Cucurbitacin B Δ23-reductase from Cucurbita maxima: I. Assay methods, isolation and purification

Abstract

1. 1.Spectrophotometric and chromatographic assay methods were developed for cucurbitacin B Δ 23-reductase (NAD(P)H:cucurbitacin B Δ 23-oxidoreductase). One spectrophotometric method is based on the decrease in absorbance at 340 mμ in the presence of NADPH or NADH as electron donor while another method is based on the decrease in absorbance at 228 mμ as a result of the reduction of the Δ 23-bond in the side chain of cucurbitacin B. 2. 2.The optimum pH of the enzyme was 6.65. 3. 3.Final proof was obtained for the reduction of the Δ 23-bond in the side chain of cucurbitacin B. 4. 4.Cucurbitacin B Δ 23-reductase was isolated from 7.1 kg of immature Green Hubbard fruits by extraction, centrifugation, alcohol fractionation, heat precipitation, pH precipitation, DEAE-cellulose chromatography and Sephadex G-75 gel filtration. The yield was 24 mg and the final enrichment 142.4. 5. 5.The results of paper electrophoresis and ultracentrifugation indicated a homogeneous preparation with a sedimentation coefficient of 2.96 S. 6. 6.Due to the fact that this enzyme falls in the class of the oxidorduetases with NADPH and NADH as the elctron donors, it is proposed that the enzyme should be called NAD(P)H:cucurbitacin B Δ 23-oxidoreductase and it would be classifiable under the number EC 1.6.99.