Isolation of adenosine diphosphoribosyltransferase by precipitation with reactive red 120 combined with affinity chromatography

✍ Scribed by Kalman G. Buki; Eva Kirsten; Ernest Kun

• Publisher: Elsevier Science
• Year: 1987
• Tongue: English
• Weight: 894 KB
• Volume: 167
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(87)90147-3

✦ Synopsis

The DNA-associating enzyme, adenosine diphosphoribosyltransferase, has been isolated from calf thymus by selective precipitation with a solution of dihydroxy Reactive Red 120, followed by extraction of the enzyme from the precipitate with 2 M KCl and an on-line train of three successive column chromatographic steps, including a final 3-aminobenzamide-Sepharose 4B affinity chromatography. The method yields 8-9 mg of more than 95% homogeneous enzyme protein per kilogram starting material and requires about 3 working days. This dye precipitation method is distinct from affinity precipitation, since it involves the binding of the dye to both nonspecific sites and the substrate and DNA sites of the transferase as indicated by enzyme inhibition by dihydroxy Reactive Red 120 at both enzyme sites.