[171] β-cyanoalanine synthase (blue lupine)

Publisher Summary This chapter focuses on β-Cyanoalanine Synthase (Blue Lupine). The assay is based on the colorimetric determination of H 2 S, one of the reaction products, by its conversion to methylene blue. Alternatively, radioactive cysteine or Hydrogen cyanide (HCN) can be used as a substrate and the β-cyanoalanine can be separated by paper chromatography and counted when the colorimetric assay cannot be used. Blue lupine ( Lupinus angustifolius L.) seed obtained from the Florida Seed and Feed Company, Ocala, Florida, is soaked for 15 hours in tap water with aeration, sown on vermiculite moistened with tap water, and germinated for 10 days in the dark at 25-28 °. The cotyledons and shoots are then harvested and 500 g of tissue is blended at full speed for 30 seconds at 0 ° with 1000 ml of 0.33 M sucrose, 0.1 M Tris-acetate buffer, pH 8.9, in a small Waring blendor. To exclude as much air as possible, the blendor is completely filled with liquid, a piece of plastic is placed over the top, and then the lid is placed on so that all the air along with a little liquid is forced out.