Steroid-protein interactions. XXXIV. Chemical modification of alpha1-acid glycoprotein for characterization of the progesterone binding site.
The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.
D001557:Benzenesulfonates; D001665:Binding Sites; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism*; D003485:Cyanates; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000032:analysis; D009574:Nitro Compounds; D009578:Nitrobenzenes; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000032:analysis
D001557:Benzenesulfonates; D001798:Blood Proteins; D003485:Cyanates; D006023:Glycoproteins; D009574:Nitro Compounds; D009578:Nitrobenzenes; D011956:Receptors, Cell Surface; D014443:Tyrosine; D011374:Progesterone; D008239:Lysine