TRAPPING OF INTERMEDIATES DURING THE REFOLDING OF RECOMBINANT HUMAN EPIDERMAL GROWTH-FACTOR (HEGF) BY CYANYLATION, AND SUBSEQUENT STRUCTURAL ELUCIDATION BY MASS-SPECTROMETRY

Human epidermal growth factor (hEGF) contains 53 amino acids and three disulfide bonds. The unfolded, reduced hEGF is allowed to refold unde r mildly alkaline conditions. The folding is quenched at different tim e points by adjusting the pH to 3.0 with an acetic acid solution of 1- cyano-4-dimethylamino-pyridinium (CDAP) which traps folding intermedia tes via cyanylation of free sulfhydryl groups. The mixture of cyanylat ed intermediates is separated by reversed-phase HPLC; the fractions co llected are identified by mass spectrometry. The disulfide structures of the intermediates are then determined by specific chemical cleavage and mass-mapping by MALDI-MS, a novel approach developed in our labor atory. The procedure of quenching and trapping of disulfide intermedia tes in acidic solution minimizes sulfhydryl-disulfide exchange, and th erefore provides a good measure of folding kinetics and preservation o f intermediate species. Our cyanylation methodology for disulfide mapp ing is simpler; faster, and more sensitive than the more conventional approach. Among 18 folding intermediates isolated and identified at di fferent time points, disulfide structures of seven well-populated inte rmediates, including two non-native isomers with scrambled disulfide s tructures, one 2-disulfide intermediate, and four I-disulfide intermed iates, have been characterized; most of them possess non-native disulf ide structures.