摘要
目的研究天然尿酸酶与PEG化尿酸酶的基本特性,为治疗高尿酸血症相关疾病药物做基础研究。方法用相对分子量5kD,活化基团为羟基琥珀亚氨的PEG作为专一性修饰剂对尿酸酶进行修饰,采用常规酶学分析方法,分别研究天然尿酸酶与PEG化尿酸酶的动力学参数,并比较修饰前后对抑制剂黄嘌呤的敏感性、最适温度、最适pH值、37℃时的稳定性以及在体半衰期等方面的差异。结果经PEG修饰后尿酸酶最适温度由30℃升为35℃,最适pH无改变仍为pH8.7;在37℃水浴下,24h后活性保留由40%增至70%,酶稳定性提高,体内半衰期由45min延长至11h;测定尿酸酶和PEG化尿酸酶的Km分别为32.40、36.34μmol/L,黄嘌呤对尿酸酶与PEG化尿酸酶抑制常数分别为4.72、11.30μmol/L。结论假丝酵母尿酸酶经PEG5kD修饰后有药用潜力。
Objective To research the basic characteristics of natural uricase and PEG-uricase,prepare PEG-uricase,modify the Candida uricase with PEG-5000 to improve the stability of the Candida uricase and extend the half-life of uricase in vivo.Methods PEG（relative molecular weight 5 kD,activation group for the NHS Asian ammonia） was used as a special modifier to modify uricase. With the conventional enzymatic analysis,some kinetic parameters of natural uricase and PEG-uricase were studied,and compared the differences of the sensitivity of the inhibitor of xanth,optimum temperature,optimum pH,the stability at 37℃ and the half-life in vivo before and after modification. Results It was demonstrated that after PEG modification the optimal temperature of the uricase was upgraded from 30℃ to 35℃,the optimum pH was still 8.7 with no changing.37 ℃ water bath,24 h post-active retention increased from 40% to 70%,the enzyme stability was improved,the half-time in vivo extended from 45 min to 90 min.The Km of uricase and PEG-uricase were 32.40 and 36.34 μmol/L,the inhibition constants of xanthine on uricase and PEG-uricase were 4.72,11.30 μmol/L. Conclusions PEG-modified uricase from the Candida utilis is promising to be a bio-drug for treating hyperuricemia.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2010年第19期2763-2766,共4页
Chinese Journal of Gerontology
基金
国家自然科学基金资助项目(30672009)
