ABSTRACT
Xanthine oxidoreductase (XDH + XO, EC 1.2.3.2) is released into the circulation from organs rich in XO activity. Herein we report the specific high affinity binding of XO to glycosaminoglycans (GAGs) and the preferential association of XO with heparin, compared with heparan sulfate, chondroitin sulfate, and dematan sulfate. The binding of XO to Sepharose 6B-conjugated heparin (HS6B) occurs at physiological ionic strength and increased with pH, with Scatchard analysis revealing a nonlinear binding pattern at pH 7.4. The dissociation constant (Kd) for XO binding was 0.4 to 1.8 x 10(-7) M, similar to the heparin-reversible binding of lipoprotein lipase to vascular endothelium. The binding energy of 9-13 kcal/mol was concordant with noncovalent electrostatic interactions. Xanthine oxidase immobilization to HS6B rendered a catalytically active enzyme from that had kinetic characteristics distinct from XO in free solution. While the Km and Ki for xanthine in phosphate buffer at pH 7.4 were 3 microM and 1.6 mM, respectively, for free XO, they were 15 microM and 2.8 mM for immobilized XO. Inhibition constants for guanine and uric acid were also increased upon XO binding to HS6B. Changes in kinetic parameters were related to a real and not apparent decrease in binding affinity for substrate and inhibitors and were not due to diffusion-controlled processes within the gel matrix. Changes in Km and Ki for xanthine also had a significant influence on the relative quantities of O2.- and H2O2 generated by a given substrate concentration. Superoxide formed by HS6B-bound XO was partially consumed within the gel microenvironment which electrostatically excluded CuZn SOD. Immobilization of XO increased the half-life of enzyme activity in buffer and in the absence of substrate from 67 to 120 h at 4 degrees C. These data indicate that binding to cell surfaces will strongly influence the catalytic properties, oxidant producing capacity, and stability of XO.