Guanidine hydrochloride

All reagents and chemicals were of analytical grade and applied without further purification. HPLC-grade deionized water was used during all experiments.
α-KG, 5-HMF, NASeLM, NALM, and glucose were supplied by CYL-Pharma (Lassnitzhöhe, Austria). NaCl, KCl, KH₂PO₄, MgSO₄, CaCl₂, NaHCO₃, and dextrose were obtained from Roth (Karlsruhe, Germany). Acetonitrile, ammonium acetate, 1-butanol, ethanol, HPLC-grade water, hydrogen peroxide, and HCl were obtained from Merck (Darmstadt, Germany). Albumin from human serum, angiotensin 1-7 acetate salt hydrate, butylated hydroxytoluene (BHT), 2,4-dinitrophenylhydrazine (DNPH), ethyl acetate, guanidine-hydrochloride, malondialdehyde tetra-butyl-ammonium salt (MDA), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (Vienna, Austria).

PA was purchased from Ultra Scientific (Philadelphia, PA 19122, USA), while fatty acid-free albumin from human serum was from Sigma-Aldrich (St. Louis, MO 63103, USA). EGCg (>95%) was obtained from Lipton Tea Co (Secacus, NJ 07094, USA). Reagent grade sodium phosphate was used to prepare 20 mM, pH 7.0 buffer used in all experiments, and guanidine hydrochloride was purchased from Sigma-Aldrich. HSA was labeled with one of two fluorescent probes, 6-propionyl-2-(dimethylamino) naphthalene (prodan) or 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CPM), for FRET distance determination. Prodan was purchased from Life Technologies (Waltham, MA 02451, USA), while CPM was obtained from Chemodex (Buckingham, Bucks MK18 1EG, UK).

Guanidine hydrochloride (5.5 M, Sigma) was added for 15 min to denature the purified re-AuFaeA or re-AuFaeA^A126C-N152C (final concentration of 2 mg/mL) [34 ]. Thiol titration was performed by incubating thirty parts of denatured re-FAE with one part of 4 mg/mL dithionitrobenzoic acid in 0.25 M Tris-HCl buffer (pH 8.0) for 15 min at 25°C, and the absorbance was measured at 410 nm in a spectrophotometer [35 (link)]. The wild-type re-AuFaeA containing one free cysteine at position 235 was used as a control. The free cysteine content of each re-FAE was calculated from a cysteine concentration-OD₄₁₀ standard formula, which was established by testing the OD₄₁₀ of cysteine concentration at a range from 0 to 0.4 mM. The number of disulfide bridges in the re-FAEs was deduced from the different value of the number of total cysteines in protein primary structure and free ones in 3D structure at the tested concentration.

Pluronic-stabilized poly(propylene sulfide) NPs were synthesized, functionalized and surface-conjugated as previously described26 (link)27 (link). For antigen conjugation, OVA and NPs were incubated overnight at room temperature in the presence of guanidine hydrochloride (Sigma-Aldrich)29 (link). For adjuvant conjugation, 5′ SPO₃-modified CpG-B was incubated overnight in endotoxin free water (B Braun, Sempach, Switzerland) at room temperature25 (link). NP-OVA and NP-CpG were purified by size-exclusion chromatography using CL6B matrix (Sigma-Aldrich), eluted and stored in PBS at room temperature. Protein concentration on NPs was determined by Pierce BCA protein assay (Thermo Scientific, Rockford, IL, USA) and CpG concentration on NPs was determined by GelRed (Brunschwig, Basel, Switzerland) assay. NP size before and after conjugation was determined by dynamic light scattering (Zetasizer, Nano ZS, Malvern, UK) to be of 30 ± 4 nm. All NPs formulations displayed endotoxin levels below 0.1 EU per administered dose, as detected with HEK-Blue hTLR4 cells from Invivogen (San Diego, CA, USA).

L-amino acid standards, norvaline, sarcosine, sodium dihydrogen phosphate (NaH₂PO₄), hydrochloric acid, sodium hydroxide, guanidine hydrochloride, 2,4-dinitrophenylhydrazine, iron (II) chloride, iron (III) chloride, ammonium thiocyanate, ortho-phthalaldehyde, 9-fluorenylmethoxycarbonyl chloride, iodoacetic acid, 3-mercaptopropionic acid, boric acid (H₃BO₃), trichloroacetic acid, and MS-grade ammonium acetate were from Sigma-Aldrich (Darmstadt, Germany). Standards for fatty acid analysis included Supelco 37 Component FAME mix (Supelco, St. Louis, MO, USA), 68D (Nu-Check-Prep, Elysian, MN, USA), and GLC-490 (Nu-Check-Prep, Elysian, MN, USA). Standards for the lipid class analysis were oleic acid, oleoyl monoacylglycerol, dioleoyl diacylglycerol, triolein, dioleoyl phosphatidylcholine, cholesteryl oleate and ethyl docosahexaenoic acid (Larodan, Solna, Sweden). All solvents used were at least HPLC grade. Water used was ultra-pure water (ELGA LabWater, High Wycombe, UK).