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The Biochemical journal1974; 143(3); 733-744; doi: 10.1042/bj1430733

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 (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH(4))(2)SO(4) the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2 degrees C than at 24 degrees 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.
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Publication Date: 1974-12-01 PubMed ID: 4462752PubMed Central: PMC1168442DOI: 10.1042/bj1430733Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article reports on a simple method to purify cholinesterase, an enzyme, from horse serum by 19000-fold. A total of 15 to 18mg of this purified cholinesterase was obtained by processing about 20 liters of horse serum.

Research Methodology

  • The method employed to purify cholinesterase from horse serum consisted of two main stages: fractionation with ammonium sulfate ((NH(4))(2)SO(4)) and ion-exchange chromatography.
  • The first stage utilizing ammonium sulfate was based on the Strelitz (1944) procedure that involves the use of an acid step at pH 3.
  • This process harnessed the stabilizing effect of 33%-saturated ammonium sulfate on the cholinesterase enzyme’s activity at a pH of 3.

Stabilizing Effect of Temperature and pH

  • The researchers observed that the cholinesterase was significantly more stable at a lower temperature of 2 degrees Celsius and a pH 3.0 compared to 24 degrees Celsius.
  • Consequently, the acid step of the procedure was performed at both these temperatures to ensure the stability of the enzyme.
  • The specific activities of the final products obtained by using the acid steps at either temperature were identical, suggesting the acid step did not damage the cholinesterase enzyme’s active sites.

Purification and Electrophoresis Results

  • The product obtained after the first two stages underwent further purification by preparative gel electrophoresis, resulting in a product that was between 85 to 90% pure cholinesterase.
  • Further analysis revealed this product was about 40% more active and contained 40% more active sites per unit weight compared to cholinesterase purified using partially purified commercial material.
  • Some uncertainty exists regarding the number of active sites per cholinesterase molecule, with estimates ranging from 3 to 4 active sites.

Composition Analysis

  • The researchers also analysed the composition of the purified cholinesterase, specifically determining the amino acid and carbohydrate composition.
  • It was found to contain 17.4% carbohydrates, inclusive of 3.2% N-acetylneuraminic acid, a type of sialic acid that plays a crucial role in biological function.
  • The partial specific volumes of cholinesterase were measured using three different methods: by a precision density meter, an ultracentrifuge, and the amino acid and carbohydrate composition; with values of 0.688, 0.71 and 0.712ml/g respectively.

Cite This Article

APA
Main AR, Soucie WG, Buxton IL, Arinc E. (1974). The purification of cholinesterase from horse serum. Biochem J, 143(3), 733-744. https://doi.org/10.1042/bj1430733

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 143
Issue: 3
Pages: 733-744

Researcher Affiliations

Main, A R
    Soucie, W G
      Buxton, I L
        Arinc, E

          MeSH Terms

          • Amino Acids / analysis
          • Ammonium Sulfate
          • Animals
          • Binding Sites
          • Carbohydrates / analysis
          • Centrifugation
          • Cholinesterases / blood
          • Cholinesterases / isolation & purification
          • Cholinesterases / metabolism
          • Chromatography, Ion Exchange
          • Densitometry
          • Electrophoresis
          • Horses
          • Hydrogen-Ion Concentration
          • Temperature

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          Citations

          This article has been cited 4 times.
          1. Burgess SK, Oxendine SL. Thermal inactivation of butyrylcholinesterase and acetylcholinesterase. J Protein Chem 1993 Dec;12(6):651-8.
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          2. Ralston JS, Main AR, Kilpatrick BF, Chasson AL. Use of procainamide gels in the purification of human and horse serum cholinesterases. Biochem J 1983 Apr 1;211(1):243-50.
            doi: 10.1042/bj2110243pubmed: 6870822google scholar: lookup
          3. Brown WE, Green AH, Cedel TE, Cairns J. Biochemistry of protein-isocyanate interactions: a comparison of the effects of aryl vs. alkyl isocyanates. Environ Health Perspect 1987 Jun;72:5-11.
            doi: 10.1289/ehp.87725pubmed: 3622443google scholar: lookup
          4. Main AR, McKnelly SC, Burgess-Miller SK. A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum. Biochem J 1977 Nov 1;167(2):367-76.
            doi: 10.1042/bj1670367pubmed: 597249google scholar: lookup
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