Enzyme concentration as an important factor in the in vitro testing of thestereospecificity of the enzymatic hydrolysis of organophosphorus compounds

A report is made of important differences in the Ca2+-dcpendent hydrolysis of the chiral phosphoramidate O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) when recorded using different quantities of hen liver microsomes. In a colorimetric microassay using the microsomes from 5 mg tissue in the pre sence of HDCP stereoisomers and 2.5 mM calcium, the R-HDCP isomer was hydro lysed at a rate similar to or slightly faster than S-HDCP isomer (14% v. 11 %), while the S-HDCP stereoisomer was hydrolysed faster than R-HDCP (17% v. 25% and 21%: v. 43%) when HDCP isomers hydrolysis was assayed in the prese nce of the microsomes from 10 or 20 mg, respectively. This stereospecific h ydrolysis was verified assaying racemic HDCP and quantities of liver micros omes from 10 to 80 mg of tissue, using a chiral chromatographic method; thu s, the increase in the ratio of remaining R-HDCP/S-HDCP was dependent on th e amount of liver microsomes (range one- to threefold). This study demonstr ates that the concentration of the subcellular fraction in in vitro assays is a critical factor to be taken into account in securing a more realistic approximation to the stereospecific enzymatic processes occurring in biolog ical systems. Our data concerning the hydrolysis of HDCP by liver microsome s at high enzyme concentrations afford a better fit to the in vivo toxicolo gical response with HDCP than assays performed with the most commonly used highly diluted preparations. (C) 1999 Elsevier Science Ltd. All rights rese rved.