Figure 6. Constitutive NADPH oxidase turnover of the purified cytochrome b₅₅₈ isolated from stimulated neutrophils on heparin affinity matrix in the presence of rS100A9-A8 full length or truncated chimeras.

Purification of cytochrome b₅₅₈ from stimulated neutrophils followed a standard protocol until it bound to the heparin affinity matrix as reported [25] and as described in Materials and Methods. The cytochrome b₅₅₈ bound to the affinity heparin matrix was washed with: (A) EBV-B lymphocyte cytosol; in some experiments, a 1/1 mixture of rS100A8 and rS100A9 was added to the Sephacryl eluted cytochrome b₅₅₈ and then the activity of NADPH oxidase was measured in the presence of (1 mM) arachidonic acid (grey square). (B) S100A9-A8; (C) S100A9-A8Δ90; (D) S100A9-A8Δ86; (E) S100A9-A8Δ57. S100 chimera proteins were preloaded with calcium (500 nM). After the washing step, eluted fractions containing cytochrome b₅₅₈ were pooled and filtrated on Sephacryl-S300. The concentration of cytochrome b₅₅₈ in the Sephacryl eluted fractions (open circle) was determined by measuring the “reduced minus oxidized” differential spectrum at 426 nm. NADPH oxidase activity of purified cytochrome b₅₅₈ was measured in a cell free assay with 0.2 pmol cytochrome b ₅₅₈/assay, in the presence of 10 µM FAD, 40 µM GTPγS, and 5 mM MgCl₂ but without arachidonic acid, and after adding 150 µM NADPH. The NADPH oxidase activity was expressed as turnover (s⁻¹) (black squares). No S100A9-A8 chimera or EBV-B lymphocyte cytosol was added to the heparin matrix, in control experiments. (F) Table representing the optimum NADPH oxidase turnovers obtained in A, B, C, D, E conditions, or by using cytosol of stimulated neutrophils instead of that of EBV-B lymphocytes [25]. Number of experiments n = 6 *p<0.05 compared to control (constitutive NADPH oxidase activity of purified cytochrome b₅₅₈ in control experiment).