Purification and Properties of Sulfite Oxidase from Chicken Liver

Abstract Sulfite oxidase (EC 1.8.3.1) has been purified from chicken liver by modification of a procedure previously applied to bovine liver. The enzyme appears homogeneous as judged by its behavior in sedimentation velocity experiments and polyacrylamide disc gel electrophoresis. A new activity stain for the enzyme on acrylamide gels is described. Despite its apparent homogeneity, the enzyme consistently behaves in a heterogeneous manner in sedimentation equilibrium experiments, suggesting an associating system whose state of association depends on both enzyme concentration and anion content. The subunit molecular weight, determined by sodium dodecyl sulfate disc gel electrophoresis, is 55,000. The amino acid composition of the purified enzyme is reported. The absorption spectrum of the enzyme reveals the presence of a b5-like cytochrome, while chemical analysis and electron paramagnetic resonance (EPR) spectra indicate that molybdenum functions as a second prosthetic group. The kinetics of the reaction of the enzyme with sulfite and cytochrome c are consistent with a ping-pong mechanism. The EPR signal from the molybdenum center of the enzyme displays a pH titration behavior similar to that of the bovine enzyme. In addition, the shape of the signal is extremely sensitive to the presence of anions. The heme EPR signal is very similar to that reported for cytochrome b5. The enzyme has also been partially purified from human liver, wheat germ, and Thiobacillus thioparus; each of these preparations displays a sulfite-dependent molybdenum EPR signal.