The Binding Of Metal Ions To CaM And The Ion Competition

Apoplast CaM has been found to exist in animals and plants, and has many biological functions. Recently its transmembrane and intracellular signal transdction mechanism has been developed. Therefore, the presence of many other metal ions in the physiological and nonphysiological environment suggests that CaM might be binding metal ions other than Ca²⁺, especially in resting cell. The capacity to substitute for Ca²⁺ is probably the similarity on ionic radii with Ca²⁺(0.99A). It is of importance to investigate the general metal ion binding properties of CaM. In this work ,we have studied the binding of various metals ions such as Cd²⁺, Pb²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Zn²⁺,Cu²⁺,La²⁺ to CaM with Linear Sweep Polarography of Cd²⁺ and Pb²⁺ ion selective electrode method . Our data show that many of these metal ions can bind to CaM but that they all bind in different order and relative affinity. The main results are as followings.Firstly, the binding of Cd²⁺ to CaM was measured at different pH through the use of Linear Sweep Polarography (LSP). The results shows that there are ten binding sites four capital sites and six auxiliary sites in CaM at pH 6.5, and only four binding sites (only four capital sites) at pH 4.5. The slope turning point in titration curve of Cd²⁺ to CaM can be used to estimate the concentration of CaM with a simple calculation C_Cam=l/10C_cd²⁺(PH=6.5) or C_cam=l/4C_cd²⁺. The detection limit of CaM with the method is of the order of 10^-8mol/L.Secondly, other metal ions binding to CaM were studied by Cd²⁺ polarography probe .The results at PH 6.5 indicated that Ca²⁺, Sr²⁺, Ba²⁺ and Mg²⁺ can only bind to the capital sites. Mn²⁺ has a preference for the A but not for the C sites. La²⁺, Zn²⁺, and Cu²⁺ can bind to two classes of the ion binding sites. The affinity of Ca²⁺ to C sites is higher than that of Cd²⁺, and the affinity of Mg²⁺, Sr²⁺, Ba²⁺ to C sites is much lower than that of Cd²⁺. La³⁺ has the highest affinity for C sites among them, La³⁺ can displace the bound Cd²⁺ inCaM.Thirdly, the binding equilibrium of Pb²⁺ to CaM was studied by the method of Pb²⁺ ion electrode , and the experimental data were solved by EXCEL programming method .The results show that Pb²⁺ has about 11 binding sites in CaM, and there are four strong binding sites (C sites) among them , suggesting that Pb²⁺ binds with an almost equal and high affinity to all four capital sites .The successive binding constants of Pb²⁺ to CaM are k₁=2.62 10⁷, k₂=3.75x10⁷, k₃=1.03xl0⁷, L₄=l.85xl0⁷, k₅=2.53xl0⁶, K₆=8.07xl0⁵, k₇=3.36x10⁵, k₈=2.76xl0⁵, k₉=2.22xl0⁵, k₁₀=3.96xl0⁴, k₁₁=3.72xl0⁴.Finally, three chmometric algorithms including EXCEL programming method , simplex method and netting calculation method have been tested to solve the Ca²⁺-CaM binding equilibrium system .The calculations showed that the accurate optimal results of parameters will not be obtained if the initial estimated value is far from the true value of the parameters, and the netting calculation method has no effect. Meanwhile the advantages of the combination of netting calculation method with simplex method or with EXCEL programming method were also discussed.