ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 339, No. 1, March 1, pp. 125–135, 1997 Article No. BB969844 Xanthine Oxidase Binding to Glycosaminoglycans: Kinetics and Superoxide Dismutase Interactions of Immobilized Xanthine Oxidase–Heparin Complexes Rafael Radi,* , † , ‡ Homero Rubbo,* , † , ‡ Ken Bush,† , ‡ and Bruce A. Freeman† , ‡ ,1 *Departamento de Bioquı´mica, Facultad de Medicina, Universidad de la Repu ´ blica, 11800 Montevideo, Uruguay and Departments of †Anesthesiology and ‡The Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233 Received November 20, 1996 in buffer and in the absence of substrate from 67 to 120 h at 4°C. These data indicate that binding to cell Xanthine oxidoreductase (XDH / XO, EC 1.2.3.2) is surfaces will strongly influence the catalytic proper- released into the circulation from organs rich in XO ties, oxidant producing capacity, and stability of XO. activity. Herein we report the specific high affinity  1997 Academic Press binding of XO to glycosaminoglycans (GAGs) and the Key Words: xanthine oxidase; free radical; superox- preferential association of XO with heparin, compared ide; superoxide dismutase; hydrogen peroxide; hepa- with heparan sulfate, chondroitin sulfate, and dema- rin; glycosaminoglycan; endothelium. tan sulfate. The binding of XO to Sepharose 6B-conju- gated heparin (HS6B) occurs at physiological ionic strength and increased with pH, with Scatchard analy- sis revealing a nonlinear binding pattern at pH 7.4. The dissociation constant (K d ) for XO binding was 0.4 In diverse forms of tissue and vascular injury, excess to 1.8 1 10 07 M, similar to the heparin-reversible bind- production and reactions of reactive oxygen species ing of lipoprotein lipase to vascular endothelium. The (i.e., superoxide (O •0 2 ), 2 hydrogen peroxide (H 2 O 2 ) and binding energy of 9 – 13 kcal/mol was concordant with hydroxyl radical ( • OH)) play a pathogenic role (1, 2). noncovalent electrostatic interactions. Xanthine oxi- The enzyme xanthine oxidoreductase (XDH / XO, EC dase immobilization to HS6B rendered a catalytically 1.2.3.2) significantly contributes to oxidant-mediated active enzyme form that had kinetic characteristics tissue pathology in various experimental models, espe- distinct from XO in free solution. While the K m and K i cially toward those species and organs rich in XDH / for xanthine in phosphate buffer at pH 7.4 were 3 mM XO specific activity (3). For example, the oxidase form and 1.6 mM, respectively, for free XO, they were 15 mM of this enzyme, XO, contributes to the oxidant stress and 2.8 mM for immobilized XO. Inhibition constants component of ischemia-reperfusion injury to intestine for guanine and uric acid were also increased upon and liver and, in some species, myocardium (4–8). En- XO binding to HS6B. Changes in kinetic parameters dogenous XO can play a critical role in vascular endo- were related to a real and not apparent decrease in thelial cell oxidant production and injury in species binding affinity for substrate and inhibitors and were having a high XO-specific activity, such as rodents (9). not due to diffusion-controlled processes within the In contrast, related investigations have shown that gel matrix. Changes in K m and K i for xanthine also had a significant influence on the relative quantities of there is no detectable contribution of XO-derived oxi- O •0 2 and H 2 O 2 generated by a given substrate concen- dants to net cellular generation of reactive oxygen spe- tration. Superoxide formed by HS6B-bound XO was partially consumed within the gel microenvironment 2 Abbreviations used: XO, xanthine oxidase; XDH, xanthine dehy- which electrostatically excluded CuZn SOD. Immobili- drogenase; O •0 2 , superoxide anion; H 2 O 2 , hydrogen peroxide; • OH, zation of XO increased the half-life of enzyme activity hydroxyl radical; • NO, nitric oxide; ONOO 0 , peroxynitrite anion; HS6B, Sepharose 6B-conjugated heparin; EDTA, ethylenediamine- 1 To whom correspondence and reprint requests should be ad- tetraacetic acid; EC-SOD, extracellular superoxide dismutase; GAG, glycosaminoglycan. dressed. Fax: (205) 934-7437. E-mail: bruce.freeman@ccc.uab.edu. 125 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.