Measurement of the refolding combination reaction between S-peptide and S-protein

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Abstract
S-Peptide combines with S-protein during the refolding of [bovine pancreas] RNase S. The kinetics of combination were now measured by a specific probe, the absorbance (492 nm) of a fluoresceinthiocarbamyl (FTC) group on lysine-7 of S-peptide. pK changes of the FTC group detect both initial combination and later, first-order, stages in folding. Combination with the slow-folding species of S-protein occurs with a half-time of 0.4 s at 50 .mu.M, whereas complete folding takes 50 s (pH 6.8, 31.degree. C). Thus combination takes place at an early stage in folding. The second-order rate constant of the refolding combination reaction (5 .times. 104 M-1 s-1) is 100-fold smaller than that for combination with folded S-protein, which probably reflects the lower affinity of S-protein for S-peptide in the initial complex. Inhibition by S-peptide of combination between FTC-S-peptide and S-protein shows that the refolding combination reaction is specific and reversible. Both the fast-folding and slow-folding species of unfolded S-protein participate in the refolding combination reaction.