Rapid estimation of avidin and streptavidin by £uorescence quenching or £uorescence polarization Gerald Kada a , Karl Kaiser a , Heinz Falk b , Hermann J. Gruber a ; * a Institute of Biophysics, J. Kepler University, Altenberger Str. 69, A-4040 Linz, Austria b Institute of Chemistry, J. Kepler University, Altenberger Str. 69, A-4040 Linz, Austria Received 9 October 1998; received in revised form 18 December 1998; accepted 22 December 1998 Abstract A new biotin^carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000^000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, 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 quenching at 8 nM ligand concentration. Adsorption of (strept)avidin to the assay tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Virtually the same specific response to avidin and streptavidin was also observed with commercial `fluorescein^biotin', except that s 10 h incubation times were required. The slow association of `fluorescein^biotin' was attributed 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 study was `biotin-4-FITC' which was analogous to `biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate residue. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avidin-bound state. In conclusion, the new ligand `biotin-4-fluorescein' appeared to be the most general and convenient probe: quenching was most pronounced and linearly dependent on (strept)avidin concentrations, the dose response for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation time. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Avidin ; Biotin ; Fluorescein ; Fluorescence ; Streptavidin 1. Introduction Avidin and streptavidin are widely used in bio- science and technology [1,2], thus speci¢c measure- ment of (strept)avidin concentrations is frequently needed. Until recently, only radioligand binding methods [3,4] or a solid phase assay with a biotiny- lated marker enzyme [5] were sensitive enough to quantitate (strept)avidin in the low nanomolar and picomolar range. However, the good performance of 0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0304-4165(98)00177-9 Abbreviations : biotin-4-FITC, 5-[2-(biotinoyl)-aminoethylthio- ureidyl]-£uorescein isothiocyanate ; biotin-4-£uorescein, 5-(and 6)-[2-(biotinoyl)-aminoethylaminocarbonyl]-£uorescein ; BSA, bo- vine serum albumin ; EDTA, ethylenediamine-N,N,NP,NP-tetra- acetic acid; `£uorescein^biotin', 5-{[N-(5-{N-[6-(biotinoyl)-ami- no]-hexanoyl}-amino)-pentyl]-thioureidyl}-£uorescein ; (strept)av- idin, streptavidin and/or avidin * Corresponding author. Fax : +43-732-2468-822 ; E-mail : hermann.gruber@jk.uni-linz.ac.at Biochimica et Biophysica Acta 1427 (1999) 44^48