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Improvement of a simple method to purify ribonucleotide reductase
Preparative Biochemistry
|January 1, 1985
Summary
Improving ribonucleotide reductase purification using ATP-agarose columns is key. Adjusting buffer conditions prevents enzyme dissociation, enhancing yield and purity by preserving essential subunits.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ribonucleotide reductase is crucial for DNA synthesis.
- ATP-agarose columns efficiently remove nucleoside diphosphate (NDP) kinase.
- Previous methods reported purification challenges.
Purpose of the Study:
- To optimize the purification of human ribonucleotide reductase using ATP-agarose chromatography.
- To investigate and mitigate enzyme dissociation during purification.
- To improve the overall yield and specific activity of the purified enzyme.
Main Methods:
- Affinity chromatography utilizing ATP-agarose columns.
- Modification of buffer ionic strength (0.5 M KCl) to prevent enzyme dissociation.
- Gel filtration for separating residual contaminants.
Main Results:
- Extending column length caused ribonucleotide reductase dissociation into subunits.
- Enzyme subunits eluted at different ionic strengths (low ionic strength buffer and 0.5 M KCl).
- Equilibration with 0.5 M KCl or specific elution strategies preserved enzyme integrity, significantly improving yield and specific activity.
Conclusions:
- Optimized ATP-agarose chromatography, with adjusted ionic strength, effectively purifies intact ribonucleotide reductase.
- Preventing subunit dissociation is critical for maximizing enzyme yield and activity.
- This refined method enhances the purification of a key enzyme for DNA synthesis.