Measurement of the refolding combination reaction between S-peptide and S-protein.
S-Peptide combines with S-protein during the refolding of ribonuclease S. The kinetics of combination have now been 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 microM, whereas complete folding takes 50 s (pH 6.8, 31 degrees C). Thus combination takes place at an early stage in folding. The second-order rate constant of the refolding combination reaction (5 X 10(4) 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.
D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D012260:Ribonucleases / Q000378:metabolism*; D013053:Spectrophotometry; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013861:Thiocyanates; C023560:ribonuclease S-peptide; D012260:Ribonucleases; C022542:ribonuclease S; D016650:Fluorescein-5-isothiocyanate