In 1959 Shore, Burkhalter and Cohn (1) reported that the reaction of o-phthalaldehyde with histamine yielded a highly fluorescent reaction product which could be used for the assay of submicrogram amounts of histamine in biological tissues. Modification of this reaction and/or the extraction techniques yielded sensitive fluorometric methods for the assay of histidine (1, 2), carnosine (2), arginine (3), agmatine (3), and several enzyme systems utilizing or forming the above amino acids and amines (2-4) *
This paper reports a further modification of the reaction procedure with o-phthalaldehyde that yields a highly specific and sensitive fluorometric assay for reduced glutathione.
MATERIALS
Glutathione
(GSH) and oxidized glutathione (GSSG) obtained from Boehringer und Soehne were used as reference standards. Other thiols, amino acids, peptides, etc. were the highest purity obtainable from either Calbiochem or Mann Research Laboratories; 6-mercaptopurine was obtained from Sigma Chemical Company and 6-thioguanine was prepared by Francis Earle Laboratories; o-phthalaldehyde was obtained from Mann Research Laboratories and was utilized without further purification.
An Aminco-Bowman spectrophotofluorometer was employed for all determinations of fluorescence spectra and intensity.