Molecule Recognition Based On The Protein Phosphorescence And Fluorescence

Fluorescence and phosphorescence analysis are the two major methods in luminescence. For its remarkable characteristics of high sensitivity, simple operation and good selectivity, these methods bring to more attention and interesting. They have been studied and applied more and more. At present, as the aggregation of dye chromophore on biological macromolecule leading to enhancement or decrease of luminescence intensity, these methods are mainly used to study biological macromolecules such as nucleic acid and proteins. Furthermore, when two ions of different charges form ion association complexes by electrostatic force, hydrophobic force or charge transferring effect, fluorescence and phosphorescence can be enhancement or decrease and the spectra characteristics can be changed. Theses methods have been used to determination of trance metal ions, nonmetal ions, organic compounds, environmental analysis and pharmaceutical analysis.Pharmaceutical analysis is an important part of analytical chemistry. Chirality is an intrinsic property of many biological systems. Most of the chemical medicines are chiral molecules, and their pharmacological functions can be acquired depending on the strict chiral matching and molecular recognition with the macromolecules in vivo. As the chiral medicine becomes more and more important, the research is necessary and it needs to develop more and more new analytical technology and analytical methods. Up to now, these are few reports about the applications of fluorescence and phosphorescence analytical method in chiral medicine analysis. Our experiments show that the fluorescence and phosphorescence of protein can be stereoselective quenched by the external chiral quencher. The researches have important application prospects and worth to further studies.The characteristics of fluorescence and phosphorescence were investigated using BSA and HSA as example in the present study, and the phosphorescence method for determining the protein was developed.In addition, the phosphorescence analytical method and its applications in the scientific research aspects were reviewed, and the theories foundation, recent study and trends envisaged for future perspectives was introduced.Under the supports of the National Natural Science Foundation of China (Grant No. 20275022), the following research systems were investigated in detail.1. Fluorescence and phosphorescence behaviors of bovine and human sera albuminThe phosphorescence analysis of bovine serum albumin (BSA) and human serum albumin (HSA) was presented. The protein phosphorescence is from tryptophan residues buried within the cores of it. Various affects including heavy atoms, pH condition and the de-oxygenation concentration were discussed and the phosphorescence analytical method of HSA and BSA was established. The phosphorescence of BSA and HSA was enhanced via the addition of iodide to introduce the so-called "heavy atom" effect and the use of sodium sulfite for chemical de-oxygenation. The phosphorescence locates 443nm when it was excited at 287 nm. And the phosphorescence has better repetition and stability under the experimental condition. The linearity and detection limit for BSA and HSA were Ixl0'6~8.6xl0"5mol/L and 2.20x10"7mol/L, 3xl0'6~13xl0"5mol/L and 4.10xl0"7mol/L, respectively. The spectra characteristics including the fluorescence/phosphorescence lifetime and the polarization were determined. The results show tryptophan residues fluorescence in BSA have two decay forms, while those in HSA have only one. Their phosphorescence belongs to long-lifetime phosphorescence and the phosphorescence spectra are incomplete polarize. The value of polarization difference between phosphorescence and fluorescence revealed that the microenvironments of the tryptophan residues are different in the two forms of luminescence. In addition, the protein conformation changes induced by surfactant and organic solvent were discussed.2. Study on the interaction between achiral molecules and sera albuminsCoporprophyrin I , III, bilirubin and biliverdin are two pairs of achiral molecules that have similar structures. The fluorescence and phosphorescence of BSA and HSA showed a gradual decrease with the addition of them as quenchers. Both dynamic and static quenching were involved in the present research, which was demonstrated by the fact that the Stern-Volmer plot slightly deviated from linearity toward the y-axis. Coporprophyrin I, III, bilirubin and biliverdin can enter the hydrophobic cavity of protein by hydrogen bond and hydrophobic interaction and interacted with the tryptophan residues buried within the cores of it. The excited singlet energy of tryptophan residues can transfer to the ground state of Coporprophyrin I, III, bilirubin and biliverdin based on the Foster's non-radiative energy transfer theory, which can influence the protein phosphorescence indirectly. So the phosphorescence intensity decreased and the lifetime shortened. There are not obvious difference among their quenching constants and the phosphorescence lifetime difference between them both closed to the experimental errors, which indicated that the interaction of them in the hydrophobic cavity are similar. In addition, the protein conformation changed when interacted with coporprophyrin I , III, while the microenvironment of tryptophan residues has not obvious changeable during the binding process with bilirubin and biliverdin.3 investigation of the chiral discrimination between the pseudo-enantiomers by sera albuminsQuinidine and quinine is a pair of pseudo-enantiomer and there are 4 chiral centers in their molecule structure. They can enter the hydrophobic cavity via hydrophobic interaction and quench the tryptophan residues phosphorescence. The excited singlet energy of tryptophan residues can transfer to the ground state of quinidine and quinine based on the Foster's non-radiative energy transfer theory, which can influence the protein phosphorescence indirectly. The quenching process is enantioselective quenching, which involved dynamic and static quenching. Proteinphosphorescence lifetime difference induced by quinidine and quinine are in the range of 14-19%, which are larger than the experimental error. That is to say molecular recognition can be obtained based on the quenching difference, which was resulted from the binding difference. In addition, the binding process resulted in the protein conformation changes.4 study on the J3-blockers enantiomers discrimination based on the sera albumins phosphorescence quenchingTwo kinds of P-blockers enantiomers including to propranolol and atenolol can enantioselective quench the phosphorescence of BSA and HSA. The enantioselective quenching of propranolol is larger than that of atenolol, and the enantiomers discrimination can be obtained based on it. The quenching mechanism is involved the non-radiative energy transfer. But for atenolol, they cannot meet the requests of non-radiative energy transfer, and the quench may result from the complex or collision. In addition, protein phosphorescence polarization increases when the small molecule binding to it, and the difference between them can also use for the enantiomer discrimination.5 study on the binaphthyl enantiomers discrimination based on the sera albumins phosphorescence quenchingThe enantiomers of l,l'-bi-2-naphthol (BNOH), l,l'-binaphthyl-2,2'-diylhydrogemphosphate (BNPO4) and l,l'-binaphthyl-252'-diamine(BNNH2) can enantioselective quench the phosphorescence of BSA and HSA and change the protein conformation. The selectivity of BNNH2 is strongest among them. The hydrogen bond and ion bond may be responsible for it. The phosphorescence of these binaphthyl compounds enhanced by BSA and HSA, which confirm the energy transfer theory and reveal that protein can provide a more hydrophobic and rigider microenvironment for phosphors. The hydrophobic cavity of protein is chiral selectivity.6. Study on the camphorsulfonic acid enantiomers discrimination based on the sera albumins luminescence quenchingCamphorsulfonic acid enantiomers enantioselective binding to sera albumins by ion bond results to the protein luminescence quenching difference, which can be used to perform the enantiomer discrimination. In addition, the binding interaction causes protein fluorescence movement, which accounts for the microenvironment of the tryptophan residues changes.