KINETIC-ANALYSIS OF THE FOLDING OF HUMAN GROWTH-HORMONE - INFLUENCE OF DISULFIDE BONDS

We report the results of a stopped-Bow kinetic evaluation of the foldi ng of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine re sidues have been reduced and alkylated to form tetra-S-carbamidomethyl ated hGH in order to elucidate the role of disulfide bonds in the fold ing reaction, Multiple detection techniques were applied to monitor bo th refolding and unfolding processes initiated by guanidine hydrochlor ide concentration jumps. Using far-UV circular dichroism (CD) detectio n to monitor folding of hGH, we find that 70% of the secondary structu re forms in a burst phase occurring within the stopped-flow dead time, Two slower phases were identified in the observable portion of the CD signal. Multiple kinetic phases were resolved when folding was monito red by intrinsic tryptophan fluorescence or near-UV absorbance as prob es of tertiary structure, and the number of time constants required to fit the data depended on the hGH concentration and nature of the dena turant jump. The associated amplitudes also displayed strong dependenc e on the final denaturant concentration. Results obtained from the tet ra-S-carbamidomethylated hGR studies demonstrate that the folding reac tions of hGH are remarkably similar in the presence and absence of the disulfide bonds. Disulfide bond reduction in hGH is proposed to affec t folding primarily by increasing the population of self-associated in termediate states in the folding pathway.