植物钙调素与Ce^(3+)离子结合反应的荧光光谱法研究

Study on Binding Reaction of Ce^(3+) to Plant Calmodulin by Ce^(3+) Fluorescence

水合Ce3+离子发射强荧光,而用Ce3+滴定植物钙调素(CaM)时,Ce3+离子荧光被完全淬灭,据此建立了测定Ce3+离子与植物钙调素结合位点数及逐级结合常数的方法。实验表明,Ce3+离子与植物钙调素的荧光激发光谱及发射光谱形状与光谱强度都有显著差别,前者的荧光强度远大于后者,故CaM存在时可以测定游离Ce3+的浓度。Ce3+离子荧光淬灭法测定结果说明,在0.5 mol.L-1KCl溶液中,CaM有4个Ce3+离子结合位点。进一步通过Ce3+滴定植物钙调素的实验,并结合Excel规划求解法,计算出Ce3+与CaM的结合常数,K1=4.17×107,K2=1.26×107,K3=1.89×106,K4=2.04×106。

The fluorescence of Ce^3＋ in aqueous solution,which results from 5d→4f transition,was observed to be more intense and sensitive than that of other lanthanide ions.The binding of Ce^3＋ to CaM was studied by Ce^3＋ fluorescence quenching method.The fluorescence properties of Ce^3＋ and CaM were studied by fluorescence spectrophotometer.The emission peak of Ce^3＋ was at 351 nm and that of CaM at 307 nm.Fluorescence quantum yield of Ce^3＋ ion is much higher than that of CaM.Ce^3＋concentration could be detected by the fluorescence intensity at 351 nm.When Ce^3＋ bound to CaM,the fluorescence f Ce^3＋ was quenched.Ce^3＋ fluorescence titration result showed that CaM can bind four Ce^3＋ in 0.5 mol·L-1 KCl solution（pH=6.5）.Using Excel programming method,the binding constants of Ce^3＋ to CaM were calculated,i.e.K1=4.17×10^7,K2=1.26×10^7,K3=1.89×10^6,K4=2.04×10^6.Our experiment results provide a molecular explanation of the mechanism for promotion of plant growth through calmodulin activation by Ce^3＋.