The inhibition of purified, human plasma cholinesterase with diisopropyl fluorophosphate.

Previous communications from this Laboratory have shown that the inhibition of cy-chymotrypsin by diisopropyl fluorophosphate (DFP) is a stoichiometric reaction that comprises the introduction of a single diisopropyl phosphate group into the molecule of cu-chymotrypsin, the elimination of fluorine as HF, and the production of an undenatured, crystallizable, inhibited enzyme protein (l-3). Several substances analogous to DFP in composition were found to inhibit oc-chymotrypsin in a similar manner. In each case the resulting inhibited protein was obtained in crystalline form and found to contain one phosphate group per mole (4). This reaction, leading to the inhibition of ar-chymotrypsin, is therefore not confined 60 DFP. There is also evidence that other esterases react with DFP in the same way as or-chymotrypsin. Thus pand y-chymotrypsins behave toward DFP in quite the same manner as the c11 enzyme (5) (which may not be astonishing in view of their general similarity) ; however, DFP also reacts with trypsin to produce a crystallizable, inhibited trypsin whose equivalent weight with respect to phosphorus is about 20,000 (5). The reaction has also been investigated with non-proteolytic esterases to an extent that at least shows their behavior to be similar to that of the proteolytic esterases. Boursnell and Webb (6) demonstrated that highly purified horse liver esterase bound phosphorus upon inhibition with radioactive DFP, and recently Michel and Krop (7) obtained a similar result with purified cholinesterase from the electric eel. In every case the inhibition of a susceptible enzyme by DFP has resulted in the introduction of the phosphorus of the inhibitor. The specific activity of cholinesterase is now a matter of considerable interest. Despite many efforts at purification, it seems doubtful whether preparations of outstanding purity have been obtained from any source. The work reported here is an attempt to deduce the specific activity of