Quenching of tryptophan fluorescence in bovine lens proteins by acrylamide and iodide

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Abstract
The microenvironments of tryptophan residues in bovine .alpha.-, .beta.H-, .beta.L and .gamma.-crystallins have been examined using acrylamide and KI quenching of fluorescence. From a consideration of the differential effects of the two quenchers, the quenching efficiencies and spectral changes, it was possible to distinguish tryptophans in different environments and to assign these to specific residues in the sequence. Two classes of tryptophan were identified in .gamma.-crystallin, one buried and one moderately accessible. The buried class contained tryptophans 42A and 125 which lie in the angles of the wedge-shaped domains of the protein. These residues, which had emission maxima at 326 nm, were not accessible to quenching by iodide. The more accessible residues, emitting at 334 nm, corresponded to tryptophans 64 and 148 which are in the widest part of the wedge-shaped subunit and close to the surface of the protein. The two .beta.-crystallins were virtually indistinguishable. They contained two buried tryptophans, probably residues 58 and 150, and three close to the surface, residues 81, 84 and 166. The quenching efficiencies for these two classes were lower than those observed with .gamma.-crystallin. Since the three-dimensional structures of the .beta.- and .gamma.-crystallins are probably very similar, this suggests that the polymeric nature of the .beta.-crystallins is responsible for the decreased accessibility of the tryptophans to the quenchers. .alpha.-crystallin demonstrated unusually high static quenching which made it difficult to distinguish different classes of tryptophan. Nevertheless, it is probable that the tryptophan found in position 60 of the B chain is in a different microenvironment from that in position 9 of both chains. The results presented differ from other observations in the literature. Possible reasons for this are discussed.