| The interaction of proteins with [Hg(SCN)4]2- and [Zn(SCN)4]2-, interaction between protein and protein, interaction between protein and sodium heparin were investigated by absorption spectra, fluorescence spectra and resonance Rayleigh scattering (RRS) spectra. The reaction mechanism, binding mode, binding sites and the main interaction forces were studied through the changes in spectra. Additional, some new methods for the determination of protein or sodium heparin were established from the investigated systems. The main work of this thesis is as follows:1. Study of the interaction between [Hg(SCN)4]2- and proteins by resonance Rayleigh scattering spectra and their analytical applications[Hg(SCN)4]2- in H2SO4 at pH 1.0-4.5 was reacted with proteins, including human serum albumin (HSA), a-chymotrypsin (a-Chy), bovine serum albumin (BSA), lysozyme (Lyso) and y-globin (y-G), to form complexes. As a result, the intensities of resonance Rayleigh scattering (RRS) were significantly enhanced. Changes in absorption, circular dichroism (CD) spectra and the fluorescence quenching of proteins were also observed. Attention was paid mainly to the RRS spectral characteristics of these complexes, suitable reaction conditions, influencing factors and a number of analytical properties. The changes in absorption, fluorescence and CD spectra for the [Hg(SCN)4]2--Lyso system were used as an example to explore the binding sites, binding model and reasons for enhanced scattering. The RRS method had a high sensitivity and the detection limits (3σ) were 4.6-10.8 ng/mL for the proteins studied. A new method is thus established herein, using [Hg(SCN)4]2- as a probe for the determination of proteins in human serum and urine samples.2. Resonance Rayleigh scattering spectral investigation of the interaction between [Hg(SCN)4]2- and hemoglobin In a slightly acidic medium of pH 1.0-4.5, anionic complex [Hg(SCN)4]2- reacted with hemoglobin to form a 10:1 complex. As a result, the intensity of resonance Rayleigh scattering and resonance nonlinear scattering such as second-order scattering and frequency doubling scattering were greatly enhanced. The discussion on the interaction, binding sites and binding model, which was based on the effect of the complexation on the resonance light scattering spectra, on the fluorescence quenching of hemoglobin by [Hg(SCN)4]2- and on the changes of absorption and circular dichroism spectroscopy, indicated that four [Hg(SCN)4]2- entered respectively the four subunits of hemoglobin to form complex with appropriate amino acid residues via hydrogen bonding, hydrophobic interaction and electrostatic forces. The enhancement of scattering was mainly due to the increase of hydrophobicity and volume of the scattering molecule, the energy transfer between fluorescence and resonance scattering, even due to the favorable conformation alteration of hemoglobin induced by [Hg(SCN)4]2-. Therefore, resonance Rayleigh scattering and resonance nonlinear scattering spectra not only can be used as a helpful tool for studying the interaction between metal complex and protein such as hemoglobin, but also can create new conditions for highly sensitive determination of trace hemoglobin.3. [Hg(SCN)4]2- quenching behavior of proteins fluorescence and its analytical application[Hg(SCN)4]2- in acidic medium of pH 1.4-3.4 was reacted with proteins, including bovine serum albumin (BSA),γ-globin (γ-G) and Hemoglobin (Hb), to form complexes. As a result, the fluorescence intensities of proteins were significantly quenched. The quenched fluorescence intensities (△F) in a certain range were linearly related to the concentration of Hg(Ⅱ), and the detection limit (3σ) were 4.4 ng/mL (BSA as a probe),6.5 ng/mL (γ-G as a probe) and 12.9 ng/mL (Hb as a probe), respectively. The effect of the interaction on the fluorescence spectral characteristics of these complexes, suitable reaction conditions and influencing factors were investigated. The quenching mechanism was studied by absorption spectroscopy, binding constant and corresponding thermodynamic parameters at different temperatures. Some potential interferents on the assay by BSA were investigated. The method displayed good selectivity and was satisfactorily applied to the determination of asceptichrome in mercurochrome solution and thiomersalatum in HB-vaccine.4. Study on the interaction between [Zn(SCN)4]2- and proteins by resonance Rayleigh scattering spectra and their analytical applications In pH 1.75-1.90 acid medium, the interactions of [Zn(SCN)4]2- with proteins, such as human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hb), a-chymotrypsin (a-Chy), lysozyme (Lyso) and y-globin (y-G), could not induce the change in absorption spectra and fluorescence spectra, but these interaction could result in the great enhancement of resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering (FDS). The maximum scattering wavelength was located at 290 nm (RRS),552 (SOS) nm and 392 nm (FDS), respectively. These six systems have different sensitivity, the detection limit (3σ) of HSA,y-G and Hb by RRS method is 4.9-7.8 ng/mL which is the highest sensitivity. The RRS spectral characteristics of these systems, suitable reaction conditions, influencing factors and effect of coexisting substances were investigated. Thus a new method for the determination of total proteins in human serum and urine was established. Additional, the binding stoichiometric ratio, the main interaction forces and possible binding mode were studied when [Zn(SCN)4]2- reacted with HSA.5. Study on the interaction between pepsin and lysozyme by resonance Rayleigh scattering and resonance non-linear scattering and its analytical applicationIn pH 4.8-5.6 acid medium, multiple negatively charged pepsin (Pep) could bind to multiple positively charged lysozyme (Lyso) to form 1:2 complex via electrostatic attraction, hydrophobic interaction and hydrogen bonding. As a result, the intensities of resonance Rayleigh scattering (RRS) and resonance non-linear scattering (RNLS), including second order scattering (SOS) and frequency doubling scattering (FDS), could be greatly enhanced. The maximum RRS wavelength was at about 290 nm where the enhanced intensity (△I) was linear to the concentration of Lyso in a certain range. The detection limit (3σ) of Lyso was 4.3 ng/mL (RRS),4.5 ng/mL (SOS) and 23.6 ng/mL(FDS). The RRS method was utilized to determine the level of Lyso in human saliva and hen egg white.6. Study on the interaction between sodium heparin and proteins by resonance Rayleigh scattering and resonance non-linear scattering and its analytical applicationIn NaAc-HCl buffer medium with proper pH, some positively charged proteins such as human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hb), Papain (Pap) and albumin egg (Alb-egg) could react with negatively charged sodium heparin (Hep) to form complexes by electrostatic attraction, hydrophobic interaction and hydrogen bonding. As a result, the intensities of resonance Rayleigh scattering (RRS), second order scattering (SOS) and frequency doubling scattering (FDS), could be significantly enhanced. The enhanced intensity (△I) was linear to the concentration of Hep in a certain range. The detection limit (3σ) of Hep by RRS was 3.1 ng/mL. The effects of coexisting substances showed good selectivity of the established method.7. Study on the interaction between sodium heparin and lysozyme by absorption, fluorescence and resonance Rayleigh scattering spectraUnder pH 7.4 physical condition, multiple positively charged lysozyme (Lyso) could bind to multiple negatively charged sodium heparin (Hep) to form 1:1 complex via electrostatic attraction and hydrophobic interaction. As a result, the change in absorption spectra, the fluorescence quenching of Lyso and the great enhancement of resonance Rayleigh scattering (RRS) intensity were observed. The RRS spectral characteristics, suitable reaction conditions, influencing factors and some analytical properties were investigated. The reaction mechanism and binding mode were studied through the changes in absorption, fluorescence, RRS and circular dichroism spectra.
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