Separation of alcohol soluble sorghum proteins using non-porous cation-exchange columns

Kafirins, the storage proteins and major protein of the cereal grain sorghum, play an important nutritional role for millions of people in parts of Africa and Asia. Kafirins are non-water soluble, being soluble only in the presence of detergents or aqueous alcohol mixtures and are among the most hydrophobic of the cereal proteins. Limited Mw heterogeneity of kafirins reduces their resolution when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Charge based separation techniques have been shown to have improved resolution of kafirins, but due to the nature of their solubility, ion-exchange (IE)-HPLC has not been widely used to separate these proteins. To overcome issues of solubility, two different mobile phases were evaluated. The first mobile phase was based on 60% acetonitrile at acidic pH using guanidine-hydrochloride (Gdn-HCl) gradients to elute the proteins from a non-porous cation-exchange column. The second mobile phase tested consisted of 60% acetonitrile using an increasing concentration gradient of a triethylamine phosphate (TEAP) buffer at pH 3.0. The type of alkylation reagent used to stabilize kafirin extracts prior to analysis was found to have an impact on the IE-HPLC separations with the reagent 4-vinylpyridine providing the best resolution. Separations of kafirins in the TEAP mobile phase system resulted in 10 major peaks being resolved. Combining IE-HPLC with reverse phase (RP)-HPLC into 2D separations revealed that the α-kafirins clustered into three major groups not readily apparent in either 1D separation.

► We optimized a method for analyzing non-water soluble sorghum proteins by ion-exchange HPLC.
► Ion-exchange separated kafirins with high resolution.
► Combining ion-exchange HPLC with RP-HPLC resulted in two-dimensional separations of kafirins.