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High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in
W D dos Santos1, Maria de Lourdes Lucio Ferrarese, O Ferrarese-Filho
1Laboratory of Plant Biochemistry, Department of Biochemistry, University of Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil.
Abstract:
This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.
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