We plan to continue the determination of the amino acid sequence of P. putida steroid isomerase. Large peptides will be generated by partial acid hydrolysis at asp-pro, cyanogen bromide cleavage at methionine and tryptic cleavage after blockade of arginine residues. With adequate amounts of protein in hand, progress on this goal should be rapid. The technique of carboxyl group modification with carbodiimide and cystamine will be tested for general applicability using several unrelated polypeptides. This technique should prove very useful for studies of the role of carboxyl groups in protein function. The steroid binding affinities of asp 38 - amidated derivatives of isomerase will be investigated. It is expected that conversion of the polar side chain of this residue to less polar amide derivatives will have a profound effect on the binding of steroids to the protein. The technique of equilibrium dialysis used by us with native enzyme will be used to measure the strength, and number of binding sites. It is planned to initiate exploratory studies of the interaction of steroids with P. testosteroni isomerase using NMR techniques as a spectroscopic probe of the binding site. A Nicolet 360 MHz FT NMR spectrometer recently acquired on this campus will be used in these studies. Since the molecular weight of isomerase is a relatively low 27,000, the molecule consisting of identical 13,500 MW subunits, fairly good resolution of resonance peaks in the aromatic proton region should be achieved.