Canavanine assay of some alfalfa varieties (Medicago sativa) by fluorescence: Practical procedure for canavanine preparation

Canavanine is extracted with 0.3 M H₂SO₄. Protein is removed by adding CHCl₃ and centrifugation. SO₄^t- is removed as BaSO₄. The canavanine is adsorbed to Dowex 50 (NH₄⁺ form) and eluted with 0.16 M NH₄OH. It is isolated by lyophilization and recrystallization from 90% ethanol. It is assayed, when present in high concentration, by weighing after lyophilization, and by the pentacyanoferrate reagent. For low concentrations, an improved fluorometric procedure is described. To 2 ml of an extract, 0.25 ml of 0.2 mM phenanthrenequinone in ethanol is added followed by 0.2 ml 0.5 M NaOH. After incubation at 37 °C for 1 hr, the cooled solution is acidified with 4 M HCl. The blank is minimized by extraction with 1 ml heptane. Excitation is at 305–308 nm, and emission is at 395–400 nm. Canavanine reduces with Pd-BaSO₄ to guanidine and homoserine. In the fluorometric procedure, guanidine has a sensitivity, 4.8 times that of canavanine on a molar basis, and 14.3 times per weight. Increase of fluorescence after reduction ascertains that canavanine is being assayed. The methods were applied to 7 varieties of alfalfa (Medicago sativa) seeds, leaves, and stems, 3 of clover seed (Trifolium repens and pratense), and the jack bean (Canavalia ensiformis). The values found for the seeds in grams/kilogram were 26 for the jack bean, from 8 to 15 for the alfalfa varieties, and from 4.1 to 4.25 for the clover. Alfalfa leaves contained 0.9-1.2 g/kg and stems 0.6-0.9 g/kg.