Probing the microenvironments of tryptophan residues in the monomeric crystallins of the bovine lens

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Abstract

Tryptophan microenvironments have been examined in bovine βs-, γII-, γIIIa-, γIIIb-, γIVa- and γIVb-crystallins by fluorescence methods. The proteins could be divided into two groups on the basis of the accessibilities of their tryptophan residues. The first group, comprising βs, γII and γIIIb, appeared to have a compact structure with none of the tryptophans accessible to KI and only moderately so to acrylamide. By contrast in γIIIa, γIVa and γIVb, all tryptophans were readily accessible to acrylamide and 70% of the fluorescence could be quenched with KI. Spectral analysis, before and after quenching, time-resolved spectroscopy and simulations of the quenching curves suggested that γIIIa, γIVa and γIVb contain two classes of tryptophan residues. One class (τ0 = 0.52 ns, ƒa = 0.3, λmax = 324 nm) which was completely inaccessible to KI and relatively inaccessible to acrylamide (Ksv = 0.25 M−1), was assigned to the topologically equivalent residues in positions 42 and 131. The other class (τ0 = 2.1–3.4 ns, ƒa = 0.7, λmax = 330 nm) was accessible to both quenchers (Ksv = 5.00–5.15 M−1 and 2.47–2.60 M−1, for acrylamide and KI, respectively) and corresponded to the tryptophan residues in positions 68 and 157. The same classes may be present in the other low molecular weight proteins (τ0 = 0.47–0.55 and 1.55–1.74) but the lower emission and low accessibilities to quenchers prevented their distinction and suggested that these proteins had more compact structures.

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