Interaction of α,β‐Diethyl Stilbestrol 4,4′‐Bisphosphate with Arginyl Substrates Resulting in Apparent Inhibition of Trypsin and Thrombin

1 α,β‐Diethyl stilbestrol 4,4′‐bisphosphate (up to 7.0 mM) had no inhibitory action on trypsin assayed with the lysyl substrate, l‐lysine nitroanilide dihydrobromide or by active‐site titration with 4‐methyl umbelliferyl‐p‐guanidinobenzoate · HCl. 2 Diethyl stilbestrol bisphosphate had an inhibitory action on the tryptic cleavage of arginyl peptides when fluorescein‐labelled soluble casein was used as a substrate. 3 This inhibitory action was caused by complex formation between diethyl stilbestrol bisphosphate and susceptible arginyl residues in the substrate, inhibition being reversed by increasing the substrate concentration or adding competitive arginine molecules. 4 This type of inhibition involving substrate modification is referred to as ‘apparent inhibition’ of trypsin and was confined to arginyl peptide bond cleavage. 5 Thrombin specifically cleaves arginyl bonds in fibrinogen to initiate clot formation by the production of fibrin monomers. Diethyl stilbestrol bisphosphate was shown to cause apparent inhibition of clot formation in whole plasma and fibrinogen solutions in the presence of added thrombin. 6 Kinetic data of clot formation demonstrated a threshold level of diethyl stilbestrol bisphosphate (approximately 5 mM) necessary to be exceeded before these apparent inhibitory effects were observed. 7 The mechanism of ‘apparent inhibition’ is brieffy discussed in relation to the more usual inhibition of an enzyme activity due to the direct action of an inhibitor with the enzyme under study.