Partial Tryptic Digestion of Cucurbitin from Pumpkin Seed

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Abstract
A stable digestion intermediate was observed when cucurbitin, the crystalline storage globulin of pumpkin [Cucurbita moschata (Duch.) Poir.] seed, was treated with trypsin. The major digestion product, termed cucurbitin-T, was isolated as a homogeneous protein of M, 285 000+ 10000 by chromatography over Sepharose CL-6B. The treatment with trypsin lowered the isoelectric point, decreased the charge heterogeneity, and increased the solubility of cucurbitin. Electrophoresis of denatured and reduced cucurbitin-T in 8 M urea at pH 8.5 suggested that the basic polypeptide chains of cucurbitin were degraded less by the enzyme treatment than were the larger acidic chains. This difference in susceptibility was confirmed by two-dimensional electrophoresis. The amino acid analyses of cucurbitin and cucurbitin-T subunit polypeptides are consistent with the changes demonstrated by electrophoresis. It is suggested that the larger fragments (M, 20000-24000) of the acidic chains found in cucurbitin-T, together with the more resistant basic chains (M, - 22000) in both forms of cucurbitin, may constitute the 12 equivalent structural domains observed for cucurbitin by X-ray diffraction analysis.