THE REFOLDING OF CHYMOTRYPSINOGEN A AS THE MIXED DISULFIDE OF GLUTATHIONE AND COMPARISON TO THAT OF THE TRYPSINOGEN MIXED DISULFIDE

WILLIAM GOWER INNES MOORE, Purdue University

Abstract

Pancreatic serine proteinases have several features that are suitable for the study of the refolding of proteins. The sequences, physical and enzymatic properties, and three-dimensional structures of the proteins are known. The amino acid sequences are homologous, and most of the homology is located within the interior of the molecules; thus these proteins are expected to have similar pathways of refolding. In my initial studies of the refolding process, Sepharose-bound trypsinogen was successfully refolded as the mixed disulfide of glutathione. The refolding was compared to the refolding of the corresponding derivative of (beta)-trypsin. The yields and rates of regeneration of the native proteins were identical, suggesting that the two proteins have similar or identical pathways of refolding. Fully reduced chymotrypsinogen was converted to the mixed disulfide of glutathione and refolded. The refolding of the soluble protein had a half-time of 56 min at 4(DEGREES), and a 48% yield. The rates of refolding of chymotrypsinogen were similar to those of trypsinogen between 4(DEGREES) and 20(DEGREES). The proteins were suggested to have a similar pathway of refolding. The temperature dependence gave activation energies of: chymotrypsinogen, 14 kcal mol('-1) and trypsinogen, 15 kcal mol('-1). Proline isomerism was ruled out as the rate-limiting step of refolding. During the refolding of chymotrypsinogen-glutathione mixed disulfide, a mixture of chymotrypsinogen and partially reduced chymotrypsinogen was produced. The modified protein was a minor product, with an 8% overall yield. The rate of formation of the modified protein agreed with the rate of formation of chymotrypsinogen. The two proteins were produced by almost the same pathway except for the production of conformational isomers. The 191-220 disulfide bond of native chymotrypsinogen was reduced and alkylated, and the mixed disulfide was prepared and refolded. The rate of refolding agreed with that of the corresponding derivative of trypsinogen. This was interpreted to mean that the role of the homologous disulfide was the same in the refolding of both proteins.

Degree

Ph.D.

Subject Area

Biochemistry

COinS