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.

Steroid-protein interactions. 40. The effect of fatty acids on progesterone binding to human serum albumin.

Human serum albumin was delipidated by solvent extraction or by treatment with charcoal. Progesterone complexes formed with these albumin preparations had higher association constants than those formed with the untreated samples. The charcoal method of delipidation resulted in somewhat higher affinity constants than extraction with chloroform/methanol. Addition of 5 mol lauric acid per mol albumin reduced the association constant of the progesterone complex by approx. 50%. Studies with lauric, myristic, and palmitic acid showed that the decrease of binding affinity for progesterone was proportional to the amount of fatty acid added to albumin, and to its chain length. These results confirm and extend our previous findings of inhibition of progesterone binding to human albumin by long-chain fatty acids.

Specificity and stability of guinea pig anti-progesterone antibodies.

Antibodies against progesterone were induced in guinea pigs of both sexes by injection of progesterone-y beta-hemisuccinate conjugated to bovine serum albumin (BSA) in a ratio of 16 moles of steroid per mole of protein. The concentration of antibody binding sites for progesterone of the animals studied ranged from 5 to 20 microM. The expected heterogeneity of binding affinity for progesterone was observed with two major populations apparently predominating. On bound progesterone with an average affinity greater than 2 X 10(9) M-1 and the other showed an average affinity less than or equal to 6 X 10(6) M-1. The antibodies were fond to be stable to extremes of pH and temperature in serum as well as in solutions of ammonium sulfate precipitates. The antibodies were not stable, however, in a more highly purified form. Attempts to obtain active preparations in high yield by purification beyond the ammonium sulfate step were unsuccessful. Competition studies and direct analysis with radiolabeled steroids showed the high-affinity population to be relatively specific for progesterone binding, whereas other steroids were bound according to the polarity rule indicating that the binding forces are predominantly hydrophobic.

Chromophore-protein interaction controls the complexity of the phytochrome photocycle.

A new protocol for the preparation of recombinant phytochromes results in significantly higher yields which, for the first time, have made kinetic studies possible. Flash photolysis with nanosecond laser excitation reveals that, in recombinant and native phytochromes, the decay kinetics of the primary photoproducts I700i and the kinetics of the formation of the Pfr form are similar. Phycocyanobilin-containing recombinant phytochrome, however, shows only a monoexponential decay of the I700 intermediate with a time constant of approximately 90 microseconds, and a biexponential formation of the Pfr form, albeit with time constants (approximately 13 and 100 ms) somewhat shorter than those from native phytochrome. Thus the seemingly small structural modification of the chromophore (substitution of the native vinyl for an ethyl group) has a profound influence on the availability of protein conformational rearrangement pathways. The result is therefore of general interest in chromoprotein dynamics.