Effect of Diisopropylfluorophosphate on Sulfhydryl Proteases

Abstract

Diisopropylfluorophosphate inhibited all the sulfhydryl proteases studied in our tests. This inhibition was most pronounced at p H 6.0. By first blocking the sulfhydryl group with p -chloromercuribenzoate, inhibition could be prevented. Neither cysteine nor choline gave appreciable reactivation of diisopropylfluorophosphate-inhibited bromelain.

Alkylphosphorylation of Stem Bromelain by Diisopropylphosphorofluoridate without Inhibition of Proteinase Activity*

“…Even in the absence of activator, DFP preparation D was found to give no inhibition (Table III). In addition, the present study has also reconfirmed the results of other investigators that some commercial preparations of DFP have inhibitory effects on plant proteinases when they are activated with cyanide (Masuda, 1959;Heinicke and Mori, 1959;Ota et al, 1961; Ebata and Yasunobu, 1963). Preparation C used in the present study gave a marked inhibition of stem bromelain, papain, and ficin in the presence of cyanide.…”

“…The reconfirmation was necessary particularly in view of the results reported by other investigators that were at variance with ours. For example, Heinicke and Mori (1959) suggested that DFP is a specific inhibitor of SH proteinases; Ota et al ( 1961) found an inhibitory effect on bromelain; Masuda (1959) and Ebata and Yasunobu (1963) isolated inactive, phosphorus-containing papain and chymopapain, respectively, after incubating the enzymes with DFP in the absence of cysteine. The conclusion we have drawn from the results of the earlier and the present experiments is that DFP per se has no inhibitory effect on stem bromelain, while impurities associated with some DFP preparations may cause an inhibition by irreversibly attacking the essential SH group of the enzyme.…”

Reaction of Ficin with Diisopropylphosphorofluoridate. Evidence for a Contaminating Inhibitor*

“…Quite coincidentally, the inhibition of ficin by the impurity also increases as the pH is raised. Those who have reported SH enzymes to be inhibited by DFP have also noted this inhibition to be pH dependent (Heinicke and Mori, 1959;Ebata and Yasunobu, 1963). It is not surprising therefore that phosphorylation and inactivation have been assumed to bear a cause-andeffect relationship to each other, an assumption which appeared to be supported by the isolation of crystalline phosphorylated derivatives of DFP-inhibited papain (Masuda, 1959) and chymopapain (Ebata and Yasunobu, 1963).…”

Mechanisms of Catalysis of Nucleophilic Reactions of Carboxylic Acid Derivatives.

“…The parallelism between the stoichiometry of the DFP reaction (equation 68) and the enzyme inactivation, and also the ability of substrates to protect against inhibition by DFP, (C8H,0)2P0F + EH -> E-PO(OC3H7)2 + HF (68) indicate strongly that phosphorylation occurs at the active site. Chymotrypsin, trypsin, papain, acetylcholinesterase, and phosphoglucomutase among others are subject to inhibition by DFP (204,237). When DFP-chymotrypsin is degraded by acid hydrolysis, serine phosphate is obtained (335).…”