Glutathione fluorescence assay

Glutathione S transferases bind bile acids in vitro but doubt has been cast over whether this happens in vivo as these enzymes were not labelled by fluorescently labelled bile acids in experiments to identify the carrier proteins but may play a role with the raised levels in cholestasis. Liver fatty-acid-binding protein has been shown to bind bile acids by using a displacement assay with fluorescent fatty-acid ligand. This work clearly showed displacement to be directly related to hydrophobicity, such that lithocholate conjugates had the greatest effect. This may indicate a mechanism to minimise toxicity within the hepatocyte. 

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

Lavigne, V., Pons, A., Dubourdieu, D. (2007). Assay of glutathion in must and wines using capillary electrophoresis and laser-induced fluorescence detection. Changes in concentration in dry white wines during alcoholic fermentation and aging. J. Chromatog. A, 1139, 130-135. 

OPA has been known to give a fluorescent adduct with most primary amines in the presence of a thiol compound, but only with several biogenic amines such as histidine, histamine, and glutathione in the absence of a thiol compound in a neutral or alkaline medium. In the case of histidine, it gradually reacts with OPA alone in an alkahne medium, to give a relatively stable fluorescent adduct showing excitation and emission maxima at 360 and 440 nm, respectively. Hakanson et al. optimized these reaction conditions and showed that the fluorescence intensity due to histidine reached a maximum 10 min after initiation of the reaction at pH 11.2-11.5, at 40°C. This fluorescence reaction is relatively selective for histidine and has been used in a batch method for the assay of histidine. "... 

Because of the high selectivity and sensitivity of the postcolumn fluorescence detection of histidine with OPA, the present HPLC method is applicable to a specific and rapid assay of histidine in human semm, blood, and urine after simple pretreatment. A recent paper demonstrated that the postcolumn detection with OPA was applicable to the simultaneous assays of histidine and its major metabolites cis- and frawi-urocanic acids) in human stratum corneum. " The postcolumn detection system was also applicable to the flow injection analysis (FIA) method for the assay of histidine in semm and urine. The FIA method enabled us to determine histidine in blood after pretreatment of the sample with A-ethylmaleimide (masking reagent of glutathione).These methods are useful in the diagnosis of histidinanemia, one of hereditary metabolic disorders characterized by a virtual deficiency of histidine ammonia-lyase. 

Biochemical status estimates are generally based upon urinary excretion or measurements of erythrocyte glutathione reductase (NADPH oxidized glutathione oxidoreductase EC 1.6.4.2) and its reactivation with flavin adenine dinucleotide (FAD) in red cell lysates. Other biochemical indices, such as plasma or red cell flavin concentrations, have been less widely used, but their potential is increasing with the advent of new assay techniques such as capillary electrophoresis with highly sensitive laser-induced fluorescence detection. Fvmctional indices... 

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