胰蛋白酶是一种丝氨酸蛋白酶,可特异切割精氨酸及赖氨酸C端肽键。重组人源阳离子胰蛋白酶(recombinant human cationic trypsin:rht1)的稳定性明显高于重组人源阴离子胰蛋白酶(recombinant human anionic trypsin:rht2)。比较ht1和ht2...胰蛋白酶是一种丝氨酸蛋白酶,可特异切割精氨酸及赖氨酸C端肽键。重组人源阳离子胰蛋白酶(recombinant human cationic trypsin:rht1)的稳定性明显高于重组人源阴离子胰蛋白酶(recombinant human anionic trypsin:rht2)。比较ht1和ht2的氨基酸序列和三维结构,二者的氨基酸序列同源性为95%,rht1比rht2多1对二硫键Cys139-Cys206。为解释该二硫键对其稳定性的作用,构建rht1的Cys139-Cys206二硫键缺失突变体rht1-dC139S-C206S和rht2的增加该对二硫键的突变体rht2-S139C-S206C。进行了重组表达和纯化,并测定rht1、rht2及两个突变体的酶学性质,对比其稳定性。结果发现,与野生型rht1、rht2相比,突变体的酶学动力学参数km和kcat值与最适pH均未有较大差异。但在pH3~12条件下的稳定性,rht1-dC139S-C206S比rht1低46.6%;rht2-S139C-S206C比rht2高30.3%。对比其热稳定性,40℃保温4 h,rht1残余活性为92.4%,而rht1-dC139S-C206S降为60%。60℃保温4 h,rht2-S139C-S206C残余活性为83.3%,而rht2完全失活。表明了该二硫键Cys139-Cys206对人胰蛋白酶稳定性的重要性。进一步进行结构模拟和分析,解释了该对二硫键对稳定性影响的机制。展开更多
Objective: To study the pharmacokinetic, distribution and elimination properties of rhTα 1 after intravenous(i.v.) and subcutaneous(s.c.)injection in mice and rats. Methods: Competition ELISA was used for testing dru...Objective: To study the pharmacokinetic, distribution and elimination properties of rhTα 1 after intravenous(i.v.) and subcutaneous(s.c.)injection in mice and rats. Methods: Competition ELISA was used for testing drug concentration in serum, urine, bile and tissue after administration of rhTα 1 in mice(0.16, 0.5, 2.5 mg/kg) and rats(0.32, 1, 5 mg/kg). Pharmacokinetic parameters were calculated by Win Nolin software. Results: Absorption of rh Tαl is rapid in both mice and rats after s.c. administration. The pharmacokinetics in mice are characterized by linear, T_(1/2) showed a prolongation with increasing dose, 1.10, 1.35, and 1.50 h corresponding to 0.32, 1 and 5 mg/kg respectively, but in rats T_(1/2) showed no difference among doses. AUC0-∞ showed a clear increase with increasing doses in mice(904.18, 2998.83, and 19001.82 h*ng/m L) and in rats(1327.56 ±237.00,2924.53 ±685.14, and 35286.26 ±5999.58 h*ng/m L). After i.v. administration of 1 mg/kg rhTα 1 in mice, the drug is seen distributed in most organs, the thymus/serum exposure ratio was higher than others at the 1 and 2 h, the accumulative urinary excretion of primary drug was 32.97% ±15.85% within 6 h. Conclusion: The results indicate that rapid absorption, extensive distribution and quick renal excretion were the basic kinetic characteristics of rh Tαl after s.c. and i.v. administration.展开更多
文摘胰蛋白酶是一种丝氨酸蛋白酶,可特异切割精氨酸及赖氨酸C端肽键。重组人源阳离子胰蛋白酶(recombinant human cationic trypsin:rht1)的稳定性明显高于重组人源阴离子胰蛋白酶(recombinant human anionic trypsin:rht2)。比较ht1和ht2的氨基酸序列和三维结构,二者的氨基酸序列同源性为95%,rht1比rht2多1对二硫键Cys139-Cys206。为解释该二硫键对其稳定性的作用,构建rht1的Cys139-Cys206二硫键缺失突变体rht1-dC139S-C206S和rht2的增加该对二硫键的突变体rht2-S139C-S206C。进行了重组表达和纯化,并测定rht1、rht2及两个突变体的酶学性质,对比其稳定性。结果发现,与野生型rht1、rht2相比,突变体的酶学动力学参数km和kcat值与最适pH均未有较大差异。但在pH3~12条件下的稳定性,rht1-dC139S-C206S比rht1低46.6%;rht2-S139C-S206C比rht2高30.3%。对比其热稳定性,40℃保温4 h,rht1残余活性为92.4%,而rht1-dC139S-C206S降为60%。60℃保温4 h,rht2-S139C-S206C残余活性为83.3%,而rht2完全失活。表明了该二硫键Cys139-Cys206对人胰蛋白酶稳定性的重要性。进一步进行结构模拟和分析,解释了该对二硫键对稳定性影响的机制。
基金Natural Science Foundation of Gansu Provincegrant number:1506RJZA278
文摘Objective: To study the pharmacokinetic, distribution and elimination properties of rhTα 1 after intravenous(i.v.) and subcutaneous(s.c.)injection in mice and rats. Methods: Competition ELISA was used for testing drug concentration in serum, urine, bile and tissue after administration of rhTα 1 in mice(0.16, 0.5, 2.5 mg/kg) and rats(0.32, 1, 5 mg/kg). Pharmacokinetic parameters were calculated by Win Nolin software. Results: Absorption of rh Tαl is rapid in both mice and rats after s.c. administration. The pharmacokinetics in mice are characterized by linear, T_(1/2) showed a prolongation with increasing dose, 1.10, 1.35, and 1.50 h corresponding to 0.32, 1 and 5 mg/kg respectively, but in rats T_(1/2) showed no difference among doses. AUC0-∞ showed a clear increase with increasing doses in mice(904.18, 2998.83, and 19001.82 h*ng/m L) and in rats(1327.56 ±237.00,2924.53 ±685.14, and 35286.26 ±5999.58 h*ng/m L). After i.v. administration of 1 mg/kg rhTα 1 in mice, the drug is seen distributed in most organs, the thymus/serum exposure ratio was higher than others at the 1 and 2 h, the accumulative urinary excretion of primary drug was 32.97% ±15.85% within 6 h. Conclusion: The results indicate that rapid absorption, extensive distribution and quick renal excretion were the basic kinetic characteristics of rh Tαl after s.c. and i.v. administration.
