ISOLATION AND PURIFICATION OF 3 ANTIFUNGAL PROTEINS FROM SORGHUM ENDOSPERM

Three proteins potently inhibitory to the growth of Fusarium moniliforme, the grain mould pathogen have been identified from sorghum endosperm, using aqueous extraction, gel filtration and ion-exchange chromatography coupled with bioassay. The aqueous extract (Tris buffer, pH 6.8) of sorghum endosperm flour was separated into two fractions by passing through Sephadex G-50. The void volume fraction, showing antifungal activity was further fractionated by ion-exchange chromatography on SP Sephadex C-50. The most potent fraction was that which bound to the SP Sephadex column at pH 6.0. A less potent fraction with an 18 kDa protein as major component was released on shifting the pH to 8.5. The bound fraction at pH 8.5 was a much more potent inhibitor of fungal growth than the unbound fraction; and it was composed of two major polypeptides with molecular weights of 26 and 30 kDa. All three polypeptides were separated on SDS-PAGE gels, transferred onto Immobilon and tested for antifungal activity. The test showed that the 26 and 30 kDa proteins retained the antifungal activity even after electrotransfer. Antibodies raised against the electroeluted proteins were shown to abolish the antifungal activity of the endosperm extracts. These proteins appeared only in the endosperm of sorghum grains as revealed by dot immunobinding assay. The 26 and 30 kDa antifungal proteins were also present in pearl millet and maize, while the 18 kDa protein was found only in sorghum. Cross-reactivity of antibodies with the aqueous extracts of wheat, rice and ragi was not seen.