Imagequant v5

DNA hydrolysis activity was analyzed using supercoiled pBluescript DNA as previously described in [25 (link),73 (link),74 (link)]. An amount of 20 μL of reaction mixture contained 18 μg/mL (6.1 nM) of supercoiled pBluescript DNA, 5.0 mM MgCl₂, 1.0 mM EDTA, 20.0 mM Tris-HCl pH 7.5, and IgG in a final concentration of 0.01 mg/mL. Samples were incubated for 48 h at 37° C. The relative amount of DNA in the supercoiled, linear, and relaxed plasmid bands was analyzed using ImageQuant v.5.2 (Molecular Dynamics, Los Angeles, CA, USA). The activity of IgG preparations was determined by the decrease in the percentage of dsDNA converted from the supercoiled form to relaxed and linear forms. Control samples did not contain IgGs. All measurements were carried out in linear regions of hydrolysis (15–40% of DNA hydrolysis), and the complete transition of the supercoiled plasmid to the hydrolyzed form was taken as 100% activity.

DNase activity of the complex was analyzed using supercoiled (sc) DNA pBluescript. The reaction mixture (20–40 µL) contained 50 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 5.0 mM EDTA, 10 µg/mL (or 3.4 nM) sc DNA, and 0.05 mg/mL (or ~25 nM) protein complex. Reaction mixtures were incubated for 1–5 h at 37 °C. The cleavage products were detected using electrophoresis in 0.8% agarose gels; DNA was colored by ethidium bromide. The ethidium-bromide-stained gels were captured using a Sony DSC-F717 camera (Sony DSC-F717 camera; Sony Centre, Berlin, Germany). The hydrolysis of sc DNA results in the formation of its relaxed form with lower electrophoretic mobility. The initial native sc DNA always contains a small amount of hydrolyzed relaxed DNA. The relative intensity of DNA in different bands was analyzed by ImageQuant v5.2 (Molecular Dynamics). The complex activities were first determined as a decrease in the percent of sc DNA converted from the initial supercoiled to its relaxed form. The distribution of DNA between these two bands in the control (incubation of the DNA plasmid in the absence of the complex) was taken into account.
pH dependencies were analyzed using different 50 mM buffers: MES-NaOH (pH 5.3–6.6), Tris-HCl (pH 6.0–8.6), and glycine-NaOH (pH 9.0–10.0). In some experiments, MnCl₂, MgCl₂, ZnCl₂, CaCl₂, and CoCl₂, (each at 0.1–25.0 mM) were added to reaction mixtures.

DNase activity of IgG preparations was analyzed as in [21 (link),33 (link)]. The reaction mixtures (20 µL) containing 20 µg/mL supercoiled (sc) DNA pBluescript, 1 mM EDTA, 3–5 mM MgCl₂, 20 mM Tris-HCl (pH 7.5), and 0.003–0.2 mg/mL IgGs were incubated for 0.5–4h (standard time, 2 h) at 37 °C. The products of DNA hydrolysis were analyzed using electrophoresis in 1% agarose gel. All initial rates of the reaction were estimated using the linear regions of the time courses and IgG concentration (20–40% of scDNA hydrolysis). The ethidium bromide-stained gels were analyzed using a Sony DSC-F717 camera (Sony Center, Berlin, Germany) and ImageQuant v5.2 (Molecular Dynamics, New York, NY, USA). The activities of IgGs were found from a decrease in scDNA corrected for the distribution of DNA between bands of initial and the relaxed form of DNA in the control (incubation of scDNA in the absence of Abs). A complete transition of the scDNA to its relaxed form after 1 h of incubation was taken for 100% activity. Finally, relative DNase activity was calculated as pmole DNA/1 mg of IgGs/1 h. DNase activity was also determined using this approach after isoelectrofocusing of IgGs.