PROPERTIES OF BETA-CAROTENE 15,15'-DIOXYGENASE STABILIZED BY LUTEIN AND DITHIOTHREITOL DURING ISOLATION

Preparations of beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) isola ted either with or without stabilization by lutein and dithiothreitol (DTT) show similar properties. The stabilized preparation has an appar ent K(m) of 17 muM, V of 2.8 nmoles retinal per h per mg protein and p H optimum of 8.1; it is sensitive to SH-reagents and is activated by F e2+, DTT, or glutathione (GSH) with the optimal effects at the concent rations of 0.5, 2, and 5 mM, respectively. At DTT concentrations excee ding 2 mM, the activity of beta-carotene 15,15'-dioxygenase decreases significantly; this fact shows the need to maintain the optimal ratio between thiol and disulfide groups in the enzyme. At 4 mM Fe2+, apo-ca rotenals are formed nonenzymatically; however, retinal is formed only after additions of commensurate amounts of thiol compounds. The enzyma tic activity depends on the concentration and nature of ions in the in cubation medium. Maximal activity is observed in 0.4 M potassium phosp hate buffer, if chloride replaces phosphate, the enzyme is partially i nhibited. The optimal reaction temperature is 45-degrees-C. Under the optimal incubation conditions (2 mM DTT, 0.4 M potassium phosphate, pH 8.1, 45-degrees-C), the yield of retinal increases 2.5-fold in compar ison with the control.