Regeneration of Bovine Pancreatic Ribonuclease A: Detailed Kinetic Analysis of Two Independent Folding Pathways

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Abstract
The regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 °C, pH 8.0, proceeds through two separate pathways in which separate nativelike three-disulfide species are populated. The populations of these two three-disulfide species during the regeneration process have been monitored directly through the use of a reduction pulse. A detailed kinetic analysis of the regeneration process using improved experimental procedures and data analysis has been carried out to obtain rate constants for disulfide interconversion among the various disulfide-bonded intermediates. This analysis indicates that these two pathways can account for essentially 100% of the native protein regenerated and that the relative amount of native protein regenerated through these two pathways is insensitive to the redox conditions used. These results indicate that the rate-determining step in both pathways involves formation of the nativelike three-disulfide species, a step in which most of the conformational folding takes place. The experimentally determined rate constants indicate that these two pathways are sufficient to explain the differences in the temperature dependence of the regeneration rate with different redox reagents. In addition, the population of a fully oxidized species that contains three native disulfide bonded pairs and a dithiothreitol bridging cysteines 65 and 72 has been observed.