Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutasefrom Nicotiana plumbaginifolia leaves

The isoenzyme I of cytosolic. Cu,Zn-superoxide dismutase (SOD) from Nicotia na plumbaginifolia (tobacco) leaves has been purified to apparent homogenei ty. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electr ophoresis shows that the enzyme is composed of two equal subunits of 16.6 k Da The isolectric point, assayed by isoelectric focusing, in the pH range o f 3.5-6.5, is 4.3. The enzyme stability was tested at different temperature s, pH, and concentration of inhibitors (KCN and H2O2). The catalytic consta nt (k(cat)) was 1.17 +/- 0.14 x 10(9) M-1 s(-1) at pH 9.9 and 0.1 M ionic s trength. The activation energy of the thermal denaturation process is 263 W mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn- SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino aci d substitution around the active sites. (C) 2001 Academic Press.