AIM:The current work is aimed at understanding the effect of pH on the thermal stability of hen egg white lysozyme (HEWL) at high concentration (200 mg/mL).MATERIALS AND METHODS:Fourier Transform Infrared (FTIR) Spectroscopy with modified hardware and software to overcome some of the traditional challenges like water subtraction, sample evaporation, proper purging etc., are used in this study.RESULTS:HEWL was subjected to thermal stress at pH 3.0-7.0 between 25°C and 95°C and monitored by FTIR spectroscopy. Calculated Tm values showed that the enzyme exhibited maximum thermal stability at pH 5.0. Second derivative plots constructed in the amide I region suggested that at pH 5.0 the enzyme possessed higher amount of α-helix and lower amount of aggregates, when compared to other pHs.CONCLUSIONS:Considering the fact that HEWL has attractive applications in various industries and being processed under different experimental conditions including high temperatures, our work is able to reveal the reason behind the pH dependent thermal stability of HEWL at high concentration, when subjected to heat denaturation. In future, studies should aim at using various excipients that may help to increase the stability and activity of the enzyme at this high concentration.

The purpose of this research is to study the thermal unfolding of high concentration bovine Immunoglobulin G (IgG) under 26 different experimental conditions by Fourier Transform Infrared spectroscopy with improved purge conditions and software calculations. When bovine IgG (25-200 mg/mL) was thermally denatured between pH 4.0 and 8.0, it was observed that at 25 mg/mL concentration, the protein exhibited maximum thermal stability at pH 6.0 and 7.0 as evident from the apparent T(m) values. Increasing the concentration from 25 to 100 mg/mL at those pH values increased the thermal resistance of the protein by 2-3 °C. But, at 200 mg/mL, IgG showed a small decrease in its transition temperature. Presence of 100 mM Trehalose enhanced the T(m) values at all conditions and possibly prevented the complete loss of IgG as insoluble aggregates at higher temperatures. Second derivative plots were constructed to explain the conformational changes of IgG during thermal unfolding.