Separation and quantitation of ribulose-1,5-bisphosphate carboxylase-oxygenase in spinach leaves by high-performance hydrophobic-interaction chromatography

Abstract

A rapid and highly efficient method is described for the separation and quantitation of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase; E.C. 4.1.1.39) in spinach leaves by hydrophobic-interaction chromatography. A TSK Phenyl 5 PW column was used for the separation of RuBPCase with magnesium sulphate solutions as the mobile phase. Different initial concentrations of the salt, pH values, buffer substances, and column temperatures were studied. After extraction of water-soluble proteins from powdered spinach leaves with 50 m M Bicine (pH 7.8) at a leaf-to-buffer ratio of 0.25 (g/ml), and after centrifugation of the homogenate, the supernatant was directly injected into the chromatographic column for the quantitative determination of RuBPCase. The chromatographic peak for RuBPCase was identified by its enzymatic activity and further characterized by stop-flow spectroscopy and by gradient polyacrylamide gel electrophoresis, with and without sodium dodecyl sulphate. The calibration curve for RuBPCase was linear for concentrations up to 300 μg of loaded enzyme. The recovery of the enzyme was greater than 90% in terms of activity.