Action of esterase in the presence of organic solvents.
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IN the method of studying esterases elaborated by the author, the active preparations are placed in contact with non-aqueous solutions of the substrates. The organic solvents used were either soluble, such as acetone, or practically insoluble in water, such as benzene, carbon tetrachloride and others. The active esterase preparations used in the majority of the experiments were obtained from pig pancreas. Prep. A (tissue pulp) was obtained by passing the fat-free pancreas three times through a mincing machine and triturating the product in a mortar. The dry prep. B was obtained by shaking tissue pulp for 1 hour with five volumes of acetone, decanting the supernatant liquid and repeating the process. After squeezing out the solvent the preparation was spread on filterpaper and air-dried. The dry preparation was then ground in a coffee mill; the final product contained 8-12 % of water. The activity of this preparation depends in great measure on the water content. When this is low, an approximate proportionality may be found between the water content and the velocity of reactions catalysed by the preparation [Sym, 1933, 1]. In the case of prep. C the aqueous extract of the esterase was obtained by shaking the dry preparation described above with 5 volumes of water for 10 min., centrifuging and passing the centrifugate repeatedly through a layer of infusorial earth. The activity of the clear extracts obtained was studied after adding 10 g. of sodium butyrate or acetate to 100 ml. of extract. Prep. D was obtained by evaporating prep. C (without sodium salt) in flasks at 20 mm. pressure until the residue had the consistency of a compact gel adherent to the bottom of the flask. The gelatinous mass so obtained is more resistant to inactivating factors than the extract from which it originated and it may be used repeatedly. The above preparations exhibit esterification activity only in the presence of water. Thus preps. A, B and D are reversibly inactivated by further dehydration, being reactivated by addition of water. Systems containing organic solvents insoluble in water, such as benzene, can, as in the case of prep. C, be of varied water content without the activity being destroyed. But when water-soluble organic solvents are used, the enzyme undergoes irreversible inactivation in presence of higher concentrations of water [Sym, 1933, 1]. The action of the enzymic preparations was studied in most cases in systems containing n-butyl alcohol (M BuOH), 0-43M butyric acid and benzene. The velocity of the reactions was studied by determining the initial velocity of esterification, v, viz. the number of millimols. of ester per litre which is formed in the non-aqueous phase during the first hour of reaction.