Research On Interaction Of Copper (â…¡), Cadmium(â…¡) And Several Small Molecules With Serum Albumin

Protein as the essential biological material in the cells has been playing many vital roles for all kinds of biological phenomena. It has many important functions such as transportation and distribution, whose biological function is dependent on its special structure. Serum Albumin (SA) is the most abundant carrier protein in blood. Serum Albumin (SA) is a small protein with a single polypeptide chain, which is largely α-helical. It consists of three structurally homologous domains: domain I , domain II and domain III. Each domain is composed of two sub-domains (A and B), which forms a columnar cavity with grooves towards each other. Site I and Site II located in domain II and domain III are known as locations of high affinity binding ability for many small molecules such as endogenous and exogenous ligands. Serum albumin has many important physiological functions. For example, by binding to chemicals such as metallic ions, fatty acids, steroids, amino acids, drugs, serum albumin realize their transportation, distribution and metabolism in the body. As a result, investigation of the interaction of serum albumin with chemicals noted previously can offer us very important information for understanding the absorption, transportation, distribution and metabolism of the chemicals in vitro.It is important to study the interaction of small molecules and metallic ion with the protein because protein-drug binding plays an important role in pharmacology, pharmacodynamics, toxicology and gene mutation. Therefore, it has been an interesting research field of life sciences, chemistry and clinical medicine. In this dissertation, on the basis of the previous research, the fluorescence spectroscopy including synchronous fluorescence and three-dimensional fluorescence combined with U-visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy were used to investigate the interaction of Cu(II), Cd(II) and several small molecules with serum albumin. The following major works were carried out:In the first chapter, there are five parts are introduced. First of all, a brief introduction of knowledge of protein and characteristics of serum albumin is offered. Secondly, common methods and instruments are elaborately introduced in this research field. Thirdly, information obtained by spectroscopy was exhibited, including binding constants K, binding sites n and major force etc. In the fourth part, current research situation was discussed. Eventually, our research contents, its innovation, purpose and theoretical significance were stated. Among them, emphasis was placed upon research methods and obtained information. In addition, future was predicted and prospected.In the second chapter, the binding of Cu(II) to bovine serum albumin (BSA) was investigated by spectroscopic (fluorescence spectroscopy, synchronous fluorescence and ultraviolet spectrum) techniques under simulative physiological conditions. Quenching for intrinsic fluorescence of serum albumin by Cu(II) was investigated in different temperatures by fluorescence spectroscopy. 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 non-fluorescence complex formed between Cu(II) and bovine serum albumin resulted in the decrease of intrinsic fluorescence of BSA. Thus static quenching predominated in the binding process. Their interaction is a spontaneous super-molecule formation (ΔS >0, ΔG <0). The hydrophobic force played a major role in the binding BSA to Cu(II). The spectral results observed showed that the binding of Cu(II) to BSA induced conformational changes in BSA. That is to say, polarity around tryptophan residues was increased and the hydrophobicity was decreased. Moreover, the interaction of Cu(II) and Cd(II) with serum albumin was investigated by fluorescence spectroscopy and the competition between Cu(II) and Cd(II) was analyzed by absorption spectra. Results showed that fluorescence emitted by tyrosyl and tryptophanyl residue could be quenched by Cu(II) and Cd(II).The quenching produced by Cu(II)was much stronger than that produced by Cd(II). Cu(II) only bound the Trp 214 in II A subdomain, but Cd(II) interacted with both Trp 214 in IIA subdomain and Trp 135 in I B subdomain of bovine serum albumin. The binding of Cu(II) with bovine serum albumin predominated in the presence of Cu(II) and Cd(II) together. In the third chapter, under the imitated physiological condition of animal body, the interactions of heteropoly salt (PM-19) with bovine serum albumin (BSA) were investigated by fluorescence spectrum and absorption spectroscopy. Quenching for intrinsic fluorescence of serum albumin by PM-19 was investigated in various temperatures by fluorescence spectroscopy. Data were handled by some equations. Thermodynamic parameters ΔH, ΔG, ΔS were calculated and their interaction forces were revealed. The distance r between donor (serum albumin SA) and acceptor (PM-19) was obtained according to fluorescence resonance energy transfer (FRET). The effect of PM-19 on the