Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)

Six fresh sheep liver samples were obtained from different male individuals in a local slaughter-house, Cairo, Egypt and stored at -40 ˚C.

The reaction mixture of XO activity assay contains 1 ml 0.05 M Tris-HCl, pH 7.6 requiring 2 mM xanthine, 0.5 mM NBT and the xanthine oxidase solution. The reaction mixture was incubated for 5 minutes at 37 ^◦C, centrifuged at 2000 rpm for 2 minutes and the absorbance was measured at 575 nm. To calculate XO units, a control reaction was done with 0.02 unit commercially available bovine milk xanthine oxidase ¹⁵.

Activity staining of xanthine oxidase was carried out by submerging the gels in 50 mM Tris-HCl, pH 7.6, 0.5 mM xanthine, 0.25 mM nitroblue tetrazolium and 630 mM TEMED. Staining of the gels was continued till the activity bands appear on the gels ¹⁶.

The acetone fraction was applied on DEAE-cellulose column (6 x 2.4 cm i.d.) equilibrated with 0.02 M Tris-HCI buffer, pH 7.6, containing 0.1 mM EDTA. The protein fractions were eluted with stepwise NaCl gradient ranging from 0 to 1 M prepared in the equilibration buffer at a flow rate of 60 ml / hour. 5 ml fractions were collected and the fractions containing XO activity were pooled and concentrated by lyophilization.

The concentrated DEAE –cellulose fractions containing XO activity were applied to a Sephacryl S-300 column (142 cm X 1.75 cm i.d.). The column was equilibrated and run with 0.02 M Tris-HCI buffer, pH 7.6, containing 0.1 mM EDTA at a flow rate of 30 ml / hour and 2 ml fractions were collected.

Native gel electrophoresis was carried out with 7% PAGE ¹⁸. SDS-PAGE was performed with 12% polyacrylamide gel ¹⁹. The subunit molecular weight of the purified XO was determined by SDS-PAGE ²⁰. Electrofocusing was performed and the isoelectric point (pI) value was calculated from a calibration curve ^{21, 22}. Coomassie brilliant blue R-250 was used to stain the proteins.

Protein content was determined by the dye (Coomassie Brilliant Blue G-250) binding assay method using BSA as a standard protein ²³.

Xanthine oxidase was a commercially important enzyme with wide area of applications ^{24, 25}. Sheep liver XO was purified using a purification scheme consisting of the following steps, mixing crude extract with n-butanol, acetone precipitation, anion-exchange chromatography on DEAE-cellulose column and gel filtration chromatography on Sephacryl S-300 column. The method is relatively short and involves only two chromatographic steps that seem to be simple and convenient method. Different purification methods of XO were reported as that of rat liver XO ^{26, 27} and buffalo milk XO ²⁸. The elution profile of DEAE-cellulose column (Figure 1a) indicated one peak containing XO activity that XO was eluted as single peak at 0.05M NaCl and designated sheep liver xanthine oxidase (SLXO) Figure 1b). After chromatography on the Sephacryl S-300 column, SLXO was purified 31.8-fold with a specific activity of 3.5 units / mg protein and 24.1% recovery (Table 1). Buffalo milk xanthine oxidase; 10.66 unit/mg ²⁸, bovine milk xanthine oxidase; 1.086 unit/mg ²⁴, 0.3 unit/mg ²⁹ and 15.8 unit/mg ³⁰, human liver xanthine oxidase; 0.00061 unit/mg ³¹, and 0.036 unit/mg ³², buffalo liver xanthine oxidase; 7.2 unit/mg ³³, rat liver xanthine oxidase; 14 unit/mg ²⁷ and Arthrobacter M3 was 8.6 unit/mg ¹³.

Figure 1.A typical elution profile for the chromatography of the sheep liver acetone fraction on DEAE-cellulose column (6 cm x 2.4 cm i.d.) previously equilibrated with 0.02 M Tris-HCI buffer, pH 7.6 containing 0.1 mM EDTA. The proteins were eluted by a stepwise gradient of NaCl ranging from 0 to 1 M in the equilibration buffer and 5 ml fractions were collected at a flow rate of 60 ml / h. (b) A typical elution profile for the chromatography of the sheep liver DEAE-cellulose fraction on Sephacryl S-300 column (142 cm x 2.4 cm i.d.) previously equilibrated with 0.02 M Tris-HCI buffer, pH 7.6 containing 0.1 mM EDTA. The proteins were eluted by the same buffer and 2 ml fractions were collected at a flow rate of 30 ml / h.

Electrophoretic analysis of n-butanol extract, DEAE-cellulose fraction and Sephacryl S-300 purified fraction of SLXO on 7 % native PAGE revealed single protein band corresponded the enzyme activity band of the purified xanthine oxidase enzyme (Figure 2a). Gel filtration chromatography depicted only one activity peak, with a native molecular mass of 150 kDa. The subunit molecular weight of SLXO was determined by SDS-PAGE (Figure 2b) to be 75 kDa which revealed that the SLXO consists of two homodimer subunits. Many XO were reported to have dimeric structure such as 300 kDa for mouse liver ³⁴, human liver ³² and rat liver ²⁶. The xanthine oxidase from the bacterium Arthrobacter M3 showed two polypeptides with SDS-PAGE of molecular weights of 35 kDa and 100 kDa ¹³. The isoelectric point of SLXO enzyme was estimated by isoelectric focusing PAGE (Figure 2c) as two major molecular species with a value of 5.6 and 5.8. Isoelectric pH of XO in buffalo liver were 6 and 6.2 ³³, mouse liver was 6.7 ³⁴ and rat liver were 6.13, 6.23 and 6.07 ²⁷.

