The purification of cholinesterase from horse serum

Abstract

A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15–18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH4)2SO4 and ion-exchange chromatography. The (NH4)2SO4 stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH4)2SO4 has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH4)2SO4 the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2°C than at 24°C, and the acid step was done at both temperatures. The specific activities of the final products obtained by way of acid steps were the same at either temperature, thus indicating that the step has not harmed the enzyme active sites. The product from the first two stages was purified over 18000-fold and was 85–90% cholinesterase. The remaining impurities were removed by preparative gel electrophoresis. The product was about 40% more active and contained 40% more active sites per unit weight than electrophoretically pure cholinesterase prepared from partially purified commercial starting material. Although the number of active sites per molecule was not determined with certainty, a value of at least 3 and possibly 4 was indicated. The partial specific volumes were determined with a precision density meter, on the ultracentrifuge and from the amino acid and carbohydrate composition. The values by these independent methods were 0.688, 0.71 and 0.712ml/g, respectively. The amino acid and carbohydrate composition was determined. The cholinesterase contained 17.4% carbohydrate including 3.2% N-acetylneuraminic acid.