Abstract
In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 μg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis–Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 μM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.
Highlights
Xanthine oxidase (XOD) is a critical cytosolic oxidase, which is widely distributed in mammalian tissues [1]
The present study aimed to develop a rapid and efficient approach based on a PDAmodified hollow fiber-immobilized XOD (XOD@PDA@HF) to screen potential XOD inhibitors from flavonoids
XOD was efficiently immobilized on a PDA-modified hollow fiber for the first time
Summary
Xanthine oxidase (XOD) is a critical cytosolic oxidase, which is widely distributed in mammalian tissues [1] It is a key enzyme of the purine pathway, which oxidizes hypoxanthine and xanthine to form uric acid while concomitantly producing hydrogen peroxide and superoxide anions [2]. Flavonoids have been reported as potential XOD inhibitors and can effectively reduce uric acid in model animals [9,10,11]. Enzyme immobilization has frequently been used for enzymatic kinetics study and inhibitor screening [19]. The present study aimed to develop a rapid and efficient approach based on a PDAmodified hollow fiber-immobilized XOD (XOD@PDA@HF) to screen potential XOD inhibitors from flavonoids. The binding sites and interactions between compounds and XOD were studied by molecular docking
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