A toxic thionin from Pyrularia pubera: Purification, properties, and amino acid sequence

Abstract

A low-molecular-weight cytotoxic protein has been purified from Pyrularia pubera Michx. (Santalaceae). By comparison with the behavior of proteins of known molecular weight during Sephadex G-75 gel filtration and denaturing electrophoresis, a molecular weight of somewhat less than 6000 is indicated. Purification involves ammonium sulfate fractionation followed by either gel filtration on Sephadex G-75 or separation on a carboxymethyl cellulose CM52 column. At concentrations of 0.04 mg/ml the protein causes visible disruption of cultured mouse B16 melanoma cells. The complete amino acid sequence has been determined. The toxin contains 47 amino acids arranged as follows: LysSerCysCysArgAsnThrTrpAlaArgAsnCys TyrAsnValCys ArgLeuProGlyThrIleSerArgGluIleCysAlaLys LysCysAspCysLys IleIleSerGlyThrThrCysProSerAspTyrProLys OH. The protein is clearly a thionin, as shown by its close resemblance to the thionins from wheat and barley, to the viscotoxins from mistletoes, and to crambin.