Degradation of recombinant porcine growth hormone in the presence of guanidine hydrochloride

This study was conducted to investigate the effect of guanidine hydrochloride (Gdn HCl) on the rate of chemical degradation of recombinant porcine growth hormone (pGH) at alkaline pH. The protein was incubated in 0.2 M Tris buffer, pH 9 at 37°C containing Gdn HCl at concentrations ranging from 0 to 6 M. The conformation of pGH under the different solution conditions was assessed by circular dichroism spectropolarimetry. 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 the protein was completely unfolded in 5 and 6 M Gdn HCl. The apparent degradation rate constant determined by RP-HPLC increased by a factor of 18 in the presence of 3 M Gdn HCl and by a factor of five in the presence of 6 M Gdn HCl relative to that in buffer alone. RP-HPLC and SEC data suggested that the product distribution also differed between buffer and solutions containing Gdn HCl. While the increased rate of degradation for the denatured state was not unexpected, the dramatic increase at an intermediate Gdn HCl concentration was surprising and indicated the importance of considering chemical instability when proteins are exposed to denaturants. Copyright (C) 1998 Elsevier Science B.V.