Rapid large-scale purification of pig heart nucleoside diphosphate kinase by affinity chromatography on Cibacron Blue 3G-A Sepharose
✍ Scribed by I. Lascu; M. Duc; A. Cristea
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 436 KB
- Volume
- 113
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A rapid procedure for the large-scale purification of pig heart nucleoside diphosphate kinase is described. The purification procedure involves extraction of the enzyme, absorption on Cibacron Blue 3G-A Sepharose, elution with ATP, ammonium sulfate precipitation, heat treatment, and rechromatography on Cibacron Blue 3G-A Sepharose. Typically, lo-12 mg of pure nucleoside diphosphate kinase is obtained from 1 kg of heart muscle (50% yield), with a purification factor of 1200 over the extract. The specific activity is 1500 units/mg at 25°C with B-bromoinosine Y-diphosphate as acceptor nucleotide. This method may be easily scaled up.
Methods
Chemicals. Sepharose 4B was obtained from Pharmacia, Uppsala, Sweden. It was crosslinked according to Kristiansen (15),