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The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers
Authors:Chang Jui-Yoa  Li Li
Affiliation:(1) Research Center for Protein Chemistry, Institute of Molecular Medicine, 2121 W. Holcombe Blvd.;(2) Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas, 77030
Abstract:The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys6-Cys14, Cys20-Cys31, and Cys33-Cys42. The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4–5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.
Keywords:EGF  urea  GdmCl  GdmSCN  denaturation  thermal denaturation  unfolding  unfolding intermediates  denaturation curves  scrambled EGF
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