光谱法研究高铁肌红蛋白活性中心与一氧化氮的配位反应

Spectral Study on Coordination Reaction between metMyoglobin and Nitric Oxide

蛋白质与配体作用的瞬时性对调节生物功能的正常发挥至关重要。采用紫外-可见吸收光谱法(UV-Vis)和圆二色光谱法(CD)研究了高铁肌红蛋白(metMyoglobin,metMb)与一氧化氮的配位反应过程,论证了metMb与NO配位反应机理以及影响配位反应的因素。结果表明:metMb能与NO发生配位反应,420,534和568nm处特征峰的出现,表明metMb与NO结合并生成一种新的配合物——亚硝基高铁肌红蛋白(nitrosylmetmyoglobin,metMbNO)。随着时间的推移,溶液中NO浓度逐渐减小,metMbNO配合物中Fe—N配位键在大量H2O分子的进攻下断裂,NO慢慢地脱离,metMbNO配合物完成解离过程并重新生成metMb。实验中还发现metMb与NO配位反应的进行受到缓冲介质类型、离子强度、pH及温度等外界因素的影响。并且在0.01mol·L-1磷酸盐缓冲液近中性条件时,metMb与NO配位反应最先达到平衡;当温度较低为280K时,更易于metMb与NO配位反应的进行。此外,CD数据表明NO只与metMb血红素中心的铁原子发生配位反应而对蛋白二级结构影响甚微。探讨metMb与NO配位反应机制对于进一步研究生物体内NO功能的发挥有着重要的指导意义。

As we all known,the instantaneous reaction between protein and ligands are very important to adjust the normal playing of biological function.And nitric oxide interactions with iron are the most important biological reactions in which NO participates.Unlike carbon monoxide or oxygen,NO can also bind reversibly to ferric iron.In this paper,UV-Vis absorption and CD spectra were used to study coordination reaction process between horse heart metMb and NO,to demonstrate the coordination reaction mechanism and to explore the influencing factors of metMb with NO.The experimental results showed that metMb could react with NO,and obtained three new peaks at 420 nm,534and 568 nm,respectively,which implied metMb and NO have reacted and generated a new complex-nitrosylmetmyoglobin（metMbNO）.Then as time went on,NO concentration decreased in the solution,and the Fe-N bond fractured under the attack of H2 O,then NO leaves slowly from metMbNO,and metMb was regenerated.In this experiment,we also found that external conditions such as buffer medium,ionic strength,pH,temperature,etc,had an important influence on the coordination reaction between metMb and NO.It was favorable for the coordination reaction,when the 0.01mol·L-1 phosphate buffer solution is near neutral condition,the temperature is 280 K,the coordination reaction could reach equilibrium at a fastest speed.In addition,the CD date show that NO only reacts with Fe atom in the center of heme and has less effect on the secondary structuers of protein.The research of metMb and NO played an important role to further study the function of NO.Especially the establish of equilibrium reaction mechanism between NO and heme protein has an important research value on maintaining the balance of NO in vivo and keeping the normal function in the body＇s cells.