苛求芽孢杆菌胞外尿酸酶的表征及其在血清尿酸测定中的应用

Characterization of an Extracellular Uricase from Bacillus fastidious and Its Application in Direct Kinetic Assay of Serum Uric Acid

目的考察苛求芽孢杆菌（ATCC 26904）分泌型尿酸酶的特性和将其应用于直接动力学尿酸酶法测定血清尿酸的有效性。方法苛求芽孢杆菌分泌的尿酸酶经DEAE-cellulose层析纯化后，用于以积分法分析尿酸酶反应曲线预测反应体系本底的直接动力学尿酸酶法测定血清尿酸。结果苛求芽孢杆菌分泌的尿酸酶只含1种肽链，分子量为34．7kD（1kD=0．9921ku），其米氏常数为（0．22±0．01）mmol／L（n=11），黄嘌呤抑制常数为（36±3）μmol／L（n=4）。用此尿酸酶，只要被分析反应曲线终点的底物浓度低于起点的30％，应用积分法就能可靠预测本底；直接测定反应前吸收，可使直接动力学尿酸酶法不受常见血清成分、酶活性变化和15μmol／L黄嘌呤等干扰。监测0．025U／ml尿酸酶反应5min的数据，其线性响应范围为1～50μmol／L；所得临床标本血清尿酸含量（Ct）与过氧化物酶偶联间接终点平衡法（Ce）正相关（Ck=-0．008＋1．046Ce，r=0．996，n=206），但Bland—Altman法分析表明两种方法不一致。结论苛求芽孢杆菌分泌的尿酸酶可用于直接动力学尿酸酶法测定尿酸，并可保障其优势。

Objective To characterize an extracellular uricase from Bacillus fastidious （ATCC 26904） and to evaluate its efficiency for direct kinetic assay of serum uric acid. Methods The extracellular uricase from Bacillus fastidious （ATCC 26904） was purified by DEAE-cellulose chromatography. This uricase was used for serum uric acid assay by predicting the background absorbance of uricase reaction solution with the integrated method. Results This uricase contained one polypeptide of 34.7 kD and its Km was （0.22±0.01） mmol/L （n=11）. The inhibition constant of xanthine was （36±3）μmol/L （n=4）. With this uricase, residual uric acid at the last datum under analysis below one-third of the first datum under analysis enabled reliable estimation of background absorbance. By directly measuring the absorbance before uricase reaction, this direct kinetic uricase method exhibited resistance to 15 μmol/L xanthine, the variation of enzyme activity and other common interferences. It gave linear response to uric acid concentration from 1 to 50μmol/L in reaction solution with 0. 025 U/ml uricase to monitor reaction within 5 rain. Uric acid concentration in clinic sera by the direct kinetic uricase method （Ck） was closely correlated to that by the equilibrium method through peroxidase-coupled assay of hydrogen peroxide （Ce） （Ck=-0. 008＋ 1. 046Ce, r= 0. 996, n= 206）, but Bland-Altman analysis indicated inconsistency between Ck and Ce. Conclusion This extracellualr uricase was desirable as a tool enzyme for serum uric acid assay by the direct kinetic uricase method.