The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers.
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.
Chang
Jui-Yoa
JY
Research Center for Protein Chemistry, Institute of Molecular Medicine, Houston, Texas, USA. Rowen.Chang@uth.tmc.edu
Li
Li
L
D000595:Amino Acid Sequence; D001685:Biological Factors / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D013820:Thermolysin / Q000737:chemistry
D001685:Biological Factors; D004220:Disulfides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D007768:Lactalbumin; D013820:Thermolysin