PURIFICATION AND CHARACTERIZATION OF XANTHINE DEHYDROGENASE FROM LOCUSTA-MIGRATORIA L

Xanthine dehydrogenase (XDH) from Locusta migratoria was purified 227-fold by ammonium sulphate precipitation, chromatography on Sephadex G-200, DEAE cellulose, and preparative polyacrylamide gel electrophoresis. The preparation appeared homogeneous on polyacrylamide disc electrophoresis. The enzyme largely resembles that of other systems in that it contained two atoms of molybdenum, eight atoms of iron and two molecules of FAD. By various criteria it has been found to have a molecular weight of approx. 280,000 and electrophoresis on sodium dodecylsulphate gels indicate that it contains subunits of 140,000. The molecular radius is 4.55 nm. Isoelectric focusing in disc polyacrylamide gels reveals an isoelectric point of 5.7. Kinetic analysis would suggest that xanthine and 2-amino-4-hydroxypteridine bind differently to the enzyme. The Km values for both substrates vary with pH such that with xanthine oxidation it is lowered to a minimum just on the alkaline side of the xanthine pK of 7.44 while with 2-amino-4-hydroxypteridine the minimum Km values occur on the acid side of the pK at 7.92. © 1979.