The purification and properties of horse liver esterase

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Abstract
An apparatus is described for the continuous potentiometric titration of the acid produced in the enzymic hydrolysis of esters. It is designed to measure low reaction rates, with the consumption of very small concentrations of substrate. A purification of horse liver esterase is described, giving a product with 140-fold greater activity/mg N than the acetone powder used as starting material. Electrophoresis of the preparation shows a major component constituting about 95% of the refractive material. The rates of hydrolysis of 3 separate esters remain in constant ratios over the last 4 stages of the enzyme purification. The progress curves of ester hydrolysis are complex in form; a simple comparison of reactions with equal initial substrate concentrations, but different enzyme concentrations, shows that at least 2 processes other than substrate depletion are involved in the decrease of reaction rate. The Michaelis relation between initial velocity and substrate concentration holds for each ester separately. Mixed substrate solutions are hydrolyzed at a rate which is much less than additive, but is significantly greater than the rate predicted on the basis of simple competition at a single type of active center. The esterase is inhibited by fluorescein, eosin, rhodamine B, o-5-acridylbenzoic acid, pyronine, and N-methyl-acridinium chloride. It hydrolyses DL-beta-butyrolactone, attacking both optical isomers. The homogeneity of the preparation, and its relation to 2 other horse liver esterase preparations are discussed.
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