All of the vital actions in cells are dependent on the expression of function of proteins, no proteins, no vital actions, a lot of biological molecules all participate in maintain and activation of proteins’ biological functions. So reveal the profound mystery of vital movement must base on deeply research on the interaction of proteins.The hemeproteins participate in important biological function in cell, such as, preserving, transport of oxygen and electron transfer, and so on, which are executors in the vital actions. Here, we have studied the interaction between hemoproteins, such as Myoglobin and Cytochrome b5and some representative ligands, such as metal ions (Cu(Ⅱ), Fe(Ⅲ)), PEG200, PEG400, under near physiological conditions in vitro.(1) The interactions between Cu(Ⅱ), Fe(Ⅲ), Fe(Ⅱ) and the water soluble fragment of cytochrome b5(cytb5) have been investigated by multi-spectroscopic techniques, such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy, time-resolved fluorescence spetroscopy and circular dichrosm(CD). The results show that metal ions, such as, Cu(Ⅱ), Fe(Ⅲ), Fe(Ⅱ) can interact with cytb5, which induce the quenching of cytb5. The quenching mechanism of cytb5has been studied by fluorescence spectroscopy in different temperatures. The quenching mechanism of cytb5is dominated by dynamic quenching induced by Cu(Ⅱ), and the quenching mechanism of cytb5induced by Fe(Ⅱ) and Fe(Ⅲ) are static quenching, the valence state of iron has little effect on the interaction between cytb5and Fe. Synchronous fluorescence and circular dichroism spectra demonstrate that Cu(Ⅱ), Fe(Ⅲ) and Fe(Ⅱ) could also influence the micro-circumatance of aromatic amino residues and secondary structure of the protein.(2) The direct interactions between the active center of myoglobin and metal ions M(Ⅱ)(Cu(Ⅱ)、Zn(Ⅱ)、Mg(Ⅱ)、Co(Ⅱ) and Mn(Ⅱ)) have been studied by ultraviolet absorbance spectra. By changing different ionic concentration [Mb:M(Ⅱ)=1:1;1:2;1:4;1:8;1:10], different temperature [277,294,310and325K] and different interaction time(2,4,6,8and10days), results show that the iron ion of myoglobin is replaced by metal ions M(Ⅱ) and the corresponding myoglobin derivatives are produced, in addition, the interaction is increased gradually with amount of external metal ions added, the temperature advanced and the time extended.(3) The interactions between Mb(K56D) and Cu(Ⅱ), Fe(Ⅲ) have been studied by UV-Vis absorption spectroscopy, fluorescence spectroscopy and circular dichrosm(CD). Quenching of Mb(K56D) were studied by fluorescence spectroscopy in different temperatures289,298and310K, respectively, and the mechanism of quenching were confirmed. The binding contant, binding site numbers were gained by Lineweaver-Burk equation and double-lg equation.The thermodynamic parameters and acting force of interaction were found by thermodynamic equation. The binding distants between protein and metal ions were achieved by the Forster theory of non-radiation energy thansfer. At the same time, how the conformation of Mb(K56D) to be affected by Cu(Ⅱ) and Fe(Ⅲ) have been reaseached by synchronous fluorescence spetra and CD. Displacement of Asp44, Asp60and Lys56amino acids on the surface of myoglobin has a certain impact on the three-dimensional structure and function of myoglobin compared with wild type myoglobin.(4) The coordination reaction between metMb(WT) and Mb(K56D) and PEG400, PEG200, PEG2000were investigated by means of UV-Vis absorption spectra, steady fluorescence spectra, circular dichroism(CD) and synchronous fluorescence spectra, respectively. The experimental results showed that the transitions of Soret and Q band shifted to lower energies due to the formation of metMb-PEG400complex. According to the fluorescence enhancement equation, the binding constants of the complex at different temperatures, actting force and the thermodynamic parameters were obtained by double-lg equation and thermodynamic equation. At the same time, how to affect on conformation of Mbs in forming the complexs has been studied by synchronous fluorescence spectroscopy and UV spectroscopy. At last, similarities and differences of coordination reaction between metMb and different PEG has been gained.(5) The unfold of cytb5and Myoglobin(WT, D44K) have been studied by the fluorescence spectra and circular dichroism spectra in different temperatures and pHs. The results show that the hydrophobic region in the interior of the proteins have been collapsed, and the conformation is changed when the temperature and pHs have been changed and the mutation of the surface-charged residue Asp44to Lys44increases the protein’s stability on the resistance to heat and acid denature.We have gained the exact and important result by studying the interaction between hemeproteins and low molecular weight’s biological molecule by the numbers.These results help to know the metabolizable course of metal ions in vitro and affect of metal ions on structure of proteins, which deeply explain the affect of metal ions on biological function of proteins; help to know the pharmacology and toxicology of neuter surfactants PEG; help to know the coordination reaction of hemoproteins and ligants, which are very important for basic investigation and application.