DEGRADATION OF RECOMBINANT PORCINE GROWTH-HORMONE IN THE PRESENCE OF GUANIDINE-HYDROCHLORIDE

This study was conducted to investigate the effect of guanidine hydroc hloride (Gdn HCl) on the rate of chemical degradation of recombinant p orcine growth hormone (pGH) at alkaline pH. The protein was incubated in 0.2 M Tris buffer, pH 9 at 37 degrees C containing Gdn HCl at conce ntrations ranging from 0 to 6 M. The conformation of pGH under the dif ferent solution conditions was assessed by circular dichroism spectrop olarimetry. Degradation was monitored by reversed phase HPLC (RP-HPLC) , size exclusion chromatography (SEC) and SDS-PAGE. An associated form of pGH was populated in the presence of 3 and 4 M Gdn HCl, whereas th e protein was completely unfolded in 5 and 6 M Gdn HCL. The apparent d egradation rate constant determined by RP-HPLC increased by a factor o f 18 in the presence of 3 M Gdn HCl and by a factor of five in the pre sence of 6 M Gdn HCL relative to that in buffer alone. RP-HPLC and SEC data suggested that the product distribution also differed between bu ffer and solutions containing Gdn HCl. While the increased rate of deg radation for the denatured state was not unexpected, the dramatic incr ease at an intermediate Gdn HCl concentration was surprising and indic ated the importance of considering chemical instability when proteins are exposed to denaturants. (C) 1998 Published by Elsevier Science B.V . All rights reserved.