Protein Labeling Fluorescent Probe Spectroscopy And Analysis Applications

Protein which maintains the metabolism of the organism is fundamental elements of life. The research on protein structure and function, and the quantitative analysis would illustrate the change in life under the physiological or pathological consideration, which is important for the clinical diagnosis and drug screening. The applications of protein marker fluorescent probes in biological analysis and proteomics become more extensive recently. The probes are used to recognize and detect the complicated conformational changes of proteins. Developing protein marker fluorescent probes and novel quantification method of proteins with high sensitivity and selectivity is important for the field of life sciences, chemistry and clinical medicine. This dissertation consists of four chapters.Chapter1:The structures, functions of proteins were briefly introduced firstly. The developments of protein marker fluorescent probes were reviewed, and the kinds of probes, marked mechanism and methods of proteins quantitative analysis were summarized.Chapter2:The interactions between sodium fluorescein (SF) and human serum albumin (HSA) were investigated by fluorescence spectroscopy. According to the modified Stern-Volmer equation, the association constants (Ka) between SF and HSA at three different temperatures were obtained to be1.34×107,1.00×107,0.80×107M-1, respectively. It is proved that the fluorescence quenching of SF by HSA is a result of the formation of SF-HSA complex. The thermodynamic parameters, enthalpy change (△H) and entropy change (AS) for the reaction were calculated to be-23.29kJ mol-1and57.09J mol-1K-1according to van't Hoff equation, indicating that the hydrophobic force was the dominant intermolecular force in stabilizing the complex. The distance r between donor (SF) and acceptor (HSA) was obtained to be5.1nm according to Forster's non-radioactive energy transfer theory. The effect of SF on the conformation of HSA was also analyzed using3D fluorescence spectroscopy and synchronous fluorescence spectroscopy. The fluorescence intensity of SF-HSA complex was proportional to the concentration of HSA, based on which, a new quantitative assay of protein was presented. The linear range was0.015～1.5μM, and the sensitivity of the method was high with detection limit was determined to be1.5×10-9M. The effects of pH and interfering substance on the detection were also investigated. The results indicated that the most of the water-soluble amino acids, metal ion and antioxidant do not interfer or only interfer slightly under the permission of±5.0%relative error, whereas SDS, Lecithin, APG, Cu2+, Fe3+, Sucrose and Tryptophane produced obvious interference. Determining serum in human serum by this method gived results which were very close to those obtained by Coomassie Brilliant Blue colorimetry.Chapter3:The photophysical parameters of probes such as quantum yield and dipole moments were calculated by salvation and quantum mechanics of second order perturbation theory. The luminiferous mechanism of probes was investigated by Density Functional Theory (DFT) and Amsterdam Density Functional (ADF) soft. According to the equations of Stern-Volmer,Scatchard and Van't Hoff, the binding parameters, binding force and binding thermodynamics parameters were calculated. Using the displacement experiments, the binding site of probes was illustrated. The effect of probes on the second structure of protein has been studied by the synchronous fluorescence and CD spectroscopy. The fluorescence of probes could be quenched by the addition of serum albumin, and the intensity of probes was linear with the concentrations of protein, based on which, a new fluorometric method for detecting protein using1,8-naphthalimide derivatives as fluorescent probes was developed. Determination of protein in human serum by this method gave results which were very close to those obtained by using Coomassie Brilliant Blue colorimetry.Chapter4:The interactions of rhapontin (RA) and ergosta-4,6,8(14),22-tetraen-3-one (ergone) with HSA were investigated by the molecular modeling and spectrua comprehensively for the first time, which applied the information about the binding domain and binding model of RA and ergone in HSA. The spectral results showed that the fluorescence of HSA was quenched by the binding of drugs, and the hydrophobic complexes were formed. The binding constant, number of binding site and thermodynamic parameters of binding interaction were calculated by different scanning calorimeter (DSC) and fluorescence spectroscopy. Three-dimensional and synchronous fluorescence spectra revealed the effect of three drugs on the second structure of HSA. Moreover, the photophysical parameters of RA and ergone were calculated by salvation and quantum mechanics of second order perturbation theory, and the luminiferous mechanism was investigated by the DFT and ADF soft. Ergone exhibited a remarkable fluorescence enhancement (EE) for serum albumin, while RA exhibited a fluorescence quenching for serum albumin, based on which, the novel sensitive quantitative determination of proteins were developed by using ergone and RHA as fluorescence probes under the physical condition. Good calibration curves of the proteins were obtained in the range of10-8～10-6M, with detection limits (3σ) of10-9M, and the effect of metal ions on the detection results were investigated. Detecting total proteins in human serum by this method gave results which were close to those obtained by using Coomassie Brilliant Blue colorimetry.In this dissertation, on the basis of the previous research, using the spectral analysis technics along with computational modeling were used to investigate the protein marker fluorescent probes and novel quantification method of proteins. The following major innovative works were carried out:1. Using SF, the novel1,8-naphthalimide derivatives, RA and ergone which are gradients of natural drug as the fluorescent probes. The photophysical parameters were calculated with quantum mechanics of second order perturbation theory, and the mechanism of photo luminescence of probes was investigated with the Density Functional Theory and ADF soft.2. The interactions of probes with HSA were studied by the comprehensive spectral analysis technology, such as the fluorescence, absorption and CD spectrum. According to the equations of Stern-Volmer, Scatchard and Van't Hoff, the binding mechanism, style of binding force and binding site was investigated.3. The interaction of RA and ergone with HSA was investigated by the molecular modeling, and the binding mode was developed, which provided the theoretical reference for the probes marked protein. The secondary structure compositions of HSA were estimated by quantitative analysis using synchronous fluorescence and CD spectroscopy selves deconvolution with second-derivative resolution enhancement and curve-fitting procedure.4. After labeling protein, the photophysical parameters of fluorescent probes changed remarkably, base on which, a new fluorometric method for detecting HSA in the HCl-Tris buffer solution (pH=7.4) was developed. The linear ranges of the calibration curves were10"8-10-6M for HSA, with the detection limits (3σ) of10-10M. The method has been applied to the determination of total serum albumin in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital and the method of G-250.