Acrylamide and iodide fluorescence quenching studies on whole human lenses and their protein extracts

Tryptophan fluorescence was monitored in whole lenses (and in soluble crystallin fractions derived from these lenses) before and after incubating them in media containing acrylamide or iodide. The effects of exposing one lens to low level broadband UV radiation were compared with the contralateral lens incubated in the dark. In addition we employed 13C labeled acrylamide in order to monitor (by NMR spectroscopy) which crystallins were most effected. These studies demonstrate an acrylamide fluorescence quenching effect in whole lenses and extracted lens proteins, which is directly age related, as is the extent of its incorporation and binding to specific lens proteins. Iodide had no effect on TRP fluorescence in whole lenses and a slight effect on the extracted crystallins. These data demonstrate differences in the microenvironment of TRP residues in the various lens crystallins and their relative susceptibility to low level UV radiation exposure at doses approaching the ambient in vivo levels in a "real life" situation. This approach helps to delineate the microenvironment of TRP residues in native proteins and the effects of UV exposure on proteins with buried versus exposed TRP residues.