This chapter discusses the determination of bovine liver crotonase. In the β-oxidation pathway of fatty acids, crotonase catalyzes the hydration of trans-α,β-unsaturated acyl-CoA derivatives to the corresponding L(+)-β-hydroxyacyl-CoA thioesters. Two spectrophotometric methods have been used to monitor this hydration continuously and quantitatively. First, addition of the elements of water across the double bond abolishes the characteristic ultraviolet absorption of the α,β-unsaturated system, which is conjugated with the carbonyl group of the thioester. This change provides the basis of the direct spectrophotometric method. Alternatively, the oxidation of the β-hydroxyacyl-CoA thioester and the accompanying reduction of nicotinamide adenine dinucleotide (NAD) may be followed in the presence of β-hydroxyacyl-CoA dehydrogenase. The spectral changes associated with NAD reduction provide the quantitative basis of this coupled assay method. In the course of purification procedure, ox liver is obtained from a local abattoir shortly after the animal is slaughtered, and is transported on ice to the laboratory. The liver is trimmed of any visible connective tissue, chopped, and wrapped in portions of convenient weight, usually 500 g, then stored in a freezer until use.
ASJC Scopus subject areas
- Molecular Biology