Fluorescence spectra of tryptophan residues in human and bovine lens proteins

Abstract

The water soluble proteins from human and bovine lenses have been studied using fluorescence spectroscopy. Excitation of aqueous solutions of α, β and γ-crystallins at 290 nm produced fluorescence emission due solely to tryptophan residues. No emission attributable to tyrosine or phenylalanine residues could be detected. The fluorescence emission maxima for all of the human and bovine crystallins in aqueous solution were in the range 332±2 nm, and the bandwidths were in the range 52±2 nm. These results indicate that the tryptophan residues in these proteins exist mainly in hydrophobic environments in aqueous solution. All of the human and bovine crystallin fractions could be denatured by addition of 8 m-urea, 5 m-guanidine hydrochloride, or 1% sodium dodecyl sulfate (SDS). The former two reagents shifted the fluorescence maxima into the range 349±2 nm, indicating that the majority of tryptophan residues are exposed to water in these solutions. Addition of SDS produced only a small spectral shift to 335±1 nm, but produced a marked spectral broadening to 59±1 nm, again indicating significant protein denaturation. The fluorescence data for lens crystallins have been compared with corresponding data for α-chymotrypsin in an effort to classify the lens crystallin data into discrete spectral classes based on specific fluorescence properties of three types of tryptophan residues.