Mode of inhibition of chymotrypsin by diisopropyl fluorophosphate; introduction of phosphorus.

It has been previously shown that the esterase and proteinase activities of trypsin and chymotrypsin (1, 2) were equally inhibited by diisopropyl fluorophosphate (DFP) (3). With chymotrypsin, 50 per cent inhibition occurred when 0.55 mole of inhibitor per mole of enzyme1 was used at pH 7.7. On the basis of a linear relationship between the-per cent inhibition and the concentration of DFP, 1.1 moles of inhibitor would be needed for complete inhibition. Crystalline DFP-inhibited chymotrypsin was obtained by the reaction of DFP slightly in excess of 1 mole to 1 mole of enzyme. This reaction product remained completely inactive either as an esterase or proteinase after recrystallization, dialysis, and lyophilization. Furthermore, it did not inhibit active chymotrypsin when mixed therewith. On the other hand, DFP was without effect on crystalline chymotrypsinogen. After treatment with DFP, this zymogen could again be crystallized and converted by the action of trypsin to chymotrypsin. The resulting enzyme was found to be just as active and just as susceptible to inhibition by DFP as that resulting from untreated chymotrypsinogen. From this it was concluded that if there was a reaction between DFP and chymotrypsinogen, it did not affect the conversion to the active chymotrypsin or the inhibition of the resulting enzyme by DFP (3). By the use of DFP containing radioactive phosphorus (P3”) it has now been shown that the phosphorus moiety of DFP is firmly attached to the crystalline DFP-inhibited chymotrypsin. Furthermore, the amount of phosphorus bound (after treatment of chymotrypsin with a small excess of DFP) in the twice crystallized, dialyzed, and completely inhibited enzyme was found to be 1.1 moles per mole of enzyme, a result which is in good agreement with the amount previously calculated on a stoichiometric basis from the 50 per cent inhibition value.