Spectrophotometric estimation of 3-OH L-kynurenine O-β-glucoside in the human lens

Abstract

A low molecular weight yellow substance with absorption maxima at 228, 263, and 365 nm was separated from the water soluble fraction in human lens by gel filtration on a Sephadex G-15 column. The absorption spectrum of the yellow substance shifted to that of 3-OH L-kynurenine (absorption maxima; 228, 267, and 368 nm) after hydrolysis with β-glucosidase [EC 3.2.1.21]. Concomitantly, its absorbances at 228 and 365 nm decreased; the spectra of both compounds intersect at 235, 249, 263, 305, and 387 nm (isosbestic points). The hydrolytic product of the yellow substance was shown to contain 3-OH L-kynurenine (by amino acid analysis) and glucose (by gas chromatography). The yellow substance was thus identified as 3-OH L-kynurenine O-β-glucoside. Since the molar extinction coefficient of authentic 3-OH L-kynurenine was found to be 3650 M-1* cm-1 at 368 nm, the molar extinction coefficient of the glucoside can be calculated to be 4340 M-1, cm-1 at 365 nm from the absorbance ratio of the glucoside at 365 nm and of 3-OH L-kynurenine at 368 nm to the absorbances at the isosbestic points.By employing the molar extinction coefficient thus obtained, the amount of 3-OH L-kynurenine O-β-glucoside isolated from human lens was estimated. The glucoside content in whole lens and the specific content, expressed as umol per g lens protein, both decrease with age. In particular, the specific content decreases linearly with age over the period from birth to the age of 30-40, and subsequently it seems to remain at a constant level. © 1981, by the Japanese Biochemical Society.