Biochemical characterization of SliC-c type lysozyme interaction.

<p>(A-E) Lysozyme activity assays with fluorescein-labeled cell walls of <i>Micrococcus lysodeikticus</i> as lysozyme substrate were used to determine the SliC-mediated inhibition of hen egg white c-type lysozyme (HEWL; A, C) and human lysozyme (HL; B-E). All assays were conducted using 96 well microtiter plates. Experimental samples containing 2.5 μM of either HEWL or HL were incubated with increasing concentrations of SliC (0–10 μM; as indicated on the right of each panel). The control wells contained HEWL, HL, or SliC alone. After incubation, the reaction was initiated by addition of lysozyme substrate and monitored for 1 h at excitation and emission wavelengths of 485 nm and 530 nm, respectively. Experiments reported in panels A and B show averages and corresponding SEMs from four independent trials. (C) Comparison of the inhibition of HEWL and HL activity by incubation with molar equivalent concentrations of rSliC (<i>n</i> = 4, mean±SEMs) was performed as described above. Protein concentrations (μM) are shown below the graph. ***<i>p</i><0.0001; not statistically significant <i>p</i> value of 0.25 (ns). (D) 2.5 μM of HL and 5 μM of either SliC or SliC* were used to test the inhibition of HL by SliC*. Assays were performed as outlined above (<i>n</i> = 3, mean±SEMs). (E) The half-maximal (50%) inhibitory concentration, IC₅₀, of SliC against the lytic activity of HL towards <i>M</i>. <i>lysodeikticus</i> peptidoglycan was determined by incubating 2.5 μM of HL with increasing concentrations of SliC (0–10 μM). (F) The binding affinity of SliC to lysozyme was assessed by biolayer interferometry. Biotinylated rSliC (20 μg/mL) was immobilized on streptavidin sensors for 10 min and incubated with increasing concentrations of HL as shown on the graph. Unloaded sensors were used as controls. After establishing the baseline, the association and dissociation steps were performed for 600 s. Experiments were performed in three biological replicates with curve fitting using a 2:1 (Heterogeneous Ligand) model. K_D value calculations were completed using Octet Data Analysis and mean±SEM is reported (version 9).