Purification of highly active milk xanthine oxidase by affinity chromatography on sepharose 4B/folate gel

Abstract

Takeshi NISHINO, Tomoko NISHINO and Keizo TSUSHIMA Department of Biochemistry, Yokohama City University School of Medicine, Urafune-cho, Minami-ku, Yokohama 232, Japan Received 9 July 1981 1. Introduction It is well known that an inactive form of xanthine oxidase, the desulfo-form, is present in milk xanthine oxidase preparations, in addition to an inactive form lacking molybdenum [1-4]. The existence of the desulfo-form in the enzyme preparations is considered to be caused by spontaneous release of sulfur during storage or purification. This inactive form is also known to be caused by treatment of the enzyme with cyanide [5]. To understand the proper mechanism of action of this enzyme, it is important to obtain an enzyme preparation essentially free of inactive forms. An affinity chromatography method for resolution of the active and inactive forms of xanthine oxidase was reported in [6]. An allopurinol analogue was used as an affinity ligand [6]. In [6], only the active enzyme, in a reduced form, was bound to the column and could be eluted after reoxidation. Despite the extreme value of affinity chromatography, prepara- tion of the enzyme by their method is not easy, because it involves difficult chemical syntheses and chromatography under anaerobic conditions. Here, we describe a much easier method for puri- fication of highly active enzyme by affinity chroma- tography, in which resolution of the sulfo- and desulfo-enzymes is achieved. We adopted folate [7], a commercially available competitive inhibitor, as a ligand instead of the allopurinol analogue. 2. Materials and methods Xanthine oxidase was prepared from cow's milk Abbreviations: DMF, dimethylformamide; EDC, N-ethyl-N'- 3 dimethylaminopropyl carbodiimide; AFR, activity flavin ratio by the method in [8] with minor modifications. Cysteine (5 mM), salicylate (1 mM) and EDTA (0.2 mM) were added to the extraction buffer of Na2HPO4 (0.2 M). Final ammonium sulfate fractionation was performed between 30-45% saturation instead of 33-42% saturation as in the original method. 2.1. Preparation of Sepharose 4B/folate gel AH-Sepharose (10 g) obtained from Pharmacia, was swollen in 0.5 M NaC1 and washed with 1 titer of 0.5 M NaC1. Washed AH-Sepharose was mixed with 60 ml 2 mM folate in 50% DMF, the pH of which had been pre-adjusted to 5.8 with dilute NaOH. Then 400 mg EDC, obtained from Fluka, was added and the mixture was gently stirred overnight at room temperature in the dark. The gel was washed sequen- tially with 200 ml of 50% DMF at pH 7.0,500 ml 0.01 M NaOH, 500 ml 0.1 M Tris-HC1 at pH 7.0, and finally with 1 liter of distilled water. The Sepha- rose 4B/folate gel was stored at 4°C in the dark. Oxipurinol was synthesized as in [9]. Allopurinol was obtained from Sigma. Enzyme activity was measured spectrophoto- metrically at 295 nm and at 25°C [4] using a Shimazu 140 spectrophotometer. Catalytic activity was expressed as AFR 25°c [10]. The following buffer mixtures were used for affinity chromatography: (A) Mixture of 20% 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA and 80% 0.05 M Tris-HC1 buffer (pH 7.8) containing 0.2 mM EDTA. (B) Mixture of 30% of 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA and 70% 0.05 M Tris-HC1 buffer (pH 7.8) containing 0.2 mM EDTA. Published by Elsevier/North-Holland Biomedical Press 00145793[81[0000-0000/$02.50 © 1981 Federation of European Biochemical Societies 369