(1) Two rare genetic variants (Ch(I)^D and Ch(I)^F) of human cholinesterase were purified by a three-step technique. The atypical cholinesterase (Ch(I)^D) was purified by a factor of 2,230 with a recovery of 11 % of the original activity. The fluorideresistant (Ch(I)^F) variant was purified 5,000-fold with a recovery of 17%. The poorer recovery is attributed to lesser stability of the unusual variants. The atypical enzyme was less stable than the normal (Ch(I)^U) in 20 mmol/l acetate buffer, pH 4.0. (2) The catalytic, physical, and immunological properties of the atypical and fluorideresistant enzymes were compared with those of the normal (Ch(I)^U). The apparent catalytical properties appeared to be unchanged by the purification procedure. The specific activity of the fluoride-resistant enzyme was about 20% of the normal and that of the atypical enzyme was about 6% for all three substrates studied. The significance of the much lower activities of the atypical variant is discussed. All three variants were equally affected by sialidase. (3) The isoelectric points were estimated to be 3.99 for the normal, 4.20 for atypical, and 3.70 for the fluoride-resistant cholinesterase. The three variants appeared to be immunologically similar and have similar molecular weights. (4) The chromatographic mobilities of the enzyme variants on a DEAE-cellulose column at pH 4.0 in 20 mmol/l acetate buffer were compared. A physical separation of Ch(I)^U and Ch(I)^F variants present in plasma from an individual heterozygous for Ch(I)^U Ch(I)^F were obtained by DEAE-cellulose chromatography.

The plasma cholinesterase activities of two individuals homozygous for the apparent Chs1 gene have been investigated by a thiocholine assay procedure using acetylthiocholine and butyrylthiocholine iodide as substrates in the presence and absence of several inhibitors, and by polyacrylamide disc electrophoresis and immunoabsorbtion test. The results have been compared with those obtained with normal plasma and red cell cholinesterase preparations. It is concluded that (1) the properties of ‘silent’ cholinesterase variants are different from those of both normal plasma and red cell cholinesterases; (2) the ‘silent’ variants are mutants of normal plasma cholinesterase, and (3) they are heterogeneous in character. Because of the complex properties of the ‘silent’ samples, the cause of heterogeneity should be treated with caution until better characterization of the purified enzyme is possible.

The existence of genetically determined human serum cholinesterase variants (acylcholine acyl-hydrolase; EC 3.1.1.8) has been recognized by the use of a short acting muscle relaxant, suxamethonium. This polymorphism is now explained by at least four alleles belonging to an autosomal locus Ch(1) or E(1) depending on the nomenclature. These are: the usual gene, the dibucaine-resistant gene, the fluoride-resistant gene and the silent gene. At present, the modes of transmission for the silent genes is confusing and lack proper understanding. Another variant, not an allele of the first group, is recognized by gel electrophoresis, belong to a second locus Ch(2) or E(2). Evidence has been presented that many more rare serum cholinesterase variants may exist. Besides these genetic variants, normal serum cholinesterase is known to exist in multiple molecular forms. The present article attempts to discuss the polymorphism of human cholinesterase in relation to their chemical and genetic characteristics and their possible function.

Purified serum cholinesterase was found to be an inhibitor of influenza virus hemagglutination with characteristics similar to serum α-inhibitors. Intratypic variation between Asian (H2) strains was observed and inhibition of all strains investigated was strongest with A/Hong Kong/68 virus. Cholinesterase treated with A/Hong Kong/68 virus maintained its enzymatic activity at an increased level on dilution and this was thought to be due to stabilization of the native molecule by the virus.

1) Triton X-100 solubilised human erythrocyte acetylcholinesterase (E 0 ), when subjected to chromatography on Sephadex G-200, showed one enzyme activity peak (Eg) and a number of protein peaks (Pg). The same sample could be separated into several subfractions of enzyme activity on DEAE-cellulose by gradient elution with increasing sodium chloride. When the gel filtered enzyme peak (Eg) alone was rechromatographed on an ion-exchange column under identical conditions, it showed only one enzyme peak. But when Eg in combination with protein peaks (Pg) without enzyme activity is rechromatographed as a physical mixture on a DEAE-cellulose column under the same conditions, the preparation could again be resolved into at least two fractions with enzyme activity.2) Disc electrophoresis of fractions from DEAEcellulose chromatography separated multiple bands which differ significantly from those produced by electrophoresis of E 0 . This suggests that E 0 had undergone some form of conformational modification during the ion-exchange chromatography. However, this modification of E 0 was avoided, if it was subjected to Sephadex G-200 chromatography before the DEAE-cellulose step.3) A three-step technique (fractionation on Sephadex G-200, DEAE-cellulose chromatography and electrofocusing) has been performed for the purification of erythrocyte acetylcholinesterase. An enzyme preaparation of high purity with a specific activity of 81 U/mg of protein was obtained. Heterogenität und partielle Reinigung der Acetylcholinesterase aus menschlichen ErythrozytenmembranenZusammenfassung: 1) Menschliche Erythrozyten-Acetylcholinesterase (E 0 ), in Triton X-100 gelöst, zeigt nach der Chromatographie an Sephadex G-200 einen Aktivitätsgipfel (Eg), aber verschiedene Proteingipfel (Pg). Nach Gradientenelution mit steigender NaCl-Konzentration an DEAE-Cellulose erhält man jedoch mehrere Fraktionen mit Enzymaktivität. Wird aber Eg unter gleichen Bedingungen an einer lonenaustauschefsäule rechromatographiert, wird nur ein einheitlicher Enzymaktivitätsgipfel erhalten. Mischt man Eg und Pg und rechromatographiert dann an einer DEAE-Cellulose-Säule, so läßt sich eine Auftrennung in mindestens zwei Fraktionen mit Enzymaktivität beobachten.