NONENZYMATIC PROTEIN-INDUCED HYDROLYSIS OF P-NITROPHENYL ACYL ESTERS IN RELATION TO LIPASE ESTERASE ASSAYS

Protein-induced hydrolysis of p-nitrophenyl acyl esters has been studi ed mainly via the reaction between bovine serum albumin (BSA) and p-ni trophenyl myristate. Tn the present study BSA caused a large increase in the amount of p-nitrophenol released (measured as increase in absor bance at 410 nm) at pH above 6.5, but at lower pH BSA had only negligi ble catalytic activity. BSA-catalysed hydrolysis of p-nitrophenyl acyl esters increased with temperature (25-80 degrees C), concentration of BSA and concentration of p-nitrophenyl acetate, but was virtually una ffected by changes in p-nitrophenyl myristate concentration. The rate of hydrolysis was highest for p-nitrophenyl acyl esters with an acyl m oiety of 8-12 carbon atoms at a p-nitrophenyl concentration (0.028 mmo l/litre) below critical micelle concentration (CMC) for all p-nitrophe nyl acyl esters tested. For a p-nitrophenyl acyl ester concentration ( 0.28 mmol/litre) above CMC (acyl moiety of greater than or equal to 6 carbon atoms) the rate of hydrolysis decreased with increasing chain l ength of the acyl moiety. The catalytic effect of BSA decreased with h eat treatment (90-95 degrees C for 15 min) of the protein solution, bu t did not disappear. Tween-20 effectively retarded BSA-catalysed hydro lysis of p-nitrophenyl myristate, and the effect was dependent on both Tween-20 and BSA concentrations. Free fatty acids displayed a similar but less pronounced inhibitory effect on the hydrolytic reaction. The inhibitory effect increased with the length of the fatty acids (C-4 t o C-10).