Spectroscopy Investigation Of Human Serum Albumin And The Photo-induced Deoxygenization Of Myoglobin

This paper reports the photo-induced deoxygenization process of Mb with firstly established fluorescence spectroscopic method. It is discovered that in some aspects fluorescence spectroscopic method is much better than that of Raman or other spectroscopy. The advantages are as follows: (1) High sensitivity. (2) There is only one peak at 597nm and no other disturbed spectrum. (3) Convenience. The spectra are characterized by the fluorescence intensity to decline gradually in every scanning time, and the fluorescence intensity of decay is very large at each time which we assign to the release of oxygen from the opening of the heme-pockets. The release of oxygen from the heme-pockets of oxy-Mb is induced by illumination. More illumination will cause more release of oxygen and if the temperature of Mb solution is increased when it is illuminated, the rate of deoxygenization will be faster. In addition, it is found that ligand-oxygen in the Fe-porphyrin can be displaced from Mb by nitrogen. This means that we can investigate the interaction between oxy-Mb and other different gases with the method of fluorescence spectroscopy. Furthermore, in our study it is implied that fluorescence spectroscopy can be employed to probe the energy transfer between tryptophan, tyrosine and Fe-porphyrin in Mb molecules. So fluorescence spectroscopy method deserves to be advocated.Next, this paper studies the photo-induced deoxygenization process of Mb(D44K) with the established fluorescence spectroscopic method. The relative deoxygenization rate of Mb(WT,D44K) are 3.48,2.05 respectively under the same conditions. The deoxygenization rate of Mb(D44K)is slower than that of Mb(WT), which means that it is harder for Mb(D44K) to release the oxygen from the opening of the heme-pockets. Furthermore,its fluorescence efficiency at 597nm is lower than that of Mb(WT) no matter what the exciting wavelength is 430nm or 409nm. The third difference is that Mb(D44K) has 628.8nm peak besides 597.9nm which assigns to H2O binging to the Mb3+. The illumination not only causes the release of oxygen from the opening of the heme-pockets, but also causes the release of H2O. The energy transfer experiment shows that the mutation (Lys44 displacing Asp44) does not influence the efficiency between tryptophan, tyrosine and Fe-porphyrin, but makes the efficiency of intrinsic fluorescence of Mb higher. In addition, in our study it implies that the maximum absorption is not always the best exciting wavelength through the fact that 430nm is better for the release of oxygen from the opening of the heme-pockets.Finally, this paper studies the binding reaction of colchicine with human serum albumin (HSA) by UV-Vis absorption, fluorescence and circular dichroism spectrometry. The results indicate that colchicine leads to the increasing of UV absorption and the quenching of intrinsic fluorescence of HSA. As the temperature increases, the quenching constant Ksv decreases. The binding constants and the numbers of the binding sites of the interaction between colchicine and HSA at different temperatures are obtained. The thermodynamic parameters, enthalpy change (?H) and entropy change (?S) are calculated to be -11.66 KJ/ mol and 51.507 J/mol·K respectively according to Van′t Hoff equation, which suggest that the main binding force between colchicine and HSA is static interaction. The protein conformation is altered (CD data) with the decrease ofα-helices in the presence of colchicine. The results show that the quenching mechanism of the combination of colchicine with human serum albumin is a static quenching procedure.