Resonance Raman Spectroscopic Investigation Of Cytochrome C–Cardiolipin Interactions

Resonance Raman spectroscopy?RRS?can selectively enhance the Raman bands of the chromophore and the groups near the chromophore and provide the molecular structural information that may not be present in the normal Raman spectra,which is a powerful tool in the study of protein structural analysis and protein-ligand interaction.Cytochrome c?Cyt c?is a heme protein located in the mitochondrial intermembrane space,which not only participates in mitochondrial respiration and regulates cellular energy metabolism,but also plays an important role in apoptosis.In this thesis,Cyt c was studied by resonance Raman spectroscopy?RRS?.The main content of this study is divided into two parts: the effects of different experimental factors on Cyt c;the interactions between Cyt c and cardiolipin?CL?liposomes.We have the following results:?1?Raman spectroscopic study on the influence of various experimental factors on Cyt c for the preparation of investigation of Cyt c-CL interaction.The effect of Raman laser lines on Cyt c was investigated.The effects of different excitation wavelengths on the Raman signas of Cyt c were studied and the results showed that the best resonance spectrum of Cyt c porphyrin rings can be obtained at the excitation wavelength of 532 nm.Then,the influence on Cyt c Raman signals was discussed by changing the laser power and illumination time of the 532 nm laser.It was found that the laser power and the irradiation time had little effect on the Raman signals of Cyt c within certain range.In addition,the effects of Fe Cl3 and Mn Cl2 on Cyt c were also discussed,and the results showed that not every heavy metal salts can denature the protein.The basic conformational transformation was investigated by comparing the Raman spectra of Cyt c between p H 7 and 12.The results showed that the porphyrin ring structure of Cyt c changed to a certain extent at about p H 10,and its sixth coordination axial ligand,Met80,was dissociated and replaced by lysine residues but remained six-coordinated low spin?6c LS?structure.?2?Resonance Raman spectroscopic investigation of ferrous/ferric cytochrome c–cardiolipin interactions clarified the major molecular form of the Cyt c that interacted with CL in the first events of apoptosis.Magnetic Ni nanowires?Ni NWs?were prepared by a dropwise method.Compared with the commonly used reducing agent sodium dithionite,we found that Ni NWs can not only reduce Fe3+cyt c,but also are non-toxic and easy to operate.Moreover,we investigated the reduction ability of Ni NWs to different heme proteins?Cyt b5,Mb,Hb?besides Cyt c.The results showed that the Ni NWs prepared have specific reduction ability to the Cyt c.In addition,we prepared CL vesicles and analyzed their UV and Raman spectra.The results showed that there was almost no absorption peak and Raman band of the CL in the solution state,which could not interfere with the signal of Cyt c.The interactions between the Cyt c in both oxidized?Fe3+?and reduced(Fe²⁺)form with CL were studied by UV-Vis and RR spectra.The data showed the interaction between Fe²⁺Cyt c and CL is much stronger than that of Fe3+Cyt c.Moreover,a surprising finding is Fe²⁺Cyt c can induce much higher peroxidase activity of Cyt c than the Fe3+Cyt c.Our study reveals that Fe²⁺Cyt c probably plays a much more important role than Fe3+Cyt c in the first events of apoptosis,which provide a new understanding of the interaction between Cyt c and CL and further more will provide a new support for exploring the mechanism of apoptosis.