The reversibility of inactivation of chymotrypsin.

Previous studies have shown that a number of factors can affect the rate, extent, or reversibility of denaturation. These factors include the nature and concentration of salt present (l-4), the concentration of protein (3, 5), the pH (6), and the nature of the denaturing agent (2). The return of enzymic activity after its loss through a process leading to denaturation is generally regarded as evidence of a return of the protein structure, or at least of that part of it concerned with the enzyme action, to its native state. In the case of chymotrypsin, it is well known that the return of catalytic activity occurs after cooling a hot acid solution (6) or after dilution of a solution in strong urea (7). Yet the reactions involved in such reversion are admittedly complex, and small (even unintentional) variations in experimental conditions may be of importance. The failure of such reversion to occur has been occasionally observed in this laboratory, and was found to be attributable to the presence of a small amount of ammonium sulfate in the system. Therefore, the present study is an attempt to correlate the effect of certain salts that prevent the reversion of denatured chymotrypsin to active enzyme. Such effects were found to require surprisingly small concentrations of some salts, the presence of which, at low levels, might well be disregarded at times. There is, moreover, no compelling reason to regard the reverted protein as necessarily the same as the original. Indeed, in the case studied here there is good evidence to the contrary, at least under the particular experimental conditions used.