CONDENSED TANNIN OF SORGHUM GRAIN: PURIFICATION AND INTERACTIONS WITH PROTEINS

The polyphenolic compounds classified as tannins are present in many plants including the important cereal crop sorghum (Sorghum bicolor (L.) Moench) and have received attention recently because of their detrimental nutritional effects. Tannins are characterized by their ability to bind and precipitate proteins, but the mechanism and specificity of their interaction with proteins are largely unknown. To facilitate studies of tannin-protein interactions, a new method for purifying large amounts of tannin with minimal protein contamination was devised. The scheme has two unique steps for reducing protein contamination: An ethanol pre-extraction of the plant tissue removes alcohol soluble protein and prevents its subsequent co-purification with tannin, and a phenol extraction removes non-covalently bound protein from the partially purified tannin. The tannin prepared by this new method contains less than 3% protein. Up to 12 mg of serum albumin can be precipitated by 1 mg of the purified tannin. Tannin specific activity, which describes the effectiveness of tannin as a protein precipitant, is defined as the ratio of the amount of protein precipitated to the amount of oxidizable material detected by the Prussian blue assay. As nontannin oxidizable material is removed during purification of tannin, the tannin specific activity increases. Tannin specific activity can be used to compare chemically diverse tannins from different sources. Three new assay methods are described here. With the first method, tannin in the precipitated tannin-protein complex is determined colorimetrically. After the precipitated complex is isolated by centrifugation, it is dissolved in an alkaline detergent solution and ferric chloride is added to develop the color. In the second method radioactively labeled protein is utilized, and the amount of protein in the precipitate is determined by its radioactivity. These two assays allow simultaneous determination of both components in the precipitated complex. With the third assay the amount of radioactively labeled protein bound by tannin in ground grain is measured. This method may be valuable for predicting the nutritional quality of tannin-containing foods. Using these assays, the interactions of tannin with various proteins were characterized. It was found that tannin-protein complexes are stabilized both by hydrogen bonds and by hydrophobic forces. Proteins were precipitated by tannin most efficiently at pH values near their isoelectric points. The affinity for tannin was inversely proportional to the size of the polymer. Peptides with less than five residues interacted very weakly with tannin. At pH 4.9, the relative affinities of a series of proteins for tannin ranged over five orders of magnitude, indicating that tannin interacts with proteins with surprising selectivity. The proteins and polymers with the highest affinities for tannin were characterized by high proline content or conformationally loose structures, while tightly coiled globular proteins had lower affinities for tannin. It was concluded that proline-rich compounds interact strongly with tannin because hydrogen bonding is enhanced by the presence of the imino acid proline. Conformationally loose compounds are probably more accessible to tannin than are tightly coiled compounds and thus interact more strongly. The unexpected specificity of interaction between tannin and protein described here suggests that tannins may be functionally similar to animal antigens