Widely employed site-specific reagents

Clearly all experiments performed with the nerve gas, diisopropyl fluorophosphate (DFP), must be done in a fume hood with the utmost care. DFP rapidly hydrolyzes in 1 N NaOH, so that all glassware which comes in contact with the nerve gas should be soaked in this alkaline solution for at least 1 hr as a precaution prior to its reuse. (Warning DFP is a potent poison. Atropine is an effective antagonist. For details of treatment, see Goodman, L. S. and Gilman, A., 1965, The Pharmacological Basis of Therapeutics, The Macmillan Co., New York, 3rd Ed., p. 454). 

Stock solutions of DFP can be conveniently prepared in isopropanol in concentrations from 0.1 M to 0.001 M. These solutions are stable for a month in the refrigerator (Jansen et al. 1949). Moon et al. (1965), in their studies of the reaction of chymotrypsin with DFP, have determined the normality of stock solution of DFP in the following manner. Roughly 0.03 moles of DFP were added to an aqueous solution of 0.17 moles of KOH in a volumetric flask. The solution was allowed to stand for 12 hr at 25°C to permit complete hydrolysis, and was titrated to pH 7.0 with 0.1 N HCl. Identical titrimetric results were obtained after 43 hr standing in alkali indicating that complete hydrolysis had taken place after 12 hr. To determine the amount of free acid, if any, in the DFP, the same amount of DFP that was used 

The derivatization of an enzyme by DFP is accomplished simply by incubating DFP with the protein. Aliquots of the incubation mixture can then be assayed for activity in the presence of DFP or after its removal by either gel filtration or diafysis. The second-order rate constant at neutral pH for the inactivation of chymotrypsin by DFP is roughly 3 x 10 sec (Main 1964). This is the most rapid rate of 

Although DFP has been and still is widely used to test if an enzyme possesses an unusually reactive serine, it should be noted that phenyl-methane sulfonyl fluoride can also be used for the same purpose ( 5.7). This reagent is in some ways preferable as it is non-volatile and relatively non-toxic. Therefore, it can be employed with fewer precautions. 

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