Determination of glutathione and its redox status in isolated vacuoles of red beetroot cells

The glutathione of the red beetroot vacuoles ( Beta vulgaris L.) was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT); the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB); the high-performance liquid chromatography (HPLC). The content of reduced (GSH) and oxidized glutathione (GSSG) differed depending on the research method. With OPT the concentration of glutathione was: GSH - 0.059 µmol /mg protein; GSSG - 0.019 µmol/mg protein and total glutathione (GSHtotal) - 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH - 0.091 µmol/mg protein; GSSG - 0.031 µmol/mg protein; GSHtotal - 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH - 0.039 µmol/mg protein; GSSG - 0.007 µmol/mg protein; GSHtotal - 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT - 3.11; with DTNB - 2.96 and HPLC - 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione.