Purification and Characterization of Two Lipoxygenase Isoenzymes from Cowpea [Vigna unguiculata (L.) Walp.]

Abstract

Lipoxygenase specific activity in 89 cultivars of cowpea [Vigna unguiculata (L.) Walp.] varied from 30 to 397 units/mg of protein. Two lipoxygenase isoenzymes L-1 and L-2 were purified from a variety UPL Cp2 by extraction with water, followed by dialysis against water, 40–60% ammonium sulfate fractionation, and DEAE-Sephadex A-50 ion-exchange and hydroxylapatite chromatography. L-1 and L-2 were both highly specific for linoleic acid and exhibited a narrow optimal activity at pH 6.2. Apparent Km values of 0.8 × 10−3 M and 0.55 × 10−4 M linoleic acid were obtained for L-1 and L-2, respectively. L-1 and L-2 had molecular weights of 68000 and 74000, respectively, by NaDodSO4 gel electrophoresis and had Rf values of 0.25 and 0.11, respectively, by regular gel electrophoresis. L-1 and L-2 isoenzymes were inhibited to varying degrees by different metal ions although, in general, L-2 was more sensitive. L-2 was also more sensitive to heat. On the other hand, L-1 was more strongly inhibited by antioxidants than L-2. The lipoxygenase isoenzymes were stable at pH 4–9. The enzyme in situ was highly stable even in seeds soaked in acidic solution at pH 2 for 10 h. However, blanching of soaked and unsoaked seeds resulted in total loss of activity. © 1982, American Chemical Society. All rights reserved.

Source or Periodical Title

Journal of Agricultural and Food Chemistry

ISSN

218561

Page

54-60

Document Type

Article

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