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 degrees 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 CaCl(2) 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.
Bosak
Anita
A
Biochemistry and Organic Analytical Chemistry Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia. abosak@imi.hr
Barlović-Tusek
Bojana
B
Reiner
Elsa
E
D002384:Catalysis; D002614:Chelating Agents / Q000378:metabolism / Q000494:pharmacology*; D004492:Edetic Acid / Q000378:metabolism / Q000494:pharmacology*; D004950:Esterases / Q000097:blood* / Q000378:metabolism; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D010648:Phenylacetates / Q000737:chemistry*
D002614:Chelating Agents; D010648:Phenylacetates; D004492:Edetic Acid; D004950:Esterases; C025136:phenylacetic acid