Probing the microenvironments of tryptophan residues in the monomeric crystallins of the bovine lens.
Tryptophan microenvironments have been examined in bovine beta s-, gamma II-, gamma IIIa-, gamma IIIb-, gamma IVa- and gamma IVb-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 beta s, gamma II and gamma IIIb, appeared to have a compact structure with none of the tryptophans accessible to KI and only moderately so to acrylamide. By contrast in gamma IIIa, gamma IVa and gamma Vb, 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 gamma IIIa, gamma IVa and gamma IVb contain two classes of tryptophan residues. One class (tau 0 = 0.52 ns, fa = 0.3, lambda max = 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 (tau 0 = 2.1-3.4 ns, fa = 0.7, lambda max = 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 (tau 0 = 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.
Augusteyn
R C
RC
National Vision Research Institute of Australia, Carlton, Vic.
Chandrasekher
G
G
Ghiggino
K P
KP
Vassett
P
P
D020106:Acrylamide; D000178:Acrylamides; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification; D011193:Potassium Iodide; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D003459:Crystallins; D011193:Potassium Iodide; D020106:Acrylamide; D014364:Tryptophan