Biochemical characterization of sorghum storage proteins (Kafirins) [Sudan]

Proximate analysis was done for sorghum seed flour 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 karfirin are reduced, the subunits correspond to Q1-Q2 and y-kafirins that on the other hand, are present in the unreduced extract as monomers. On the contrary, B-kafirin 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 alcoholresembling the classical unreduced prolamins. Therefore, it seems that the extractability of a part of the kafirins is actually due to their degree of polymerisation 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-HPLIC 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 siz polymers. From the comparison of the proteins extracted with 60 tr-butanol and those extracted with sonication, it seems that Q2- and Y-kafirins are present in all the polymers and oligomers, whereas B-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 B-kafirin is present in the residue remained after sonication, which can consider as containing the polymers with the high molecular weight. The results presented here provide evidences that B-kafirin is directly related to the molecular size of the polymers, whereas Y-kafirin despite its very high number of cysteine residues is distributed, along with Q1-kafirin in all the different sized polymers Q2-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 Q2-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 polymerised 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 polymerised 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 B-kafirin was 60 (v/v), whereas for dimers was 80, which reflects their strong hydrophobic nature. On the other hand for the free Q- and Y-kafirin, 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 evidences which stated that the degradation of the kafrirn 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 appreeiol compared to pepsin digestion and germination. The improvement of sorghum protein digestibility by fermentation 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 polymers were formed during cooking through disulphide bonding. On the contrary the cross-linked kafirin extracted with 60 tr-butanol containing 5 ME and total protein extracted with 2 SDS 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 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 indicated that, there was a qualitative difference between the two fractions