硫酸阿托品拮抗次乌头碱毒性的荧光光谱研究

Study on antagonism between atropina sulfate and hypaconitine by fluorescence spectroscopy

目的研究次乌头碱(HA)与牛血清白蛋白(BSA)非共价结合特征,并进一步探讨硫酸阿托品(AT)对HA与BSA结合的影响。方法模拟人体生理pH条件,应用荧光光谱及紫外光谱技术,确定HA与BSA作用方式、作用力类型及AT对它们结合的影响。结果 HA通过动态猝灭机制导致BSA荧光猝灭。HA与BSA表观结合常数K2b98K=1.10×102、K3b10K=3.18×105L.mol-1,结合位点数n298K=0.59、n310K=1.24;结合距离4.64 nm;主要作用力为疏水力;HA与BSA的结合反应是一个高温自发过程;HA与BSA结合后色氨酸残基所处微环境疏水性增强。AT使HA与BSA的Kb和n均减小。结论 HA与BSA作用形成一种超分子。AT能降低二者的结合程度,通过减少HA在生物体内的积累,加快代谢,发挥解毒作用。

Aim To study the characteristics of noncovalent binding between Hypaconitine（HA） and bovine serum albumin（BSA）,and discuss the effect of atropine sulphate（AT） on the binding of BSA and HA.Methods The interaction mode and predominant intermolecular forces of HA binding to BSA,and influence of AT on the binding were studied in simulating physiological condition（pH 7.40） by ultraviolet absorption and fluorescence spectra.Results HA quenched the endogenous fluorescence of BSA via a dynamic quenching procedure.Apparent binding constants（Kb）were 1.10×102 L·mol-1（298K） and 3.18×105 L·mol-1（310K）,and the number of binding-sites（n）was 0.59（298K） and 1.24（310K）,which increased with a rise in temperature.The distance between the BSA and HA was 4.64 nm,and predominant intermolecular forces between them were hydrophobic interactions.The negative value of free energy change was taken as the evidence for the spontaneity of the binding of HA to BSA.The polarity around the Trp residues was decreased and the hydrophobicity was increased by synchronous fluorescence techniques.AT acted competitively with HA in binding to BSA and decreased apparent binding constant and the number of binding sites of HA to BSA.Conclusions HA and BSA form supramolecules.AT can increase the content of unbound HA,shorten the residence time of HA in blood and accelerate the metabolism.