Abstract
The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half‐width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8__M__) solutions, and in aqueous guanidine hydrochloride (0–6.4__M__) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin.
The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure‐breaking action of urea on water in the cleft. 4__M__ Urea is sufficient for this reaction.
Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6__M__ and 3.2–4.8__M__ guanidine hydrochloride ranges demonstrated two guanidine hydrochloride‐induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4__M__ guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.