Specific Assay of Medium-Chain Acyl-CoA Dehydrogenase Based on the Spectrophotometric Measurement of Product Formation

✍ Scribed by K.W. Yao; H. Schulz

• Book ID: 102967564
• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 546 KB
• Volume: 214
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1534

✦ Synopsis

A spectrophotometric method for assaying medium-chain acyl-CoA dehydrogenase is described. The assay measures at 308 nm the formation of cinnamoyl-CoA from 3-phenylpropionyl-CoA in the presence of phenazine methosulfate as electron acceptor. Apparent kinetic constants (Km, Vmax) determined with 3-phenylpropionyl-CoA are similar to constants obtained with octanoyl-CoA, the preferred substrate of this enzyme. The assay is specific for medium-chain acyl-CoA dehydrogenase because long-chain and short-chain acyl-CoA dehydrogenases exhibit little or no activity with 3-phenylpropionyl-CoA as substrate. Since absorbance changes at 308 nm caused by other reactions are less than 5% of the absorbance change due to cinnamoyl-CoA formation catalyzed by medium-chain acyl-CoA dehydrogenase, the assay can be used to measure the activity of this enzyme in crude tissue homogenates. Specific activities of medium-chain acyl-CoA dehydrogenase determined by use of this assay in homogenates of rat liver, heart, and leukocytes were found to be 29, 68, and 2.1 mU/mg of protein, respectively.