木犀草素对酪氨酸酶活性抑制及构象诱导作用

Effect of Luteolin on Activity and Conformation of Tyrosinase

在37℃、pH 6.8的磷酸盐(100 mmol/L)缓冲体系中,采用酶抑制动力学方法,研究了木犀草素对酪氨酸酶的抑制作用、抑制类型和抑制动力学常数,并探讨了木犀草素对酪氨酸酶表面疏水性和二级结构的影响。结果表明,木犀草素是一种可逆的非竞争型酪氨酸酶抑制剂(半抑制率IC50为55.35±1.51μg/m,抑制常数Ki为63.57±2.12μg/mL);失活动力学时间进程分析表明木犀草素能快速与酪氨酸酶发生作用并迅速降低酶的活性;荧光光谱分析显示木犀草素能显著增强酪氨酸酶的表面疏水性;圆二色谱分析表明木犀草素诱导酪氨酸酶的构象伸展和解折叠,随着木犀草素浓度的增加,α-螺旋含量由31.3%逐渐增加到41.6%,由此可见木犀草素能够诱导酪氨酸酶的构象发生部分改变,使酶结构变化而不利于其形成活性中心,进而降低酪氨酸酶的活力。

The effects of luteolin on inhibitory action, inhibitory type and inhibitory kinetics of tyrosinase were investigated using the method of enzyme inhibition kinetics in 37 ℃, pH 6.8 sodium phosphate buffer（100 mmol/L） system, the changes of surface hydrophobicity and secondary structure of tyrosinase were also probed. The results showed that luteolin was a reversible and noncompetitive inhibitor（the half inhibition concentration（IC50） and inhibition constant（Ki） were obtained to be 55.35 μg/mL and 63.57 μg/mL, respectively）. The time-courses analysis of inactivation kinetics indicated that luteolin interacted with tyrosinase quickly and inhibited the acticity of enzyme rapidly. Fluorescence spectrum analysis showed that luteolin significantly increased the surface hydrophobicity of tyrosinase. Circular dichroism spectra analysis showed the binding of luteolin to tyrosinase induced the unfolding of conformation and the gradual increase of α-helix content of tyrosinase（from 31.3% to 41.6%）. The results of spectra analysis indicated that the binding of luteolin to tyrosinase altered the conformation of tyrosinase which was not conducive to the formation of active center, thereby reducing the activity of tyrosinase.