The Research To The Unfold Of Hemoproteins

In all of the cellular components, protein is the most abundant and polyfunctional organic material. It plays an executive role in the activities of life.Protein has spatial structures, right folding conformation is the foundation of its complete life functions. So the researchs of protein such as the prediction of protein structure and the problem of protein folding are the important issues in the field of current biology, which is also one of the core problems in life science research. The research contents in this thesis as flollows:⑴In this experiment,we have collected large amounts of cells for engineering bacteria, and used two-steps(NH4)2SO4 precipitation, DEAE ion-exchange column chromatography, Sephadex-G100 gel fitration to purify the myoglobin.At last, we get pure protein of purity requirement above 96%.⑵ The paper studied the unfolding process of Mb induced by different p H and determined the spectroscopy characteristics of myoglobin in the different p H, by using of three kinds of spectroscopy methods, the ultraviolet absorption spectra,fluorescence spectra, and circular dichroism spectra(CD). Experiment results show that with the increased acidity of the protein solution,Mb tends to unfold, and the process of its degeneration correspond to the "binary" mechanism. Uv-visible absorption spectra show that the Soret band of 409 nm and the Q band of 503 nm,630 nm reduce and disappear gradually, while the 370.5 nm, new absorption peaks appeared at 513 nm and 644 nm; the values of characteristic peak for fluorescence spectra increase, these results show that peptides stretch gradually; the result of CD also indicated that the unfolding process of Mb occurs in different p H, the percentages of α-helical content is decreased, which means the secondary structure collapses.⑶ In this paper, we use the UV absorption spectra, fluorescence spectra, to study the biological characteristics of the guanidine hydrochloride-induced denaturation of Myoglobin. The results show that along with the increment of the guanidine hydrochloride, the protein is unfolded. When the guanidine hydrochloride is 1.93mol/L, the protein is mid-denaturature. Myoglobin’three-dimensiona structure will stretch, and secondary structure collapse.⑷ In this paper, we use the UV absorption spectra, fluorescence spectra,synchronous fluorescence spectroscopy and circular dichroism(CD) spectroscopy to study the biological characteristics of the urea-induced denaturation of hemoglobin.The results found that along with the increment of the urinary, Hemoglobin decreases in the OD value of 406 nm, while the absorption band is moved to longer wavelengths at the time. With the increase of the concentration of urine, the secondary structure of the most closely alpha-helical content decreases gradually,therefore the role of the denaturant causes the whole protein from close natural structure to loose stretched state. According to add β-mercaptoethanol, the unfolding of hemoglobinsolution more quickly,,which descript β- mercaptoethanol which were added to the hemoglobin has obvious effect in the folding process.The research, concerning the unfolding process of Mb induced by different p H,guanidine hydrochloride and urea will help us for the understanding of protein space structure and protein function in living organisms and provide us with some experimental basis in the studying of protein folding, as well as the research of fold and unfold diseases.