Cereal Chem 62:327-331   |  VIEW ARTICLE
Improved Separation and Toxicity Analysis Methods for Purothionins.

B. L. Jones, G. L. Lookhart, and D. E. Johnson. Copyright 1985 by the American Association of Cereal Chemists, Inc. 

A high-performance liquid chromatography (HPLC) method has been developed for separating alpha1-, and alpha2-, and beta-purothionins from bread wheat. The separation utilized C-18 reversed-phase columns and maximal resolution occurred at ice-water temperature. Up to 4 mg of an alpha1- and alpha2-purothionin mixture could be separated in one run, using a preparative column. Separation of a 4-mg sample yielded alpha2-purothionin that was 97.4% pure, whereas HPLC separations of lesser amounts (up to 2 mg/run) gave essentially 100% pure material. Beta-Purothionin was completely separated from alpha2-purothionin and was partially separated from alpha1-purothionin by the HPLC method. A fast, reliable, and easily quantitated method for measuring the toxicity of purothionin to cultured mosquito and spruce budworm cells is reported. The test showed that cultured cells became more resistant to purothionin poisoning as they became older, and that 50% of fresh (three days old) budworm cells were killed by about 20 micrograms/ml of each of the three purothionin forms. Older budworm cells were less sensitive to beta-purothionin than to either of the alpha-purothionins, indicating there may be some difference in the ways the alpha- and beta- purothionins interact with the cells. Mosquito cells also showed LC50 values of about 20 micrograms/ml for all three purothionins. Subjecting alpha1- and beta-purothionins to HPLC separation conditions (pH 2.8, 7.3M acetonitrile) did not affect their toxicities to cultured cells.