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Home / Equine Research Bank / Biochem J / Use of procainamide gels in the purification of human and ho...
The Biochemical journal1983; 211(1); 243-250; doi: 10.1042/bj2110243

Use of procainamide gels in the purification of human and horse serum cholinesterases.

Abstract: Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the relative binding of human and horse cholinesterases to procainamide-, methylacridinium (MAC)-, m-trimethylammoniophenyl (m-PTA)- and p-trimethylammoniophenyl (p-PTA)-Sepharose 4B gels were measured, by using two approaches. In one, binding was measured by a procedure involving equilibration of pure cholinesterase in a small volume of diluted gel slurry (4%, v/v). A partially purified preparation of Electrophorus acetylcholinesterase was included. Pure human cholinesterase bound consistently more tightly to each of the gels than did horse cholinesterase, and the acetylcholinesterase appeared to bind the gels 10-100 times more tightly than did the non-specific cholinesterases. The order of binding for the cholinesterases, beginning with the tightest, was: procainamide-Sepharose 4B, MAC-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. For the acetylcholinesterase the order was: MAC-Sepharose 4B, procainamide-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. The second approach involved passing native sera or partially purified sera fractions through 1 ml test columns of each of the four affinity gels to determine their retention capacity for the cholinesterases. With these impure samples, the MAC-Sepharose 4B gels proved superior to the procainamide-Sepharose 4B gels at retaining human cholinesterase, but the opposite was true for the horse cholinesterase.
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Publication Date: 1983-04-01 PubMed ID: 6870822PubMed Central: PMC1154348DOI: 10.1042/bj2110243Google Scholar: Lookup
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Summary

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The research article discusses the development of two methods using procainamide-Sepharose gels for the purification of human and horse serum cholinesterases, enzymes involved in nerve function. The study concludes that these methods are more effective for purifying horse cholinesterase than human cholinesterase.

Methods Developed

  • The researchers developed two large-scale purification methods primarily using procainamide-Sepharose gels.
  • The first method (Method I) employs procainamide-Sepharose 4B gel in the initial purification step to handle and process large volumes of serum.
  • The second method (Method II) uses the same gel but in the final step to obtain a pure enzyme.

Effectiveness of Methods

  • Both methods were capable of producing electrophoretically pure cholinesterase preparations in good yields.
  • However, they were significantly more efficient at purifying the enzyme from horse serum compared to human serum.

Experimentation with Different Gels

  • In the interest of investigating this disparity, they carried out tests measuring the relative binding of human and horse cholinesterases to four different gels: procainamide-Sepharose 4B, methylacridinium (MAC)-Sepharose 4B, m-trimethylammoniophenyl-Sepharose 4B, and p-trimethylammoniophenyl-Sepharose 4B.
  • Pure human cholinesterase bound consistently more tightly to each of the gels than its horse counterpart. Furthermore, an enzyme called acetylcholinesterase also appeared to bind these gels 10-100 times more tightly than the non-specific cholinesterases.

Binding Rankings

  • The researchers established a binding order for each enzyme according to the tightness of their bonding, with procainamide-Sepharose 4B appearing to be the strongest for cholinesterases but the MAC-Sepharose 4B proving to be the preferred gel for acetylcholinesterase.

Comparison of the Gels

  • Additionally, using a second approach which involved testing impure samples, the results indicated that MAC-Sepharose 4B gels were superior to procainamide-Sepharose 4B gels at retaining human cholinesterase. Yet, the procainamide-Sepharose 4B gels worked better for horse cholinesterase.

Cite This Article

APA
Ralston JS, Main AR, Kilpatrick BF, Chasson AL. (1983). Use of procainamide gels in the purification of human and horse serum cholinesterases. Biochem J, 211(1), 243-250. https://doi.org/10.1042/bj2110243

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 211
Issue: 1
Pages: 243-250

Researcher Affiliations

Ralston, J S
    Main, A R
      Kilpatrick, B F
        Chasson, A L

          MeSH Terms

          • Acetylcholinesterase / isolation & purification
          • Animals
          • Chemical Phenomena
          • Chemistry
          • Cholinesterases / blood
          • Cholinesterases / isolation & purification
          • Chromatography, Affinity / methods
          • Horses
          • Humans
          • Procainamide

          Grant Funding

          • ES-00044 / NIEHS NIH HHS
          • ES-07046 / NIEHS NIH HHS

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