Loss of milk clotting activity when rennin was treated with urea at several concentrations and at different pH values was compared with the change in UV absorption of the protein. The change in absorption at 287 and 293 nm did not correlate with loss in activity, indicating that the loss of enzymic properties was not related to the extent of unfolding of the molecule but was related to the reaction of susceptible groups to the environmental conditions.
Reactions of modifying agents, for example N-bromoacetamide, 2-hydroxy-5-nitrobenzyl bromide and mercuric ions with tryptophan groups and the relationship of these changes to enzyme activity suggested that tryptophan was not specifically associated with enzyme action but was important in maintaining tertiary structure.
Rennin was not inhibited by the pepsin inhibitors p-bromophenacyl bromide and α-diazo-p-bromoacetophenone which suggested that the active site did not possess a reactive carboxyl group as in pepsin. However, the reversible inhibition with 1-cyclohexyl-3(2morpholinoethyl)-carbodiimide metho p-toluenesulphonate together with the results of oxidation by N-bromoacetamide indicated that a tyrosine group or groups was important in enzyme activity.