Slow refolding kinetics in yeast iso-2 cytochrome c

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Abstract
In refolding of iso-2 cytochrome c from Saccharomyces cerevisiae, there are two slow folding reactions, .tau.1a and .tau.1b. The slower of the slow reactions .tau.1a = 100-200 s, is observed only by absorbance changes, while .tau.1b (10-20-fold faster) is detected by fluorescence changes. The temperature dependence of the rates of these reactions has been measured: for kinetic experiments ending below the folding-unfolding transition zone (pH 7.2, 0.3 M guanidine hydrochloride, 5-30.degree. C), the activation enthalpies are .DELTA.H.dbldag. = 27 kcal/mol for .tau.1a and 21 kcal/mol for .tau.1b. Double-jump (unfolding, then refolding) experiments demonstrate that the two sets of species responsible for the slow folding reactions are generated slowly but at different rates under unfolding conditions (3 M guanidine hydrochloride, pH 7.2, 20.degree. C). Finally, as a test for changes in the population of the slow refolding species under different unfolding conditions, the amplitudes for slow refolding have been measured as a function of the initial unfolding conditions with the final refolding conditions held constant. Over the range accessible to measurement in the absence of interference from other reactions, the amplitudes for fluorescence-detected (.alpha.1b) and absorbance-detected (.alpha.1a) slow folding are independent of guanidine hydrochloride concentration and pH in the initial conditions. Although a full description requires a more complex explanation, may of the properties of the slow folding species are those expected for proline imide bond isomerization.