Lipolysis of fluorescein and eosin esters. Kinetics of hydrolysis

Abstract

A series of fluorescein and eosin esters have been prepared for use in the assay of the activity of lipase and acylase: the diacetyl, dipropionyl, dichloropropionyl, dibutyryl, divaleryl, and dicaproyl esters of fluorescein, and the diacetyl, dipropionyl, and dibutyryl esters of eosin. The eosin esters were found to hydrolyze extremely slowly in the presence of enzyme, even at pH's as high as 10.0. The rate of hydrolysis of the fluorescein esters by all enzymes except porcine pancreas lipase was found to decrease in the order: acetyl > propionyl > butyryl > valeryl > caproyl. With porcine pancreas, the divaleryl and dicaproyl esters of fluorescein had a higher rate of hydrolysis than the dibutyryl ester. Steapsin, porcine pancreas, and wheat germ lipase, as well as acylase, catalyze the hydrolysis of fluorescein esters to fluorescein. The order of activity is porcine pancrease > acylase > steapsin > wheat germ lipase. The kinetic and thermodynamic constants ( K′ m, k 3, ΔE ∗, ΔH′, ΔF′, ΔS′ ) for the hydrolysis of the various esters by four different enzymes are reported. Sodium taurocholate was found to increase the rate of chymotrypsin hydrolysis of the fluorescein esters. A decrease in K m , ΔE ∗ , Δ H, Δ F, and Δ S and an increase in k 3 were observed when this bile salt was added to the chymotrypsin reaction solutions. No effect on lipase was observed, thus indicating that the probable effect of taurocholate on lipolytic hydrolysis is in the solubilization of the substrate.