Characterisation of Cucurbitin from Various Species of the Cucurbitaceae

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Abstract
There have been many studies of cucurbitin, the major seed storage globulin which can be isolated in crystalline form from several genera of cucurbits. However, recent literature reports have suggested a wide range of molecular weights for oligomeric cucurbitin at neutral pH in buffered aqueous salt solutions. We have measured the molecular weight of oligomeric cucurbitin isolated from mature pumpkin seeds by several methods in the analytical ultracentrifuge. The molecular weights of the major and minor components present are 325000 and 630000 respectively. In combination with dodecylsulphate gel results, these values show that cucurbitin is a hexamer composed of subunits of molecular weight 54000, while most preparations contain several percent of a dodecamer composed of the same subunits. The molecular weight of the major component was 320000 from analytical gel filtration on Sepharose CL‐6B.Aqueous salt solutions of cucurbitin isolated from mature seeds of marrow, cucumber, rock melon, water melon, and ornamental gourds are also composed of major (hexamer) and minor (dodecamer) components. The major fractions have similar elution volumes following chromatography over Sepharose CL‐6B, sedimentation coefficients in the range 12.7–12.9 S and molecular weights in the range 324000–327000 from meniscus depletion sedimentation equilibrium measurements. Although there is limited size heterogeneity between the cucurbitin subunits, there is considerable charge heterogeneity. This has been demonstrated by electrophoresis on cellulose acetate membranes in 8 M urea at pH 8.5 with and without reduction of disulphide bonds. The existence of two classes of subunit polypeptide (acidic Mr∼ 33000 and basic Mr∼ 22000), comparable to those for glycinin (soybean) or legumin (pea, broad bean), has been shown by two‐dimensional electrophoresis.