Structural requirements for steroid binding and quenching of albumin fluorescence in bovine plasma albumin.

1. Steroids interact with bovine plasma albumin at a binding region that involves tryptophanyl, tyrosyl, arginyl and lysyl residues. The function of the tryptophanyl residues is demonstrated by: (a) the decrease of albumin binding affinity after modification of one tryptophanyl with 2-nitrophenylsulfenyl chloride; (b) steroid quenching of albumin tryptophanyl fluorescence; and (c) steroid quenching of 1-anilinonaphth-alene-8-sulfonate fluorescence, when it is excited by energy transfer from excited tryptophanyls. The function of tyrosyl residues is demonstrated by the decrease of albumin binding affinity after nitration of 30% tyrosyls with tetranitromethane, or deprotonation of tyrosyls by variation of pH. The function of arginyl and lysyl residues is demonstrated by the decrease of binding affinity after modification of these residues with glyoxal, formaldehyde or acetic anhydride. The presence of both apolar (Trp, Tyr and Lys (deprotonated)) and polar (Arg and Lys(protonated)) residues at the steroid binding site fits in well with the site relative apolarity, when expressed on the Kosower scale (Kosower, E.M. (1958) J. Am. Chem. Soc. 80, 3253-3260). 2. The contribution of specific amino acid residues to steroid binding depends to some extent on the steroid structure, as exemplified by the quantitatively different role of arginyl (or lysyl) residues in albumin interaction with testosterone acetate and epitestosterone, respectively, or that of tyrosyl residues in albumin interaction with 11-deoxycorticosterone and epitestosterone, respectively. 3. The concerted action of polar and apolar amino acid residues is an essential requirement for steroid binding, since unfolding of albumin polypeptide chain by guanidine-HC1, urea, or by reduction of disulfide bridges with 2-mercaptoethanol, strongly decreases steroid binding to albumin while, conversely, reoxidation and refolding of the unfolded polypeptide chain restore albumin affinity for steroids. 4. Parallel determinations of steroid binding constants by equilibrium dialysis and fluorimetric titration, as well as the general pattern of the pH and temperature effects on steroid quenching of albumin fluorescence, confirm the validity of the fluorescence quenching titration as an effective method for measuring albumin-steroid molecular interactions.