A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site.
The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.
Mock
D M
DM
Department of Pediatrics, University of Iowa, Iowa City 52242.
Lankford
G
G
Horowitz
P
P
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D011485:Protein Binding; D012995:Solubility; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D001360:Avidin; C047136:2-anilinonaphthalene-6-sulfonic acid; D001710:Biotin