β-Carotene-15,15′-dioxygenase (EC1.13.11.21) isolation reaction mechanism and an improved assay procedure

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Abstract
-dioxygenase (EC 1.13.11.21; -14C2l- or [15,15-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled -carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining -carotene consumed confirm the central cleavage mechanism for the enzyme's action. More -carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formedlmg protein per h. The formation of a lipid-protein aggregate containing the β-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal serapings were homogenized in buffer at a proportion of 1:4 (w/v).