Isolation and characterization of sorghum kafirin proteins and the development of ELISAs for the quantitation of alpha-kafirin, beta-kafirin and gamma-kafirin
Abstract
Alpha-kafirin, beta-kafirin and gamma-kafirin were isolated from grain sorghum by a combination of differential solubility, electrophoresis and column chromotography. These proteins were subjected to amino acid analysis. Alpha-kafirin was high in leucine and glutamine and low in both sulfur containing amino acids. Beta-kafirin was high in both cysteine and methionine, while gamma-kafirin was high in cysteine and histidine. All of the kafirin proteins were low in lysine. Amino acid contents of alpha-kafirin, beta-kafirin and gamma-kafirin resembled those of maize alpha-zein, beta-zein and gamma-zein, respectively. Structural similarity between gamma-kafirin and maize gamma-zein was demonstrated through an immunological study showing cross-reactivity of gamma-kafirin protein and gamma-zein anti-serum. Specific polyclonal antibodies were produced against alpha-kafirin, M$\sb{\rm r}$ 20,000 beta-kafirin and gamma-kafirin by injecting the isolated proteins into hens. The antibodies were partially purified from the eggs and used in the development of enzyme-linked immunosorbent assays (ELISA) for the quantitation of alpha-kafirin, M$\sb{\rm r}$ 20,000 beta-kafirin and gamma-kafirin. These ELISA assays, along with the Landry-Moureaux fractionation procedure and SDS-PAGE analysis were used to investigate qualitative and quantitative differences in the protein compositions of vitreous and opaque endosperms of sorghum. Vitreous endosperm contained 1.5-2% more protein than opaque endosperm of the same variety. This was due to the higher prolamin content of vitreous endosperm. Opaque endosperm contained more albumin/globulin type protein, while the amount of glutelin protein is similar in both vitreous and opaque endosperm. Quantitative differences were noted in the compositions of the protein fractions. The albumin/globulin fraction of opaque endosperm contained some higher molecular weight bands not seen in the albumin/globulin fraction of vitreous endosperm, while vitreous endosperm contained some lower molecular weight bands which did not appear in the opaque endosperm extracts. Differences were also noted in the kafirin fractions. Both the SDS-PAGE gel of a total kafirin extract and data from the ELISAs indicate that the kafirin fraction of opaque endosperm has a higher percentage of M$\sb{\rm r}$ 20,000 beta-kafirin and gamma-kafirin and a lower percentage of alpha-kafirin compared to the kafirin fraction of vitreous endosperm.
Degree
Ph.D.
Advisors
Kirleis, Purdue University.
Subject Area
Food science
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