,8-ANS for the same binding sites in microsomes. On the other hand, 1,8-ANS lowered the rate of drug metabolism when present in the incubation mixture.In vitro treatments of microsomes with trypsin, phospholipase A or digitonin altered the binding properties of 1,8-ANS and ethidium bromide to microsomes.It is concluded that the binding sites of 1,8-ANS in microsomes are important for the activity of drug-metabolizing enzymes. The mechanisms of dietary cholesterol in enhancing the drug metabolism and the role of microsomal phospholipids in regulating the activity of drug-metabolizing enzymes are discussed.     

Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.     

Fluorescence energy transfer between heterologous active sites of affinity-labeled aspartokinase of Escherichia coli.

The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates. The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling. Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide. Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8. The distance between the fluorescent probe and the quencher was 29 +/- 4 A. In the presence of threonine, this distance increased to 36 +/- 5 A. Threonine binding either increased the intersite distance by ca. 7 A or caused a reorientation of the probe at the dehydrogenase site.     

Probing the adenosine receptor with adenosine and xanthine biotin conjugates

Biotin-containing analogs of a potent agonist (N6-phenyladenosine) and a potent antagonist (1,3-dipropyl-8-phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para-position of the phenyl ring. Two biotin conjugates of N6-phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter-chain derivative was more potent in inhibiting binding of N6-[3H]cyclohexyladenosine to rat cerebral cortical membranes (Ki of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3-dipropyl-8-phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.