Identification of two C-zeins ("15" kDa or beta zeins) by RP-HPLC and SDS-PAGE. --C. M. Wilson C-zein is a high-methionine alcohol-soluble protein in corn endosperm protein bodies. Based on its mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it is also known as 15 kDa-zein, although its amino acid composition, derived from the sequence of a cDNA, indicates a molecular mass of 17.3 kDa (K. Pedersen et al, J. Biol. Chem. 261:6279, 1986). C-zeins from different inbreds differ slightly in relative SDS-PAGE mobilities (C. M. Wilson, Plant Physiol. 82:196, 1986). Paulis and Bietz (J. Cereal Sci. 4:205, 1986) found C-zein in the first protein peak eluted from a C18 reversed-phase high-performance liquid chromatography (RP-HPLC) column by an acetonitrile gradient.

When alcohol extracts were assayed by a modified RP-HPLC gradient, peak 1 from some inbreds eluted at 25.79 min, while that from other lines eluted an average of 0.37 min later. The early-eluting C-zein came from inbreds with the slow SDS-PAGE variant. C-zeins from two pairs of inbreds, B57-N28 and Oh43-W64A, and from their reciprocal F1 hybrid seeds were assayed by RP-HPLC. As expected, double peaks occurred in the F1 extracts, with the higher peak corresponding to that of the maternal parent. N28 and W64A have the slow SDS-PAGE C-zein band and the first HPLC peak, while B57 and Oh43 have the fast SDS-PAGE band and the second HPLC peak. Other inbreds with the slow SDS-PAGE variant include B37, BSSS-53, B84, L289, Oh45, Pa91, R802, Va35, and WF9; the fast variant also occurs in A619, B14A, B68, B73, M14, Mo17, R801, and W22.

HPLC is thus a convenient assay for identification of C-zein variants among inbreds and segregating populations. A manuscript is in preparation describing how the same HPLC system has identified additional variation among major A- and B-zeins, which were previously identified by IEF and SDS-PAGE.

A single nucleotide change in the DNA for C-zein could convert glutamine into a hydrophobic leucine residue. Such a substitution can increase SDS-PAGE mobility of a 20 kDa protein by 3% (W. W. deJong, Biochem. Biophys. Res. Commun. 82:532, 1978), probably because leucine binds more tightly to SDS. I suggest that such an amino acid replacement in C-zein might cause the modified molecule to move faster by SDS-PAGE, without changing molecular mass. Increased hydrophobicity of the modified C-zein would, however, cause it to bind more tightly to the RP-HPLC column and thus elute later.

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