Avidin. 5. Quenching of fluorescence by dinitrophenyl groups

Abstract

The positions of the fluorescence maxima of denatured avidin (350 m[mu]), native avidin (338 m[mu]) and the avidin-biotin complex (328 m[mu]) confirmed the conclusion drawn from ultraviolet difference spectra that the polarity of the tryptophan environment decreases on going from denatured avidin, via native avidin, to the avidin-biotin complex. From the relation between extent of dinitrophenylation and fluorescence it was shown that a single DNP group could quench at least 90% of the fluorescence of an avidin molecule. The fluorescence was also quenched when the dinitrophenylhydrazide of biotin was bound. From the shape of the fluorescence-titration curve it was shown that to a first approximation the three binding sites of avidin were independent and combined at random with biotin. The fluorescence of avidin was quenched by bound lipoic acid, as a consequence of its weak absorption band at 330 m[mu].