THE REFOLDING OF BOVINE TRYPSINOGEN: IDENTIFICATION OF INTERMEDIATES AND THE CHARACTERIZATION AND REFOLDING OF NEOTRYPSINOGEN (SIZE-EXCLUSION HPLC, SERINE PROTEINASE, PROTEIN STRUCTURE)
Abstract
A partial separation and characterization of a large number of trypsinogen refolding intermediates was accomplished using size exclusion high performance liquid chromatography and isoelectric focusing techniques. Intermediates were identified as components with greater apparent molecular weights and more acidic isoelectric points relative to native trypsinogen. These intermediates appeared and subsequently disappeared as they went on to form products. Based on a kinetic analysis of the appearance/disappearance of these intermediates, trypsinogen does not appear to refold by a simple sequential build up of native structure. The large number of conformationally distinct intermediates at early stages of refolding suggests that, at least initially, the refolding is a more random process than that observed for BPTI. Trypsinogen appears to refold by way of a mechanism more closely resembling that of RNase. Two modified forms of trypsinogen called neotrypsinogen were identified in commercial samples. Valine-neotrypsinogen contained a single peptide bond cleavage at Arg 105-Val 106 and Serine-neotrypsinogen contained a cleavage at Lys 131-Ser 132. These two-chain forms of trypsinogen were activatable and showed enzymatic properties similar to the intact zymogen. Subtle differences in their molecular properties allowed them to be purified by HPLC. These autolytic variants of trypsinogen were excellent candidates for studying the refolding of trypsinogen at the domain level. The kinetics of the Serine-neotrypsinogen refolding process support the notion that the two domains of trypsinogen are capable of first refolding independently of each other before interacting and associating properly to form the native zymogen. A comparison of the kinetic data obtained for Serine-neotrypsinogen to that obtained for Threonine-neochymotrypsinogen suggests that members of the pancreatic serine proteinase class of enzymes refold by similar mechanisms.
Degree
Ph.D.
Subject Area
Biochemistry
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