Chicken egg lysozyme

5 mg chicken egg lysozyme (Sigma-Aldrich) was dissolved in 950 μl of 0.1 mM NaHCO₃ buffer, pH 8.3. 1 mg azide-PEG2-NHS ester (BroadPharm, San Diego) was dissolved separately in 50 μl DMSO. The two solutions were mixed together and incubated for 2 h at 22°C with gentle rotation of the tube. 50 mM ethanolamine was then added and the reaction solution incubated for a further 0.5 h at 22°C to quench unreacted NHS ester. The reaction mixture was then passed through Mirocon-10k centrifugal filters (10k Da size cut-off, Cedarlane, Burlington). Two further washes, each with 400 μl ddH₂O, were carried out. The concentrated protein was dissolved in about 100 μl 10 mM HEPES buffer (pH 7.4). The concentration of protein was determined by the BCA assay (BCA protein assay kit, Thermo Fisher Scientific, Toronto), and mass spectrometry was carried out to verify the azide-labeling.

The turbidimetric method of Litwack^{67 (link)} was used to measure the amount of lysozyme in blood plasma. Micrococcus lysodeikticus bacterium standard solution was prepared by adding 0.6 mg of M. lysodeikticus for every millilitre of phosphate buffer (0.05 M Na₂HPO₄⋅2H₂O: disodium hydrogen phosphate dihydrate, in 1000 ml distilled water; pH 6.2). A 100 µl of blood serum was taken into a cuvette on a spectrophotometer set at 450 nm. The cuvette was filled with 650 µl of M. lysodeikticus solution. After 30 s of combining the bacterial solution and serum, the initial absorbance reading was taken. The absorbance was recorded for 3 to 5 min at 30-s intervals. The absorbance was put on the standard curve's Y-axis, and the lysozyme concentration was calculated by back-calculation. The standard curve was plotted by chicken egg lysozyme activity against the bacterial solution. The powdered form of chicken egg lysozyme (Sigma Chemical Company, St. Louis, USA) was diluted into a stock solution of 40 ml by adding 1 mg of lysozyme powder to the buffer. The following standard solutions were used: 1, 2, 4, 5, 8, and 10 µg/ml). The standards were reacted with the bacterial solution. A standard curve was plotted to put the optical density along the Y-axis, with the standard concentration along the X-axis.

DNase I, NAD⁺, p-iodonitrotetrazolium violet (INT), L- and D-glutamic acid, L-glutamate dehydrogenase, diaphorase, isopropyl-β-D thiogalactopyranoside (IPTG), bovine serum albumin (BSA), chicken egg lysozyme, kanamycin were purchased from Sigma-Aldrich. Other chemicals were reagent grade.

Glycation induced protein cross-linking inhibitory effect of COS was assessed using the method described by Perera and Ranasinghe [37 ]. Briefly, chicken egg lysozyme (Sigma) was incubated with fructose in the presence or absence of 250 μg/ml or 2 mg/ml COS extracts for 14 days. Other conditions were as described in the fructosamine assay and incubated for 14 days. Aliquots were collected at day 6 and 14 and analyzed for the appearance of high molecular weight products using sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was carried out with the Enduro Vertical Gel Electrophoresis system- E2010-P according to the standard Laemmli method using 12 % SDS-polyacrylamide gels [36 (link)]. Gels were stained with Coomassie brilliant blue. Appearance of high molecular weight products of lysozyme in the aliquots was compared. Experiments were repeated three times.