2.2. Succinylation of lysozyme

Succinylation of LYS was performed essentially as reported previously [22] with minor alterations. The succinylation reaction was conducted at four different theoretical molar ratios of succinic anhydride to LYS, i.e. 100, 10, 5 and 0.5, respectively, and the resulting LYS analogues are denoted LYS-100, LYS-10, LYS-5 and LYS-0.5. Unmodified lysozyme is simply denoted LYS. LYS was dissolved in sodium phosphate buffer (100 mM, pH 8) to a concentration of 20 mg/ml. Depending on the desired molar ratio, succinic anhydride was dissolved in 250 (LYS-10 (70 mg/ml), LYS-5 and LYS-0.5 (35 mg/ml)) or 500 (LYS-100 (340 mg/ml)) µl DMSO, respectively. The succinic anhydride solutions was added to the protein solutions (12.5 ml) in small aliquots over a period of 60 min under constant stirring. For the LYS-0.5, only 25 µl succinic anhydride and DMSO solution (35 mg/ml) was added over a period of 10 min, resulting in a succinic anhydride to lysozyme molar ratio of 0.5. The pH was maintained at 8–9 by adjusting the pH with 0.1 M NaOH solution. The reaction was allowed to proceed for 30 min at 4 °C. The LYS analogues were dialyzed for two days against deionized water using a volume corresponding to at least 200 times the sample volume in Slide-A-Lyzer™ Dialysis Cassettes (12–30 ml, Thermo Fisher Scientific, Waltham, MA, USA). The analogues were filtered through 0.2 nm Minisart® filters (Sartorius Stedim Biotech Göttingen, Germany). The final protein concentration was determined by UV spectroscopy at 280 nm using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific).

The theoretical pI values of the succinylated LYS analogues were estimated from the amino acid sequence of hen egg white lysozyme [23]. The pI values were calculated using the online ProtParam tool program [24] applying a molar extinction coefficient, ε, of 37,370 M⁻¹ cm⁻¹ (A_0.1% = 2.653 mg ml⁻¹ cm⁻¹), by replacing lysine (K) and tyrosine residues (Y) with glutamate (E) residues (Supplementary Fig. 1). ProtParam does not allow for replacement of the N-terminal amine group, hence succinylation of this group was not accounted for in the calculations.