聚乙二醇化尿酸酶体内外稳定性研究

目的研究聚乙二醇化尿酸酶体内外稳定性。方法以酶活为指标,考察聚乙二醇修饰尿酸酶和尿酸酶的温度稳定性(4~80℃)、酸碱稳定性、抗胰蛋白酶水解能力和小鼠体内半衰期。结果4~60℃条件下,修饰的尿酸酶的稳定性大于尿酸酶,在70℃时,两者活性均迅速降低。pH 5.2~6.0及pH 9.2~10.0之间,尿酸酶活性迅速降低,而修饰的尿酸酶却保留了较高的活性。抗胰蛋白酶水解中,尿酸酶在作用200 min后,活性降至最高值的20%;而修饰的尿酸酶仍保留70%的活性。体内稳定性试验表明,修饰的尿酸酶和尿酸酶的半衰期分别为1 530和45 min。结论聚乙二醇修饰可以增加尿酸酶的稳定性和抗胰蛋白酶水解能力,延长体内半衰期。

Study on stability of N-terminal site-specific PEGylated uricase

Purpose The research was conducted to evaluate the stability of N-terminal site-specific PEGylated uricase. Methods The enzyme activity is used as an index to evaluate the thermal stability (at 4-80 ℃) , the pH stability, the ability of avoiding the trypsin digestion and half-life in vivo of PEG-uricase, then it is compared with uricase. Results PEGylated uricase is thermally more stable than uricase between 4 ℃ and 60 ℃, but at 70 ℃, the enzyme activity of both PEG-uricase and uricase decreases sharply. In the test of pH stability, the curve of uricase shows an obvious decrease of enzyme activity at 5 .2-6.0 and 9.2-10.0. The behavior of PEG-uricase indicates that the destabilizing was prevented effectively by PEGylation. The test of anti-trypsin digestion suggests that PEG-uricase retains 70% of its enzyme activity,but uricase only 20% ,200 minutes after being reserved in trypsin solution. Stability in vivo indicates that the half-life of PEG-uricase is 1 530 min and uricase, only 45 min. Conclusion PEGylated uricase has improved thermal stability, the pH stability, ability of protecting from trypsin digestion and stability in vivo.