SUNFLOWER 11S-GLOBULIN, SUSCEPTIBILITY TO PROTEOLYTIC CLEAVAGE OF THE SUBUNITS OF NATIVE HELIANTHININ DURING ISOLATION - HPLC FRACTIONATION OF THE SUBUNITS

The subunits of helianthinin, the major sunflower seed globulin, are susceptible to proteolytic cleavage or nicking of the acidic polypeptide chains by endogenous seed proteases during isolation. The course of the degradation was determined using reducing and non-reducing SDS-PAGE. This degradation can be partly retarded by including high salt and the protease inhibitor, phenylmethylsulphonyl fluoride in the extraction buffer. Separation of the globulins and albumins stopped the degradation of the helianthinin subunits. Rapid precipitation of the sunflower globulins with 60% methanol was the most effective way of isolating helianthinin with undegraded subunits. Native helianthinin was also extensively degraded by trypsin and chymotrypsin. Helianthinin was dissociated into its component subunits in 0.1 % trifluoroacetic acid and these were separated by reversed-phase HPLC. Eleven discrete fractions were obtained and analysed by SDS-PAGE. Six distinct subunits of Mr $ ̃56 000 and $ ̃52 000 and one subunit of $ ̃46 000 were identified. Three fractions contained essentially a single subunit, composed of two disulphide-linked polypeptide chains, suitable for further structural analysis. The amino acid compositions of the separated subunits of helianthinin are presented. © 1990.