Figure 2.(a) Protein and XO isoenzyme pattern of sheep liver xanthine oxidase (SLXO) on 7 % native PAGE: (1) n-butanol extract, (2) DEAE-cellulose fraction, (3) Sephacryl S-300 purified fraction, and (4) SLXO isoenzyme pattern. (b) Subunit molecular weight determination by electrophoretic analysis of SLXO on 12 % SDS-PAGE: (1) Molecular weight marker proteins and (2) Purified SLXO. (c) Isoelectrofocusing: (1) isoelectric point (pI) marker proteins and (2) The purified sheep liver xanthine oxidase SLXO.

The effect of pH on the activity of sheep liver xanthine oxidase SLXO was examined in 0.05 M potassium phosphate buffer, pH (5.8-7.0) and 0.05 M Tris–HCl buffer, pH (7.2-9.0). The pH profile of SLXO displayed an optimum activity at pH 7.6 (Figure 3a). The optimum pH of the rabbit liver XO was found at pH 8.1 ³⁴. The Km value was calculated from the Lineweaver-Burk plot for the reciprocal of the reaction velocity (1/v) and substrate concentration (1/(S)) (Figure 3b) using xanthine as substrate. SLXO enzyme has a Km value of 0.9 mM (Figure 3b) indicating the high affinity of SLXO toward xanthine. Various Km values for XO were reported as; 1.1 mM xanthine for buffalo liver ³³, 3.4 µM xanthine for Mouse liver ³⁴, 22 µM for rabbit liver ³⁵ and 53 µM for rat liver XO was ²⁷.

Figure 3.(a) Effect of pH on the purified sheep liver xanthine oxidase SLXO using 0.05 M potassium phosphate buffer, pH (5.8-7.0) and 0.05 M Tris–HCl buffer, pH (7.2-9.0). (b) Lineweaver-Burk plot relating the reciprocal of the reaction velocity of the purified SLXO to xanthine concentration in mM.

The effect of metal compounds on the purified SLXO activity was examined (Table 2). Divalent cations, such as CuCl₂, MgCl₂, MnCl₂, CaCl₂, CoCl₂ and ZnCl₂ inhibited the activity of sheep liver xanthine oxidase (SLXO) whereas FeCl₂ and NiCl₂ increased its activity. These results were consistent with that of cow milk xanthine oxidase which was inhibited by Cu⁺², Hg⁺² and Ag⁺ ions ³⁶.

Furthermore, the inhibition of purified SLXO activity by several inhibitors was studied (Table 3). Pre-incubation of the inhibitors for 5 min at 37˚C were carried out and the inhibition % was concluded as a proportion of a non-inhibited control. The purified SLXO was not inhibited with the serine protease inhibitor PMSF indicating that the active site of this isoenzyme doesn't contain a serine residue. No inhibition of SLXO by B mercaptoethanol and dithiothretol was observed indicating that no role of -SH groups in the enzyme activity. Iodoacetamide inhibited the purified SLXO activity which indicates that methionine, cysteine and histidine residues play important role in the structure and activity of the enzyme. The inhibition of the purified SLXO isoenzyme by the metal chelator EDTA indicates that SLXO isoenzyme is metalloenzyme. The inhibition of SLXO activity with K₂Cr₂O₇ was probably due to strong oxidizing power of K₂Cr₂O₇ that may cause oxidation of metal prosthetic groups in enzyme that important to enzyme activity. Allopurinol was found to be the most potent inhibitor of the purified SLXO. The effect of allopurinol concentrations on the purified SLXO indicated that 50% inhibition (I₅₀) is caused by 0.1 mM allopurinol and the maximum inhibition of the enzyme (95.8%) was achieved by 1 mM allopurinol. However, full exploitation of these data required knowledge of the number of inhibitor molecules bound per enzyme molecule. Therefore, from the titration curve data (Figure 4a), a linear relationship was observed by constructing the Hill plot for the inhibition of the purified SLXO by allopurinol (Figure 4b). The slope of the Hill plot was found to be 1.19 indicating the existence of one binding site for allopurinol on the purified SLXO. The type of inhibition of the purified SLXO by allopurinol was found to be competitive type, whereas the presence of allopurinol did not alter the Vmax value but increased the Km value (Figure 4c). For the determination of the Ki value, the slopes of the reciprocal plots lines were plotted against the allopurinol concentration. The Ki value of the SLXO inhibition by allopurinol was found to be 0.06 mM directly from the intercept of the X axis of the plot (Figure 4d) indicating the potency of the allopurinol as inhibitor of XO. It is well known that, allopurinol is used as a drug for treating the elevated levels of uric acid in human by inhibiting xanthine oxidase and preventing the conversion of hypoxanthine and xanthine into uric acid.

Figure 4.(a) Titration curve for inhibition of the purified SLXO by varying concentrations of allopurinol. (b) Hill plot for inhibition of the purified SLXO by varying concentrations of allopurinol. (c) Lineweaver-Burk plots showing the type of inhibition of the purified SLXO by allopurinol. (d) Determination of the inhibition constant (Ki) value for the inhibition of the purified SLXO by allopurinol.