Computational and experimental approaches for assessing the interactions between the model calycin beta-lactoglobulin and two antibacterial fluoroquinolones.

Norfloxacin and levofloxacin, two fluoroquinolones of different bulk, rigidity and hydrophobicity taken as model ligands, were docked to one apo and two holo crystallographic structures of bovine beta-lactoglobulin (BLG) using different computational approaches. BLG is a member of the lipocalin superfamily. Lipocalins show a typical b-barrel structure encompassing an internal cavity where small hydrophobic molecules are usually bound. Our studies allowed the identification of two putative binding sites in addition to the calyx. The rigid docking approximation resulted in strong repulsive forces when the ligands were docked into the calyx of the apo form. On the contrary, hindrance was not experienced in flexible docking protocols whether on the apo or on the holo BLG forms, due to allowance for side chain rearrangement. K(i) between 10(-7) and 10(-6) M were estimated for norfloxacin at pH 7.4, smaller than 10(-5) M for levofloxacin. Spectroscopic and electrophoretic techniques experimentally validated the occurrence of an interaction between norfloxacin and BLG. Changes in chemical shift and dynamic parameters were observed between the (19)F NMR spectra of the complex and of the ligand. A K(i) (ca 10(-7) M) comparable with the docking results was estimated through a NMR relaxation titration. Stabilization against unfolding was demonstrated by denaturant gradient gel electrophoresis on the complex versus apo BLG. NMR experimental evidence points to a very loose interaction for ofloxacin, the racemic mixture containing levofloxacin. Furthermore, we were able to calculate in silico K(i)'s comparable to the published experimental values for the complexes of palmitic and retinoic acid with BLG.

Reference maps of mouse serum acute-phase proteins: changes with LPS-induced inflammation and apolipoprotein A-I and A-II transgenes.

We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.