Mode of inhibition of chymotrypsin by diisopropyl fluorophosphate. II. Introduction of isopropyl and elimination of fluorine as hydrogen fluoride.

Considerable practical importance is attached to the mode of inhibition of chymotrypsin by diisopropyl fluorophosphate (DFP), because it is evident that this inhibition reaction, fairly general among esterases, may furnish a clue to the nature of the active grouping in this enzyme, and may also explain the mechanism of the toxicity of DFP together with a number of related substances whose industrial applications are becoming fairly wide-spread, and therefore a matter of some public concern. Trypsin and chymotrypsin are both inhibited by small amounts of DFP (1). The esterolytic and proteolytic activity of each enzyme is inhibited at the same rate and to the same extent, thus indicating the probability that the two activities reside in the same “active centers.” Approximately 1 mole of inhibitor is required for the complete inhibition of chymotrypsin, whereas trypsin requires more inhibitor. The reaction time is very brief. An enzymically inert, crystalline protein has been obtained as a reaction product of a-chymotrypsin with slightly more than 1 mole of DFP per mole of enzyme. By the use of DFP containing radioactive phosphorus, it was found that phosphorus had been introduced into the crystalline, inhibited protein to the extent of 1.1 moles per mole of enzyme (2), a value in excellent agreement with that calculated from the amount needed for 50 per cent inhibition. These equivalents are calculated with 27,000 as the molecular weight of cr-chymotrypsin, determined by light-scattering measurements’ in this Laboratory on carefully purified samples of the enzyme. The molecular weight of the inhibited protein as determined by osmotic pressure and the light-scattering method was found to be substantially the same as that of the original enzyme.