两种多酸型酪氨酸酶抑制剂的性能研究

doi:10.7503/cjcu20131010

摘要/Abstract

摘要：

以H₃PW₁₂O₄₀和H₄SiW₁₂O₄₀(简写为PW₁₂和SiW₁₂)为效应物, 测定其对酪氨酸酶活力的抑制作用. 通过非变性聚丙烯凝胶(Native-PAGE)电泳确定酪氨酸酶是多家族基因编码, 其分子量为3×10⁴~3.4×10⁴, 4.2×10⁴~4.6×10⁴, 6.4×10⁴~6.8×10⁴, 且均具有活性, 测定PW₁₂和SiW₁₂对酪氨酸酶的抑制效果, 并结合酶动力学法研究其抑制机理. 结果表明, 当PW₁₂和SiW₁₂浓度分别达到13和25 mmol/L时, 酪氨酸酶的活力完全被抑制, 即PW₁₂和SiW₁₂对酪氨酸酶二酚酶具有不同程度的抑制效果. 当所加酶量为0.0173 mg/mL时, PW₁₂和SiW₁₂对酪氨酸酶二酚酶活力的半抑制率 (IC₅₀)分别为1.57和2.31 mmol/L, 它们对酪氨酸酶二酚酶的抑制均为可逆过程. 其中, PW₁₂对二酚酶的抑制类型为混合型, 其K_I及K_IS分别为0.34和0.43 mmol/L, SiW₁₂对二酚酶的抑制类型表现为竞争型, 其K_I为0.59 mmol/L. 综合考虑IC₅₀值和抑制常数等参数, PW₁₂对酪氨酸酶二酚酶的抑制能力优于SiW₁₂.

关键词: 多金属氧酸盐, 酪氨酸酶, 非变性聚丙烯凝胶电泳, 抑制作用

Abstract:

H₃PW₁₂O₄₀ and H₄SiW₁₂O₄₀(abbreviated as PW₁₂ and SiW₁₂) were taken as inhibitors and their inhibitory effects and mechanisms on mushroom tyrosinase were evaluated by Native polyacrylamide gel electrophoresis(PAGE) electrophoresis method and kinetic enzymatic method. The Native-PAGE electrophoresis results show that tyrosinase are encoded by a multigene family, their molecular weight are mainly 3×10⁴—3.4×10⁴, 4.2×10⁴—4.6×10⁴ and 6.4×10⁴—6.8×10⁴, which were all active.The inhibitory effects of Kiggin-type polyoxotungstates, H₃PW₁₂O₄₀ and H₄SiW₁₂O₄₀ on mushroom tyrosinase were evaluated, and then the inhibitory mechanisms were further studied combined with the kinetic enzymatic method. The results indicated that the enzyme was inactive when the concentration of PW₁₂ and SiW₁₂ was 13 and 25 mmol/L, respectively. More-over, the kinetic enzymatic method suggested that both PW₁₂ and SiW₁₂ exhibited potent inhibitory activities with IC₅₀ value of 1.57 and 2.31 mmol/L, respectively. PW₁₂ was found to be a mixed-type inhibitor with K_I=0.34 mmol/L and K_IS=0.43 mmol/L, while SiW₁₂ was suggested to be competitive inhibitor of tyrosinase with K_I of 0.59 mmol/L. Based on a comprehensive consideration of IC₅₀ and inhibitory constants, PW₁₂ was more effective against mushroom tyrosinase than SiW₁₂.

Key words: Polyoxometalate, Mushroom tyrosinase, Native polyacrylamide gel electrophoresis(PAGE), Inhibition effect

中图分类号:

O614.5
Q55

TrendMD:

郑阿萍, 王芳, 邢蕊, 蒋爱华, 王力. 两种多酸型酪氨酸酶抑制剂的性能研究. 高等学校化学学报, 2014, 35(3): 476.

ZHENG Aping, WANG Fang, XING Rui, JIANG Aihua, WANG Li. Functionality Research of Keggin-type Polyoxotungstate as Potent Mushroom Tyrosinase Inhibitors^†. Chem. J. Chinese Universities, 2014, 35(3): 476.

图/表 6

Fig.1 Electrophoresis analysis of protein mole-cular weight and species of mushroom tyrosinaseLane M: protein molecular weight markers; lane 1: tyrosinase stained with coomassie blue; lane 2, 3: tyrosinase stained with L-DOPA.

Fig.2 Native-PAGE analysis of Inhibitory effects of PW12(A) and SiW12(B) on the mushroom tyrosinaseConcentrations of PW12 for lanes 1—10 were 0, 5, 10, 11, 12, 13, 14, 15, 16, 18 mmol/L, respectively. Concentrations of SiW12 for lanes 1—10 were 0, 5, 10, 15, 16, 17, 18, 19, 20, 25 mmol/L, respectively.

Fig.3 Inhibitory effects of PW12(a) and SiW12(b) on the mushroom tyrosinase

Fig.4 Inhibitory mechanism of PW12(A) and SiW12(B) on the mushroom tyrosinase (A) Concentrations of curves a—c were 0, 0.67 and 1 mmol/L, respectively; (B) concentrations of curves a—c were 0, 1.67 and 3.33 mmol/L, respectively.

Fig.5 Inhibitory types and the inhibitory constants of PW12 on mushroom tyrosinase for the catalysis of L-DOPA(A), the secondary plot of the slope vs. the concentrations of PW12 for determining the inhibitory constant KI(B), the secondary plot of the intercept vs. the concentrations of PW12 for determining the inhibitory constant KIS(C)(A) Concentrations of PW12 for curves a—d were 0, 0.67, 1 and 1.33 mmol/L, respectively.

Fig.6 Inhibitory types and the inhibitory constants of SiW12 on mushroom tyrosinase for the catalysis of L-DOPA(A) and the secondary plot of the intercept vs. the concentrations of PW12 for determining the inhibitory constant KIS(B)(A) Concentrations of SiW12 for curves a—d were 0, 0.67, 1.33 and 2 mmol/L, respectively.

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