| Abstract: | Refolding of the reduced pancreatic trypsin inhibitor has been investigated using thiol-disulphide exchange with various disulphide reagents to regenerate the three disulphide bonds. Essentially quantitative renaturation was routinely achieved. The refolded inhibitor was indistinguishable from the original protein in interaction with trypsin and chymotrypsin, electrophoretic mobility, and nature of disulphide bonds.The kinetics of refolding using oxidized dithiothreitol to form the disulphide bonds have been studied in some detail. The renaturation reaction is usually of second-order, being first-order in both inhibitor and disulphide reagent concentrations. A short lag period in the appearance of inhibitor activity and the inhibition of the rate, but not the extent, of renaturation by low levels of reduced dithiothreitol suggest the accumulation of metastable intermediates. In addition, heterogeneity of the refolding reaction is apparent at high concentrations of disulphide reagent, with a fraction of the material being only slowly renatured. |
