Fluorescence lifetimes of chromophores in intact human lenses and lens proteins

Abstract

The time-dependence of fluorescence decay has been measured for whole human lenses and for some lens protein fractions, using a pulsed nanosecond light source. Two fluorescence regions were monitored: First, an ultraviolet fluorescence band characterized by 296nm excitation/332nm emission maximum, attributed to tryptophan residues. Second, a visible fluorescence band characterized by 359 nm escitation-435 nm emission maximum, attributed to age-dependent fluorescent chromophores in the lens. Both the ultraviolet and the visible fluorescence decays were complex, consisting of more than one exponential component: but to a first approximation, the data were analyzed as single exponentials and yielded effective-average lifetimes for the various fluorescence decays. Treated in this way, the visible fluorescence (detected at 435 nm) from several intact human lenses had a lifetime of 3·8±0·2 nsec, independent of lens age or cataractous condition, and independent of the anatomical region of the lens excited. This lifetime was compared with lifetimes measured for several model compounds previously proposed to be active fluorescent species in the lens. For the ultraviolet fluorescence band (detected at 332 nm), the effective lifetime in normal human lenses was found to vary from 1·8±0·1 nsec at the edge of the lens (cortex only) to 2·7±0·2 nsec at the center of the lens (cortex plus nucleus), independent of age. (Cataractous lenses showed minor variations from these values.) The effective lifetimes of the ultraviolet fluorescence bands of some lens protein fractions were also meaured. The soluble fractions α- and β-crystallin had lifetimes of 1·8±0·1 and 1·9±0·1 nsec respectively, while γ-crystallin had a lifetime of 2·4±0·1 nsec and the insoluble protein fraction had a lifetime of 2·6±0·1 nsec, all detected at 332 nm. Comparison of the whole lens and lens protein data suggests that the cortex is relatively richer in α-and/or β-crystallin, while the nucleus is relatively richer in γ-crystallin and/or insoluble protein. This does not mean, however, that there is no γ-crystallin whatsoever in the cortex or that there is no α-, β-crystallin in the nucleus.