Differentiation of EDTA-sensitive from EDTA-insensitive human serum esterases hydrolysing phenylacetate

The aim of this study was to differentiate the EDTA-sensitive from the EDTA-insensitive human serum esterases by evaluating their catalytic constants, K_M and V_m, for the hydrolysis of phenylacetate (PA). Measurements were done at 37°C in 0.1 M Tris/HCl buffer pH 7.4 and 8.4. The K_M,sen and K_M,ins constants were significantly different, 0.97 and 2.7 mM respectively, confirming that two esterases hydrolyse PA. The pH of the medium had no effect on K_M values, and also no effect on V_m,sen while V_m,ins was two fold higher at pH 8.4 than at 7.4 further confirming the existence of two different enzymes. The stability of the esterases in aqueous media was also studied. EDTA-sensitive activity in buffer without CaCl₂ was extremely unstable; the time-course of inactivation followed a two-phase reaction kinetics, indicating that two EDTA-sensitive esterases hydrolyse PA. The EDTA-insensitive activity remained constant in aqueous media under the same experimental conditions.