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 lM) 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 L-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine AE HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of L L-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

“…In contrast, HEWL aggregation is facile and spontaneous with rise in pH as shown previously [13], [14]. Subsequent work from our lab has revealed that aggregation of HEWL at pH 12.2 is a slow (0—12 hours) but concentration dependent spontaneous process at 298 K [15]. This aggregation was instantly triggered by negligible net charge and increased exposure of hydrophobic surfaces in HEWL at high pH.…”

“…HEWL was covalently labeled with dansyl chloride (2-dimethyl aminonaphthalene-6-sulfonyl chloride) following the protocol recommended by Molecular Probes with minor modification as reported previously[15]. For labeling with dabcyl (4-((4-(dimethylamino) phenyl) azo) benzoic acid succinimidyl ester), the protocol suggested by Molecular Probes was followed.…”

“…Steady state fluorescence intensity was measured after excitation at 380 nm (slit  =  1 nm). Emission (slit  =  16 nm for 0.3 µM, 8 nm for rest) was collected in the range 400-600 nm as reported previously [15]. For experiments related to binding of ANS to HEWL aggregates transferred to pH 7, 10 µM ANS was employed in all samples, while identical slit widths were maintained throughout.…”

“…The steady-state fluorescence anisotropy, r ss was measured after G-factor correction and dark counts subtraction as described previously [15], [16]. For HEWL monomer concentrations 3—120 µM, the concentration of dansyl-conjugated HEWL was 0.6 µM, while remaining protein was unlabeled.…”

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

“…At pH levels of between 9 and 11, native lysozyme showed a substantial decline in activity, while the derivative LZ-mPEG-SS 5000 retained between 70% and 80% of its activity. As noted by Homchaudhuri et al (2006), native LZ forms aggregates at pH 12.2 that can lead to the loss of enzymatic activity at 25°C.…”