Manipulation of the observed kinetic phases in the refolding of denatured ferricytochromes c.
The refolding of guanidine hydrochloride-denatured horse heart ferricytochrome c at pH 7.0 and 23 degrees C occurs in three kinetic phases as observed by stopped flow measurements using changes in Soret absorbance or in tryptophan fluorescence. The three kinetic phases have time constants of 10 +/- 5 ms, 240 +/- 30 ms, and 13 +/- 3 s accounting for 15 +/- 5%, 70 +/- 5%, and 15 +/- 5% of the total reaction, respectively. The intermediate kinetic phase can be selectively eliminated by conducting the refolding measurements at pH 5.0. Both the intermediate and slow kinetic phases can be eliminated by conducting the refolding measurements either at pH 7.0 or at pH 5.0 in the presence of an excess of an extrinsic ligand for an axial position of the heme iron. Similar results are obtained using tuna heart ferricytochrome c except that the fractional reaction in the fast and intermediate kinetic phases at pH 7.0 in the absence of extrinsic ligand are 29 +/- 2% and 58 +/- 2%, respectively. We suggest that both the intermediate and slow kinetic phases are generated by proline peptide isomerization occurring during and prior to the refolding procedure, respectively, and that their occurrence is dependent upon the conformation of the denatured protein and upon the ligation of methionine 80 in the folded product, respectively.
D000818:Animals; D002175:Candida; D002642:Chick Embryo; D003574:Cytochrome c Group / Q000378:metabolism*; D005399:Fishes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae; D013045:Species Specificity; D014413:Tuna
D003574:Cytochrome c Group; D006146:Guanidines; D019791:Guanidine