Reoxidation of reduced bovine growth hormone from a stable secondary structure

• Published in: Biochemistry (Easton) Vol. 25; no. 22; pp. 6907 - 6917
• Main Authors: Holzman, Thomas F, Brems, David N, Dougherty, John J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 04.11.1986
• Subjects: Animals; Biological and medical sciences; Cattle; disulfide bonds; Drug Stability; Fundamental and applied biological sciences. Psychology; growth hormone; Growth Hormone - metabolism; Kinetics; Molecular biophysics; Oxidation-Reduction; Protein Conformation; secondary structure; Spectrophotometry, Ultraviolet; Structure in molecular biology; Tridimensional structure; Tyrosine; Refolding; STH; Ox; Secondary structure; Protein hormone; Vertebrata; Mammalia; Disulfide bond; Reduced state; Oxidation; Artiodactyla; Ungulata; Conformational transition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00370a026

In order to determine solution conditions appropriate for reoxidizing reduced bovine growth hormone (bGH), we have examined the possibility of using a particular denaturant concentration to poise the secondary and tertiary structure of the reduced protein in a stable, nativelike state. It was envisioned that the structure of the reduced molecule would differ from that of the final oxidized molecule solely by the absence of disulfide bonds. Dilution of concentrated samples of reduced and unfolded protein from 6.0 M guanidine into 4.5 M urea followed by air oxidation indicated it was possible to induce refolding and reoxidation to an oxidized monomeric species in high yield (approximately 90%). The choice of solution conditions was based on comparison of urea equilibrium denaturation data for native oxidized protein to those for completely reduced protein and to protein in which sulfhydryl groups had been either partially or completely reduced and subjected to modification with iodoacetamide or methyl methanethiolsulfonate. The denaturation behavior of these species supports the existence of equilibrium folding intermediates for bovine growth hormone and demonstrates that chemical modification of the protein is capable of inducing differences in the denaturation behavior of these intermediates. The changes in the protein absorption spectrum and helix-related circular dichroism signal, along with direct titration of protein sulfhydryl groups, indicated that the refolding/reoxidation of bGH is a multistate process. The ordered nature of the kinetic changes in these probes during reoxidation indicates that disulfide formation is a sequential process, with little mispairing in 4.5 M urea, and that it proceeds through one or more obligatory kinetic folding events. The equilibrium denaturation behavior of the oxidized molecule and the various chemically modified forms, together with the reoxidation data, indicated that the protein maintains a high degree of secondary structure without intrachain disulfide bonds. The formation of these disulfide bonds is a discrete process which occurs after a framework of protein secondary structure is established.