engleski

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, KM and Vm, for the hydrolysis of phenylacetate (PA). Measurements were done at 37 oC in 0.1 M Tris/HCl buffer pH 7.4 and 8.4. The KM, sen and KM, 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 KM values, and also no effect on Vm, sen while Vm, ins was two times 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.

arylesterases; catalytic constants; Km; Vm; esterase stability; phenylacetate hydrolysis measurement

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