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A simple method to purify ribonucleotide reductase.

T Spector, D R Averett

    Analytical Biochemistry
    |October 15, 1983
    PubMed
    Summary
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    Researchers developed a new method to purify ribonucleotide reductase, an enzyme crucial for DNA synthesis. This technique effectively separates it from interfering enzymes, enabling more accurate studies of DNA repair and cell growth.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • Ribonucleotide reductase is essential for DNA synthesis and repair.
    • Assays are often complicated by nucleoside diphosphate kinase activity, which depletes the substrate CDP.
    • Efficient purification of active ribonucleotide reductase is critical for biochemical studies.

    Purpose of the Study:

    • To develop a method for separating nucleoside diphosphate kinase from ribonucleotide reductase.
    • To enable accurate and stable assays of ribonucleotide reductase activity.
    • To provide a generalizable purification strategy for ribonucleotide reductase.

    Main Methods:

    • Chromatography using an ATP-agarose column to separate enzymes.
    • Fractionation of cell extracts using ammonium sulfate precipitation.

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  • Enzyme activity assays and stability testing under different buffer conditions.

    • Main Results:

    • Greater than 99.9% of nucleoside diphosphate kinase was removed using ATP-agarose chromatography.
    • Ribonucleotide reductase was recovered with high yield (>200%) and stability.
    • HEPES-Na buffer enhanced enzyme activity 2.5-fold compared to Tris-HCl.

    Conclusions:

    • ATP-agarose chromatography is an effective method for purifying ribonucleotide reductase.
    • The developed purification strategy minimizes substrate depletion and enhances enzyme stability.
    • This method has broad applicability for isolating ribonucleotide reductase from various sources.