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New assay for uronosyl 5-epimerases.

P Campbell, D S Feingold, J W Jensen

    Analytical Biochemistry
    |May 1, 1983
    PubMed
    Summary
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    New assays simplify the study of uronosyl 5-epimerases involved in heparin and dermatan sulfate biosynthesis. These methods accurately quantify tritium-labeled water generated during enzymatic reactions, improving efficiency.

    Area of Science:

    • Biochemistry
    • Glycosaminoglycan biosynthesis

    Background:

    • Heparin and dermatan sulfate biosynthesis involve two key uronosyl 5-epimerases.
    • Previous assays for these enzymes were complex and time-consuming.

    Purpose of the Study:

    • To develop simpler and more efficient assays for uronosyl 5-epimerases.
    • To quantify tritium-labeled water generated during enzymatic epimerization reactions.

    Main Methods:

    • Enzymatic preparation of tritium-labeled substrates (D-glucuronosyl and L-iduronosyl residues).
    • Quantitation of 3H2O using a liquid-liquid extraction method (Pollard et al., 1981).
    • Assay validation through linearity with time and enzyme concentration.

    Main Results:

    • A simplified assay was developed for heparosan N-sulfate D-glucuronosyl 5-epimerase and chondroitin D-glucuronosyl 5-epimerase.

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  • The new method avoids distillation, significantly reducing assay complexity.
  • Assays demonstrated linearity, confirming reliability.

    • Conclusions:

    • The developed assays are simpler and more efficient for studying uronosyl 5-epimerases.
    • This method is applicable to other enzymatic reactions forming 3H2O from 3H-labeled substrates.
    • These assays will facilitate research on glycosaminoglycan biosynthesis.