Slow aggregation of lysozyme in alkaline pH monitored in real time employing the fluorescence anisotropy of covalently labelled dansyl probe

Abstract

The onset of hen egg white lysozyme aggregation on exposure to alkaline pH of 12.2 and subsequent slow growth of soluble lysozyme aggregates (at 298 K) was directly monitored by steady-state and time-resolved fluorescence anisotropy of covalently attached dansyl probe over a period of 24 h. The rotational correlation time accounting for tumbling of lysozyme in solution (40 μM) increased from ∼3.6 ns (in pH 7) to ∼40 ns on exposure to pH 12.2 over a period of 6 h and remained stable thereafter. The growth of aggregates was strongly concentration dependent, irreversible after 60 min and inhibited by the presence of 0.9 M l-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine · HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of l-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.