Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization

A new biotin-carboxyfluorescein conjugate has been presented in the accompa nying study (G. Kada et al., Biochim. Biophys Acta 000 (1999) 000-000) whic h contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-flu orescein showed exceptionally fast and tight binding to avidin and streptav idin, and binding was accompanied by strong quenching. In the present study the specific quenching of 'biotin-4-fluorescein' was utilized to measure ( strept)avidin concentrations (0.2-2 nM) by the extent of fluorescence quenc hing at 8 nM ligand concentration. Adsorption of (strept)avidin to the assa y tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Vi rtually the same specific response to avidin and streptavidin was also obse rved with commercial 'fluorescein-biotin'. except that > 10 h incubation ti mes were required. The slow association of 'fluorescein-biotin' was attribu ted to the anti-cooperative binding which is due to the much longer spacer as compared to 'biotin-4-fluorescein'. The third ligand tested in this stud y was 'biotin-4-FITC' which was analogous to 'biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate resi due. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avid in-bound state. In conclusion, the new ligand 'biotin-4-fluorescein' appear ed to be the most general and convenient probe: quenching was most pronounc ed and linearly dependent on (strept)avidin concentrations, the dose respon se for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation tim e. (C) 1999 Elsevier Science B.V. All rights reserved.