Slow Refolding Kinetics in Yeast Iso-2 Cytochrome c†

John J. Osterhout, Barry T. Nall

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

In refolding of iso-2 cytochrome c from Saccharomyces cerevisiae, there are two slow folding reactions, τ1a and τlb. The slower of the slow reactions, τla = 100-200 s, is observed only by absorbance changes, while τ1b (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 °C), the activation enthalpies are ΔH† = 27 kcal/mol for τla and 21 kcal/mol for τlb. 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 °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 (7alpha;lb) and absorbance-detected (αla) slow folding are independent of guanidine hydrochloride concentration and pH in the initial conditions. Although a full description requires a more complex explanation, many of the properties of the slow folding species are those expected for proline imide bond isomerization.

Original languageEnglish (US)
Pages (from-to)7999-8005
Number of pages7
JournalBiochemistry
Volume24
Issue number27
DOIs
StatePublished - Dec 1 1985
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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