The properties of the guanidine hydrochloride induced unfolding transition of iso-2 cytochrome c (iso-2) from Saccharomyces cerevisiae have been investigated by using kinetic and equilibrium techniques and have been compared with previously published studies of horse cytochrome c, which differs from iso-2 by 46% in amino acid sequence. Measurements of absorbance in the ultraviolet and visible spectral regions as a function of guanidine hydrochloride concentration give superimposable equilibrium transition curves with a midpoint of 1.15 M at pH 7.2 and 20 degrees C. A two-state analysis of the equilibrium data gives a Gibbs free energy of unfolding of 3.1 kcal/mol at 20 degrees C in the absence of denaturant. This agrees well with the predicted difference in stability between S. cerevisiae iso-2 and horse cytochrome c estimated from the free energies of transfer of buried hydrophobic groups. Three kinetic phases associated with folding can be detected throughout most of the transition zone. Two of the phases are detected by stopped-flow mixing experiments. The third phase is over within the mixing time of the flow experiments but is detectable by temperature jumps. At 20 degrees C, pH 7.2, the slowest phase (T1) is in the 20-100-s time range, the middle phase (T2) is in the 0.1-3-s range, and the fastest phase (T3) is on the order of 1 ms. For the reactions observed in the stopped flow (T1 and T2), a simplified three-state mechanism can be used to predict quantitatively the relative amplitudes of the phases and the equilibrium unfolding curve from the observed time constant data. Previously this same mechanism has been successful in describing the folding reactions of horse cytochrome c [Hagerman, P. J. (1977) Biopolymers 16, 731]. We suggest that the qualitative features of protein folding reactions may be conserved among homologous proteins.

• PMID: 6271187
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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Nall BT, Landers TA

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