【作者】 赵运胜；

【导师】 廖飞；

【作者基本信息】 重庆医科大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 测定血清尿酸是临床检验常规工作之一。利用尿酸酶的高专一性进行酶法分析测定血清尿酸含量,即尿酸酶法,是目前常规测定血清尿酸的主要方法。现有尿酸酶法中,过氧化物酶偶联尿酸酶的间接终点平衡法最常用,但此终点平衡法受血清中还原物质和过氧化物,及黄嘌呤等的显著干扰。本实验室在国家自然科学基金(30200266)资助下,建立了血清尿酸含量测定的专利方法(zl 03135649.4),此专利方法对血清中常见干扰物质不敏感;与其它尿酸酶法相比,相同效率下其成本最低,线性范围最宽,对黄嘌呤抗性最强。已报道尿酸酶法中,测定尿酸酶作用下紫外吸收总变化的直接终点平衡法对常见干扰抗性最强。本实验室的专利方法与直接终点平衡法一致,但与最常用的过氧化物酶偶联尿酸酶间接终点平衡法不一致。可见此专利方法可行且可靠。但此专利方法需要K_m较高且对黄嘌呤抑制不敏感的工具尿酸酶。目前市售尿酸酶K_m都较低且对黄嘌呤敏感。另一方面,尿酸酶也是当前治疗高尿酸血症相关疾病的理想药物。苛求芽孢杆菌依赖于高浓度尿酸而生长,其可表达高比活性的胞内和胞外尿酸酶;此类尿酸酶K_m较高,最接近我们专利方法要求;但至今未见关于此物种的任何基因序列信息报道。为获得廉价且符合要求尿酸酶以促进测定血清更多还原

【Abstract】 Serum uric acid assay is a routine work in medical laboratory. The indirect equilibrium method by peroxidase-coupled assay of hydrogen peroxide in common use suffers from interferences from reducing agents and xanthine. Recently, a kinetic uricase method was established for serum uric acid assay by predicting background absorbance with an integrated method (China patent zl 03135649.4). The use of uricase of higher Michaelis-Menten constant (Km) and lower sensitivity to xanthine improved its resistance to xanthine. However, most commercial uricases show higher sensitivity to xanthine besides lower Km. Herein, uricases from Bacillus fastidious were investigated to seek for a desirabel tool enzyme. These works were reported.1. Extracellular uricase was prepared from the medium of Bacillus fastidious (ATCC 26904) by fractionation with ammonium sulfate, and further purified by DEAE-cellulose chromatography. This uricase suited for serum uric acid assay by direct assay of initial absorbance before uricase action and predicting the background absorbance of uricase reaction solution with the integrated method. This uricase was composed of single更多还原