Biochemical Characterization of Sorghum Storage Proteins (Kafirins)

Abstract

Proximate analysis was done for sorghum seed and defatted seed flour of Dabar variety. The protein present in sorghum endosperm in terms of solubility, molecular size, aggregation behaviour and subunits composition have been characterised. The solubilization of sorghum flour with 60% tr-butanol allows the recovery of a fraction of protein in which more than 75% are in the form of S-S linked (cross-linked) subunits. When kafirins are reduced, the subunit corresponds to α1-, α2 and γ-kafirins that on the other hand, are present in the unreduced extract as monomers. On the contrary, β- kafirins do not participate to the cross- linked aggregates extracted with aqueous alcohol, although this protein seems to be present only in trace amounts in the polymers with the higher molecular size. Sonication was also used to extract the kafirin that remained in the residue after alcohol extraction. Sonicated kafirin remains soluble in aqueous alcohol resembling the classical unreduced prolamins. Therefore, it seems that the extractability of a part of the kafirins is actually due to their degree of polymerization via S-S bonds. In fact non-covalent bond is unlikely, since the extraction with 2% SDS did not allowed the recovery of any protein. The polymers extracted by sonication and present in the SE-HPLC excluded peak representing more than 80% of the total kafirin extracted after sonication , are likely to be fragments deriving from the rupture of some of the S-S bonds present in the original unextractable high molecular size polymers. From the comparison of the proteins extracted with 60% tr-butanol and those extracted with sonication, it seems that α2-and γ- kafirins are present in all the polymers and oligomers, whereas β- kafirin seems to be present only in the polymers of high molecular weight. This protein, in fact present in the polymers extracted with sonication, whereas is absent in those extractable with alcohol alone. Moreover a very high quantity of β-kafirin is directly related to the molecular size of the polymers, whereas γ-kafirin, despite its very high number of cysteine residues is distributed, along with α1-kafirin in all the different sized polymers. α2-kafirin is mainly represented in the alcohol-extractable oligomers and, among the latter , as a part of those has a low molecular size. So it seems that α2-kafirin has a tendency to dimerize, but not to participate to the formation of large S-S bonded structures. It can be concluded that the majority of the prolamins are present in sorghum endosperm as polymerized proteins, although some monomeric kafirins are also detected. The distribution between these two molecular states can not be based on extractability because a large proportion of polymerized proteins can be solubilized, along with the monomers, without the aid of a reducing agent. However, the subunit composition of the differently sized polymers is variable according to their different extractability. Different sorghum kafirin aggregates can be extracted with different tr-butanol concentrations ranging from monomers up to polymers. The optimum tr-butanol concentration to extract the maximum quantity of polymers, trimers and β-kafirin was 60% (v/v), whereas for dimers was 80%, which reflects their strong hydrophobic nature. On the other hand for the free α-and γ-kafirins, more or less the same quantities of protein can be extracted with 60 and 80% tr-butanol. The effect of pepsin digestion, germination and fermentation on kafirin aggregates, have been studied using SDS-PAGE and HPLC. The degradation of the aggregates by the enzymes produced during these processes took place with the same mechanism, but differed in the degree of the effectiveness. The results supported the evidence which stated that the degradation of the kafirin aggregates initiated progressively from the surface of the protein bodies. In fact the degradation started with the high molecular sized aggregates and then the other types of protein were degraded. It was noted that, the effect of fermentation on kafirin aggregates was not appreciable compared to pepsin digestion and germination. The improvement of sorghum protein digestibility by fermentation may take place mainly by reducing tannin contents since the results presented here indicated that it has no significant effect on prolamins degradation. After cooking sorghum kafirin became less extractable and its extractable quantity decreased as the cooking times increased, simply because an enzymatically resistant protein polymer was formed during cooking through disulphide bonding. On the contrary the cross-linked kafirin extracted with 60% tr-butanol containing 5% ME increased as the cooking times increased. This postulated that cooking caused the formation of protein polymers which can not be extracted without the use of reducing agent. Literally the reduced kafirin (60% tr- butanol extract protein) and cross- linked kafirin (fraction extracted with 60% tr-butanol containing reducing agent) contain mostly the same proteins with quantitative difference in their amounts. Using SDS-PAGE, acid –PAGE AND 2-dimensional –PAGE (A-PAGE X SDS-PAGE) the results presented here indicate that, there was a qualitative significant difference between the two fractions.