Identification and localization of kafirins in sorghum (Sorghum bicolor (L.) Moench) endosperm

Jeannette Christine McLaughlin Shull, Purdue University

Abstract

Kafirins are the storage proteins of sorghum found in protein bodies of the seed endosperm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, differential solubility, and Western blot analysis were used to demonstrate similarities between maize zeins and sorghum kafirins. Sorghum kafirins were separated into three overlapping groups based on molecular weight and solubility differences: Mr 25,000 and 23,000 (extractable in 40-90% t-butanol plus 2-ME), Mr 20,000, 18,000, and 16,000 (extractable in 10-60% t-butanol plus 2-ME), and Mr 28,000 (extractable in 10-80% t-butanol plus 2-ME). Reactions were observed on Western blots between $\alpha$-zein antiserum and Mr 25,000 and 23,000 kafirin polypeptides, $\beta$-zein antiserum and Mr 20,000 kafirin polypeptide, and $\gamma$-zein antiserum and the Mr 28,000 kafirin polypeptide. A nomenclature for sorghum kafirins was proposed based on similarities in molecular weight, extractability and structure to zeins. Immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. Protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed predominantly of $\alpha$-kafirins with minor portions of $\beta$- and $\gamma$-kafirins. The darkly stained inclusions do not contain $\alpha$-kafirins, but do not include $\beta$- and $\gamma$-kafirins. Sorghum decreases in protein digestibility after cooking, and the predominant indigestible proteins are kafirins. Transmission electron microscopy and immunocytochemistry were used to examine how cooking and the treatment with sodium bisulfite affect protein body microstructure. Protein bodies in uncooked samples were digested by pitting from the outer surface. In contrast, the protein bodies from the cooked sorghum did not exhibit any pitting, but did become ellipsoidal. Protein bodies in cooked samples which had been soaked in sodium bisulfite did exhibit shallow pits suggesting a reversal in the reactions that took place during cooking. In all treatments, light staining regions of the protein body were digested first leaving a dark staining remnant that was more resistant to digestion.

Degree

Ph.D.

Advisors

Kirleis, Purdue University.

Subject Area

Food science

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