Studies On The Interaction Of Myoglobin And Its Mutants With Metal Ions By Spectral Technology

Protein as the essential biological material in the biosystem has been playing many vital roles for all kinds of biological phenomena. Myoglobin (Mb) has many important functions, such as oxygen storage and facilitated oxygen diffusion. Myoglobin has been used as a model protein in structural and binding studies. The interaction of protein with metal ions is a very critical theme for chemistry. When entering into animal body, metal ions interact with protein following by change of protein structure and its function. In this dissertation, the interactions of myoglobin and its mutants Mb(D44K),Mb(D60K) with metal ions (Cu²⁺,Fe³⁺) were studied in vitro using ultraviolet absorption, fluorescence and circular dichroism (CD) techniques.1. Under simulative physiological conditions of animal body, the interaction of Mb with Cu²⁺ was investigated by fluorescence spectroscopy in various temperatures (298,310,320 and 328K). Data were handled by using Stern-Volmer and Lineweaver-Burk double reciprocal equations. The major force between them was obtained by thermodynamic parameters. It was found that static quenching predominated in the binding process and non-fluorescence complex formed between Mb and Cu²⁺ resulted in the decrease of intrinsic fluorescence of Mb. Electrostatic force played a major role in the binding Cu²⁺ to Mb. The binding distance r between Mb and Cu²⁺ was 2.56 nm based on F?rster theory of non-radiation energy transfer. The change of microenvironment of tryptophan residues in protein and the secondary structure conformation of protein in the absence and presence of Cu²⁺ were investigated by using ultraviolet absorption, synchronous fluorescence and CD spectra. It was found that the polar of microenvironment of tryptophan residues increased, but the hydrophobic decreased. The contents of alpha helices changed from 64.0% to 61.8% .2. The interaction of Mb with Fe³⁺ was investigated by spectroscopic (fluorescence, synchronous fluorescence, ultraviolet absorption and CD) techniques under simulative physiological conditions. Data which were investigated in different temperatures (289, 298 and 310K) by fluorescence spectroscopy were handled by using Stern-Volmer and Lineweaver-Burk double reciprocal equations. The results showed that static quenching was predominant. Electrostatic force played an important role in the binding reaction and hydrophobic interactions also played a role in stabilizing the complex. In addition, the binding instance r was 3.18 nm. Synchronous fluorescence spectram indicated that the polarity around the tryptophan residues increased and the hydrophobicity decreased. The contents of alpha helices changed from 64.4% to 62.0% .3. With fluorescence spectroscopy, synchronous fluorescence and CD spectra, the interactions of Mb mutants Mb(D44K),Mb(D60K) with metal ions (Cu²⁺,Fe³⁺) were studied. Quenching of intrinsic fluorescence of protein were investigated at different temperatures. Stern-Volmer, Lineweaver-Burk double reciprocal equations and double logarithm equations were used to study various binding parameters. The thermodynamic parameters had also been calculated. The binding average distance r was determined based on the F?rster theory. Spectral results revealed that the binding of metal ions induced conformational changes in protein, and resulted in the decrease of a-helical. The experimental data demonstrated that the mutation of the surface-charged residue altered spatial structure of myoglobin.