Purification by Affinity Chromatography of 2,4-Dienoyl-CoA Reductases from Bovine Liver and Escherichia coli

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Abstract
1 Dye‐ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL‐6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2′,5′‐ADP (2′,5′‐ADP‐ Sepharose 4B) and immobilized 3′,5′‐ADP (3′,5′‐ADP‐ Agarose) were employed for purification of NADPH‐dependent 2‐enoyl‐CoA reductase and 2,4‐dienoyl‐CoA reductase from bovine liver (formerly called 4‐enoyl‐CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533–544], as well as 2,4‐dienoyl‐CoA reductase from Escherichia coli. 2 The NADPH‐dependent 2‐enoyl‐CoA reductase from bovine liver mitochondria was separated from 2,4‐dienoyl‐CoA reductase by dye‐ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2′,5′‐ADP ‐ Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3 The NADPH‐dependent 2,4‐dienoyl‐CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL‐6B, Matrex gel red A, and 2′,5′‐ADP–Sepharose 4B chromatography. 4 The bacterial 2,4‐dienoyl‐CoA reductase was completely purified by ion‐exchange chromatography on DEAE‐cellulose followed by a single affinity chromatography step employing 2′,5′‐ADP ‐ Sepharose 4B and biospecific elution from the column with a substrate, trans, trans‐2,4‐decadienoyl‐CoA. 5 The application of dye‐ligand and general ligand affinity chromatography for purification of NADPH‐dependent 2,4‐dienoyl‐CoA reductases taking part in the β‐oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.

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