Conventional biotin-fluorophore conjugates with approximately 14 atom spacers are strongly quenched when bound to avidin or streptavidin, whereas fluorescence becomes insensitive to receptor binding if typical fluorophores are linked to biotin via poly(ethylene glycol) (PEG) chains (Gruber et al., see the second of three papers in this issue). In the present study the antagonism between PEG-PEG repulsion and fluorophore interaction was examined more closely, using biotin-PEG-pyrene conjugates as model compounds. The antagonistic tendencies between hydrophilic PEG chains and hydrophobic pyrene labels were about balanced in the PEG1900 derivative since quenching was approximately 50% in 4:1 complexes with avidin or streptavidin. In contrast, strong quenching and concomitant excimer fluorescence was seen with the biotin-PEG800-pyrene conjugate, providing for a new fluorescence assay to accurately measure avidin and streptavidin concentrations at > or = 40 and > or = 10 nM, respectively. Association/ dissociation kinetics were analyzed from pyrene fluorescence changes, and dissociation constants were deduced. About 3-fold affinities were observed for streptavidin as compared to avidin, and little influence of PEG chain length was seen. All affinities were increased by a factor of approximately 3 when biotin-PEG-tetramethylrhodamine conjugates were used. The observed effect of fluorophore variation upon biotin binding is unexpectedly small; thus, the kinetic/thermodynamic data appear to be representative for biotin-PEG conjugates in general.