conformation of serum albumin has been analyzed by synchronous fluorescence spectroscopy. It was proved that the fluorescence quenching of SA by PM-19 is mainly a result of static quenching and non-radiation energy transfer. The binding constants of the complex formed by PM-19 and SA is 10⁴-10⁶L·mol^-1, which illustrated that the complex is more stable. It indicated that PM-19 can be stored and transported by SA. Electrostatic force played an important role in the binding reaction; hydrogen bonds and van der waals interactions play a major role in stabilizing the complex. In addition, the binding instance r is 4.14nm (BSA-PM-19) and 4.21nm (HSA-PM-19) respectively. Furthermore synchronous fluorescence spectra showed that the binding sites for PM-19 is close to tryptophan residue (Trp 212) of BSA and Trp 214 of HSA. The microenvironment of the tyrosine and tryptophan residue has not obvious changed, which indicated that the interaction of PM-19 with SA does not affect the conformation of serum albumin.In the fourth chapter, the interaction between 4-nitroaniline and serum albumin (SA) has been studied by fluorescence spectroscopy, absorption spectra, synchronous fluorescence and three dimensional fluorescence spectra. Quenching of intrinsic fluorescence of SA was investigated at different temperature. Stern-Volmer and double logarithm equations were used to study various binding parameters. The thermodynamic parameters had also been calculated. The binding average distance, r between the donor (SA) and acceptor (4-nitroaniline) was determined based on the F(o|¨)rster's theory. The effect of 4-nitroaniline on the conformation of serum albumin has been analyzed by means of synchronous and three dimensional fluorescence spectroscopy. A strong fluorescence quenching reaction of 4-nitroaniline to SA was observed and the quenching mechanism was suggested as static quenching and non-radiation energy transfer. In addition, for BSA, at higher ligand concentration, the Stern-Volmer plot exhibits an upward curvature, concave towards the y-axis. It illustrated both dynamic and static quenching was involved. There is about one class binding sites for binding of 4-nitroaniline to SA. Thermodynamic analysis showed that electrostatic and hydrophobic interactions were the mainly binding force Negative gibbs free energy (ΔG), positive entropy (ΔS) and negative enthalpy (ΔH) values indicated the interaction between 4-nitroaniline and SA is spontaneous. The binding instance r is 3.87nm for BSA and 3.76nm for HSA respectively. Furthermore synchronous fluorescence spectra showed that the binding sites for 4-nitroaniline is close to tryptophan residue of SA. It is also indicated that the polarity around the tryptophan residues were decreased and the hydrophobicity was increased. The three dimensional fluorescence spectra proved that 4-Nitroaniline destroyed ordered structure of SA, resulting in formation of more folding of SA.In the fifth chapter, absorption, fluorescence, synchronous fluorescence and three dimensional fluorescence spectra were used to investigate the interaction of 2,4-dinitroaniline with BSA. Data was treated by several equations. The distance r between acceptor and donor was calculated using F(o|¨)rster's theory. In addition, the impact of ligand on conformation of BSA was studied. Results revealed that static quenching occurs together with non-radiation energy transfer. As a result, 2,4-dinitroaniline caused the fluorescence quenching of BSA by the formation of complex. The number of binding sites is 1.17 and the distance r is 3.13 nm. Fluorescence originating from tryptophan residues and tyrosine residues can be quenched. It indicated that the polarity around the tyrosine residues was decreased and the hydrophobicity was increased. The three dimensional fluorescence spectra proved that 2,4-dinitroaniline destroyed ordered structure of SA, resulting in formation of more folding in SA.In the sixth chapter, the interaction of hexadecyl trimethyl ammonium bromide (CTAB) and sodium dodecyl benzene sulfonate (SDBS) with Bovine serum albumin was investigated by fluorescence spectroscopy at different temperature respectively. The effect of SDBS and CTAB on conformation of protein was discussed. It was found that the intrinsic fluorescence can be quenched by either CTAB or SDBS. Quenching by SDBS is stronger than that by CTAB. There are two types of sites in the binding of surfactant to BSA. The fluorescence quenching for the first type binding site was proved that Static quenching is predominant. Hydrogen bonds and van der waals interactions play a major role in stabilizing the complex. Negative entropy (ΔS) and negative enthalpy (ΔH) indicated that the interaction of CTAB or SDBS is spontaneous process. Polarity around tryptophan residue was decreased and hydrophobicity was increased by addition to CTAB or SDBS. Infrared spectral results revealed that the binding of CTAB or SDBS induced conformational changes in BSA, resulting in the decrease of a-helical.Total conclusions were given in the seventh chapter.