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

A report is made of important differences in the Ca(2+)-dependent 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 5mg tissue in the presence of HDCP stereoisomers and 2.5mM calcium, the R-HDCP isomer was hydrolysed 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 presence of the microsomes from 10 or 20mg, respectively. This stereospecific hydrolysis was verified assaying racemic HDCP and quantities of liver microsomes from 10 to 80mg of tissue, using a chiral chromatographic method; thus, the increase in the ratio of remaining R-HDCP/S-HDCP was dependent on the amount of liver microsomes (range one- to threefold). This study demonstrates 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 biological systems. Our data concerning the hydrolysis of HDCP by liver microsomes at high enzyme concentrations afford a better fit to the in vivo toxicological response with HDCP than assays performed with the most commonly used highly diluted preparations.