The Estimation of Thiols and Disulphides in Barley

Methods are described for the extraction and quantitation of endogenous barley thiols and disulphides. Thiols and disulphides, together with added internal standards, are extracted from ground barley tissues (embryos or degermed grains) and the thiols in the extract are trapped on columns of beaded p-hydroxymercuribenzoyl-agarose (p-HMB-agarose). After elution they are derivatised with 5,5′-dithiobis (2-nitrobenzoic acid) and the derivatives are separated and quantified by hplc. The disulphides in the effluent from the p-HMB-agarose column are collected on a silica-based ion exchange material. After elution they are reduced to thiols and, after derivatisation, they are assayed by hplc. Dry barley embryos contain substantial quantities of glutathione, progressively lesser quantities of oxidised glutathione, cystine and cysteine and traces of γ-glutamyl cysteine and cysteinyl glycine.