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Immunology1979; 37(3); 517-527;

Purification of the subunit Clq from the first component of equine complement.

Abstract: Initial separation and concentration of Clq from fresh, normal equine serum was accomplished by precipitation in 0.02 M acetate buffer, pH 5.5, at 4 degrees for 24 h. The re-dissolved precipitate was clarified by centrifugation at 80,000 g for 1 h and then dialysed against Tris-HCl buffer (0.05 M, pH 8.0) containing 10-3 M EDTA. The clarified dialysate remained biologically active at 5 degrees for at least 4 weeks. Biological activity of equine Clq was determined by assay of its ability to agglutinate sensitized sheep erythrocytes (EA). Following ammonium sulphate fractionation, Sepharose 4B gel filtration yielded three major peaks. Two protein bands were demonstrated on analysis of the second Sepharose peak by disc acrylamide electrophoresis, pH 8.3. Elution of the protein bands showed EA-agglutinating activity only in the band which migrated furthest toward the cathode. Equine Clq isolated by this method yielded an approximate forty-fold purification in specific activity. Some properties of equine Clq were characterized. Equine Clq was heat-labile, as shown by loss of its EA-agglutinating activity after heating 58 degrees for 15 min. Moreover, storage at 4 degrees and freeze-thaw cycles greatly reduced EA agglutination. Preliminary determination of the sedimentation coefficient indicated that it was comparable to that reported for human and rabbit Clq.
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Publication Date: 1979-07-01 PubMed ID: 91570PubMed Central: PMC1457737
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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.

The study details a method for isolating the Clq subunit from the equine (horse) immune system’s first component, observing its behavior and characteristics. The researchers achieved approximately forty-fold purification and found certain attributes of the equine Clq.

Research Methodology and Findings

  • The researchers began their study by separating and concentrating Clq from fresh, normal horse serum. This was achieved by causing it to precipitate in an acetate buffer at certain conditions (pH 5.5, 4 degrees for 24 hours).
  • This precipitate was then redissolved and clarified by high-speed centrifugation. The clarified solution was dialysed against a Tris-HCl buffer with EDTA included. Importantly, the equine Clq retained biological activity at a certain temperature (5 degrees) for a significant period (at least 4 weeks).
  • Biological activity of the equine Clq was measured by examining its ability to agglutinate, or bind together, pre-made sensitized sheep erythrocytes (red blood cells), denoted as EA.
  • Using further chemical separation techniques, including ammonium sulphate fractionation and Sepharose 4B gel filtration, they obtained three major peaks. Two protein bands were identified in the second Sepharose peak via disc acrylamide electrophoresis.
  • Only one of these bands showed EA-agglutinating activity, demonstrating that the active portion of the equine Clq was located here.

Characteristics of Equine Clq

  • On further examination, the equine Clq was found to be heat-labile, as heating at 58 degrees Celsius for 15 minutes saw it lose its EA-agglutinating activity.
  • Equine Clq was also observed to be sensitive to storage at 4 degrees and to freeze-thaw cycles, as these conditions greatly reduced its ability to agglutinate EA.
  • A preliminary estimate of the equine Clq’s sedimentation coefficient – a measure of particle size in solution – was comparable to that reported for human and rabbit Clq.

In summary, the researchers successfully developed and carried out a method for purifying the Clq subunit from the first component of the equine immune system, determining its nature, function, and several of its characteristics in the process.

Cite This Article

APA
McDonald TL, Burger D. (1979). Purification of the subunit Clq from the first component of equine complement. Immunology, 37(3), 517-527.

Publication

Immunology
ISSN: 0019-2805
NlmUniqueID: 0374672
Country: England
Language: English
Volume: 37
Issue: 3
Pages: 517-527

Researcher Affiliations

McDonald, T L
    Burger, D

      MeSH Terms

      • Amino Acids / analysis
      • Animals
      • Centrifugation, Density Gradient
      • Chromatography, Gel
      • Complement C1 / isolation & purification
      • Electrophoresis, Polyacrylamide Gel
      • Epitopes
      • Horses / immunology
      • Immunoelectrophoresis
      • Molecular Weight
      • Preservation, Biological

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      Citations

      This article has been cited 2 times.
      1. Yonemasu K, Sasaki T. Purification and characterization of subcomponent C1q of the first component of mouse complement. Biochem J 1981 Feb 1;193(2):621-9.
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        pubmed: 6977483
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