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.

[Plate 5]Some of the constituent amino-acids of fibroin (degummed silk) are determined. Special attention is directed to histidine, owing to its use in the calculation of the molecular weight of fibroin. A value of 0*45 % has been found by methods in which the histidine is isolated as nitranilate or di-(3:4-dichlorobenzenesulphonate). Other values obtained are serine 12*6%, threonine 1-5 %, tyrosine 10*6 %, and proline 1*5 %. Hydroxyproline appears to be absent, but the presence of small amounts of some hydroxyamino-acid other than serine and threonine is indicated.The mean residue weight of fibroin is determined by three methods, one of which is a new method based on analysis of the complex formed between fibroin and cupri-ethylenediamine. This method gives a Cu:fibroin-N ratio of 1: 1*92 and, if allowance is made for co-ordination with the tyrosine hydroxyl group, an equivalence of 1*964 atoms of peptide-nitrogen to 1 atom of copper is obtained. The three methods give an average value of 78*0 for the mean residue weight of fibroin. This value, together with the most acceptable data for amino-acid constituents, indicate that the unidentified anhydro-residues (about 20%) have a mean residue weight of about 107.Evidence is presented that fibroin contains no amide-nitrogen. Methods for the deter mination of amide-nitrogen at present in use, which indicate a content of 1 to 2 %, are considered to be unreliable.Fibroin dissolved in cupri-ethylenediamine gives, on neutralization and dialysis of the resulting solution, a water-soluble protein. The production of this water-soluble protein is attended by little or no degradation of the original fibroin as shown by determinations of fluidity, amino-nitrogen, and acid-and alkali-combining power.The water-soluble protein is precipitated by the normal protein-precipitating reagents, but in every instance examined the precipitated material exhibits an insolubility com parable with that of the original fibroin.Factors responsible for the solubilization process are investigated and data for molecular weight, titration values, ultra-violet absorption spectra, reducing activity, optical rotation, tryptic hydrolysis, and viscosity for both soluble and dispersed fibroin are given. Soluble fibroin has [a]^5 -53*1° and dispersed fibroin [a]*® -58*9°, both in aqueous media.The preparation and properties of films and filaments of fibroin are described. Films of fibroin can be prepared that are water-soluble. On stretching, these films show strainbirefringence, acquire considerable tensile strength, and become insoluble in water, but X-ray examination gives the /?-keratin pattern for both the stretched and unstretched films.Reasons are advanced for considering the water-soluble form of fibroin to be the native or renatured protein and the original protein to be the denatured form.The denaturation of fibroin is discussed on the basis that denaturation is essentially an unfolding of a coiled long-chain molecule. The subsequent aggregation of the uncoiled molecules to give an insoluble pr...

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Abstract. The thermal melting of bovine serum albumin (BSA) in the presence of excipients like ethanol and sucrose was studied by circular dichroism spectroscopy at physiological pH in phosphate buffered saline. Calculated apparent Tm values were used to assess the thermal stability using two state fitted experimental curves. 0.5 M sucrose stabilized the BSA indicated by the increase in Tm of ∼8 • C when compared to the Tm of the same solution measured in the absence of sucrose. Conversely, in the presence of varying concentrations of ethanol (2-20%), the protein was destabilized by a decrease of ∼3-10 • C of Tm. In the binary mixture of sucrose and ethanol, the Tm values showed that ethanol dominantly destabilized BSA in the presence of sucrose, possibly by weakening the hydrophobic interactions in the protein.