Fluorometric Quantitation of Cytochrome C Oxidase Activity in Cultured Crystalline Lenses

Abstract

Abstract: : Purpose:The role of cytochrome c oxidase in the synthesis of ATPs in the lens is important particularly for the differentiation of secondary lens fibre cells from the epithelium. This study was designed to develop a new approach for quantitative biochemical evaluation of mitochondrial cytochrome c oxidase activity in UV treated cultured lenses over a time period. Methods:Porcine lenses were maintained in cultured chambers containing M199 medium supplemented with 1% antibiotic. At one week of preincubation at 37°C and 4% CO₂, the lenses were randomly assigned to untreated control and 0.48 J/cm² UVB treated groups. The pre and post UV treatment fluorescence readings of both control and treated lenses were obtained at various intervals for 4 weeks using the alamarBlue assay and a plate reader. Results:Analyses show statistically significant (p < 0.05) decrease in fluorescence capability in the UV treated lenses beginning from 96 hr after UV treatment. In contrast, no decrease in fluorescence was obtained in the control lenses through the duration of experiment. Conclusions:The interaction of the nonfluorescent alamarBlue dye with the cytochrome c oxidase enzyme in the mitochondria of the lenses yielded fluorescent emission in numerical values for quantitative comparison and statistical analysis. The decrease in fluorescence obtained in the UV treated group exemplifies UV impairment of cytochrome c oxidase activity in lenses. The results show that this approach would be suitable for in vitro assessment of cytochrome c oxidase activity in crystalline lenses without the risk of contamination.

Keywords: mitochondria • radiation damage: light/UV • enzymes/enzyme inhibitors