A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from the mold Aspergillus giganteus MDH 18894 has been developed. alpha-Sarcin and AFP were purified simultaneously by carboxymethylcellulose 52 cation-exchange chromatography and Blue Sepharose CL-6B affinity chromatography. By this method, 4.2 mg of alpha-sarcin and 6.8 mg of AFP were obtained from 2 liters of medium. Compared with other purification methods such as gel-filtration chromatography, this procedure was simple and specific. The purified alpha-sarcin and AFP were homogeneous characterized on SDS-polyacrylamide gel. The enzymatic activity of several ribosome-inactivating proteins such as alpha-sarcin, trichosanthin, and cinnamomin was significantly inhibited by the dye Cibacron blue F3GA. In 50 microliter of reaction mixture, 10 microM of the dye could inhibit 50% activity of cinnamomin (7 x 10(-9) M), whereas 50% inhibition of the enzymatic activity of trichosanthin (7 x 10(-9) M) and alpha-sarcin (1 x 10(-7) M) required 100 and 50 microM of the dye, respectively.
Copyright 1998 Academic Press.