Hew lysozyme

Low-viscosity Ch (~ 80% DD; 20–200 mPa s, Fluka, Germany) was purified as reported elsewhere (Hashemi Doulabi et al. 2013c (link)). Hen egg white (HEW) lysozyme (46,400 U/mg) and glutaraldehyde were purchased from Sigma-Aldrich (Germany). PEGF (M_w: 19, M_n: 16 kDa) was synthesized in-house previously by condensation polymerization of Fumaryl chloride with polyethylene glycol (PEG) diol M_w = 3 kDa, Merck Chemicals, Dusseldorf, Germany) in the presence of propylene oxide (Hashemi Doulabi et al. 2008 (link)). Fumaryl chloride and propylene oxide were obtained from Aldrich, Milwaukee, MN, USA. All other chemicals used were of reagent grade.

Hen egg-white lysozyme (HEW lysozyme, E.C. number: 3.2.1.17, lyophilized powder, L 6876, 50,000 units mg⁻¹ protein) and other chemicals (Thioflavin T, HCl) were purchased from Sigma-Aldrich Chemicals Company (St Louis, MO, USA). The ionic liquids (ILs), 1-ethyl-3-methylimidazolium tetrafluoroborate (>98%) (EMIM-BF₄) and 1-ethyl-3-methylimidazolium acetate (95%) (EMIM-ac), were purchased from IoLiTec (Germany). For all measurements, lysozyme powder was dissolved in aqueous solution containing 0%, 0.5%, 1% and 5% (v/v) ILs to a final lysozyme concentration 2 mg/mL (140 μM). The pH was adjusted to 2.5 by a small amount of 1 M HCl before each experiment and carefully checked at every experimental step. The protein concentration was determined spectrophotometrically (UV/Vis JASCO V-630 spectrophotometer) using an extinction coefficient at 280 nm equal to 38,940 M⁻¹ cm⁻¹. Ultrapure deionized water (Milli-Q) was used for the experiments.

ITC analysis was performed as previously described [12 (link)]. Briefly, 6xHis-2xFlag-RsiV^59–285 and mutants were purified as described above and buffer matched with HEW lysozyme (≥98% pure, Sigma Aldrich) by dialysis into 50 mM Na₂HPO₄, 200 mM NaCl, and pH 7.0 for 24 h at 4°C. Final protein concentrations as determined by absorbance at OD₂₈₀ were adjusted to 6xHis-2xFlag-RsiV^59–285 (0.01 mM) and HEW lysozyme (0.1 mM) with filtered dialysate. The protein samples were degassed and ITC measurements recorded using a MicroCal VP-ITC System (GE Healthcare) with HEW lysozyme as the injected sample and 6_XHis-2_XFlag-RsiV^59–285 as the cell sample. 21 injections of HEW lysozyme were used, with 180 seconds spacing between events. The chamber was kept under constant stirring at 350 rpm and all experiments were performed at 25°C. The binding reaction reached saturation during the experiment and control experiments where HEW lysozyme was injected into buffer showed that the heats of dilution were constant across all injections. The constant heat of dilution, as determined by the average of the last 3–5 injections, was subtracted and the data are analyzed using the single site binding model provided in the ITC analysis package. The values for affinity were averaged from three separate runs from two different protein preps.