Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Abstract

Soluble invertase was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, DEAE-Sepharose column, Con-A- and Green 19-Sepharose affinity columns, hydroxyapatite column, ultra-filtration, and Sephacryl 300 gel filtration. The purified soluble acid (SAC) and alkaline (SALK) invertases had a pH optimum of 5.3 and 7.3, respectively. The temperature optimum of two invertases was 37 degrees C. The effects of various concentrations of Tris-HCl, HgCl2, and CuSO4 on the activities of the two purified enzymes were examined. Tris-HCl and HgCl2 did not affect SAC activity, whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK activity by about 50%. SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 by 50%, respectively. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM, respectively. The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa. The molecular mass of SALK was 30 kDa. Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7, respectively.