Carboxymethylation of Cysteines Impedes Aggregation of Hen Lysozyme in Alkaline pH

Abstract

In our previous work, we have shown that hen lysozyme spontaneously forms globular and fibrillar aggregates which are stabilized later through formation of intermolecular disulphide bond in pH 12.2. Therefore, focus of the present work was on suppression of HEWL aggregation by carboxymethylation of free -SH groups with iodoacetamide that inhibits further formation of disulphide bonds. Changes in structure and dynamics of aggregates were monitored using various biophysical techniques like fluorescence spectroscopy and atomic force microscopy (AFM). Thioflavin T (ThT) fluorescence showed that in comparison to free thiol containing HEWL control, carboxymethylated HEWL was unable to form fibrils. However presence of moderate ThT intensity in modified HEWL indicated presence of oligomers. Increased fluorescence intensity and marginal red shift of ANS spectra in modified HEWL compared to unblocked control revealed that modified HEWL possessed exposed hydrophobic residues compared to free -SH containing HEWL. Fluorescence anisotropy of carboxymethylated dansyl-labeled HEWL were significantly lower compared to control. The fluorescence anisotropy decay kinetics revealed that carboxymethylated HEWL-dansyl conjugates possessed shorter global rotational correlation time (∼11.7 ns) compare to control (∼22 ns) after 30 hours of incubation in pH 12.2 at room temperature. Taken together, these investigations suggest that blocking thiol groups in HEWL suppresses formation of amyloid fibrils and bigger aggregates but not oligomers, in pH 12.2 at room temperature. This work can be helpful for food industries in making aggregate-free lysozyme preservatives in future.Authors acknowledge Department of Science and Technology, New Delhi and University Grants Commision New Delhi for financial support.