Gene Expression, Purification And Functions Studies Of Human Cytoglobin

Cytoglobin(Cygb) is the recently discovered fourth member of the vertebrate globin family. Although Cygb exhibits a traditional globin fold with a three-over-threeα-helical sandwich, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb. A stereo view of the protein matrix cavity system recognized next to heme, which as temporary docking stations for small gaseous ligands. Although distributed in almost all human tissues, Cygb has not been ascribed a specific function. Some hypotheses have been suggested, such as O2 storage and delivery, cytoplasm of fibroblast-like cells, most prominent role in cells of the fibroblast lineage, and so forth, whereas, the exact biological functions has been unclear. So, this thesis studied the expression, purification and spectra characterization of recombinant human Cygb, in addition, its essential biochemical characters and its potential biological functions were investigated. The main contributions of the thesis are as follows:1. Expression, purification and spectral characterization of human Cygb. The pET3a plasmid with the gene of human Cygb was transformed to E.coli BL21 (DE3) plys cells. Human Cygb has expressed in soluble form (Scygb) and inclusion bodies form (Icygb). Scygb was purified by ammonium sulfate precipitation, Hiprep 16/10 Q FF anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and CM Sepharose Fast Flow cation exchange column. Icygb was purified by dissolving in 6 mol/L guanidinium chloride, renatured with haemin solution and chromatography.Electrospray ionization mass spectrometry results indicated that the molecular weight of Scygb is 21403.8 Da and Icygb is 21556.8 Da. UV-spectra indicated that the absorption peaks of Scygb and Icygb are similar either in their ferrous form or in their ferric form. The ferric form has a strong absorption peak at 416 nm and a wider and weaker absorption peak between 500 nm and 600 nm. While, the ferrous form has a strong absorption peak at 428 nm, and two weak peaks at 531 nm and 561 nm. But the ratio of A428(R) and A416(O)is different between Scygb and Icygb. The maximal emission wavelength of Scygb and Icygb is 340 nm. But the intensity of Icygb is only half of the intensity of Scygb in the same concentration. Circular dichroism spectra of Scygb and Icygb showed that theα-helix content of their secondary structure are 64.4% and 62.0%. The ferrous forms of Scygb and Icygb both have absorption peaks at 424 nm and 555 nm and the ferric forms both have absorption peaks at 257 nm, 412 nm and 285 nm. However, compared with Icygb, the CD spectra of Scygb also has a peak at 425 nm in the ferrous form and a peak at 453 nm in the ferric form. Scygb and Icygb exist difference in their thermal, acidic and alkaline stability. By comparison, Scygb is close to the original nature, so the Cygb of the soluble form was detailedly investigated.2. Fluorescence and CD spectra studies on Cygb. The fluorescence properties of Cygb and the influence of pH and 2-mercaptoethanol (2-ME) on Cygb conformation were investigated by the methods of fluorescence quenching, fluorescence probe and synchronous fluorescence. Synchronous fluorescence spectra of Cygb indicated the change of Trp and Tyr exist difference at different protein concentrations whenΔλ= 20 nm andΔλ= 80 nm, but they are similar at different pH value. The influence of pH on Cygb by 8-Anilino-1- naphthalene-sulfonic acid (ANS) probe studies indicated that, ANS can bind to Cygb at low pH because pH value influences the local circumstances of the protein. The fluorescence quenching studies showed that the fluorescence intensity of Cygb can be quenched by CsCl, KI and acrylamide to different degree. Stern-Volmer constants determination indicated the fluorescence quenching of Cygb by CsCl, KI and acrylamide are all dynamic process. Stern-Volmer constants have increased in the presence of 2-ME. The effects of environmental factors on secondary structure of Cygb were detailedly investigated by using far-UV CD. The results showed that Cygb contains moreα-helices at lower concentration. Theα-helix content of Cygb decreases with increasing temperature, but over 20% ofα-helices can be kept at 368 K. Cygb loses itsα-helical secondary structure in either acidic or alkaline solution to some extent. Theα-helix content of Cygb in methanol and ethanol is obviously higher than that in water. Therefore, methanol and ethanol can induce the formation ofα-helix structure of Cygb.3. The unfolding investigation of Cygb. The effects of acid on Cygb stability has been investigated by UV spectra, fluorescence and CD spectra. The results showed that with the acidity increasing, the heme group disassociates from the protein chain and the protein's fluorophores exposed to more polar environment, which result in the enhancement of the fluorescence intensity. And the secondary structure of Cygb is also destroyed in acidic media, but the changes of the effects of acid on Cygb are not complete.The unfolding processes of Cygb in urea and guanidine hydrochloride (GdnHCl) with or without 2-ME were investigated by using UV spectra, fluorescence,"Phase Diagram"method of fluorescence and CD spectra. The results showed that Cygb has a strong resistance to the denaturing action by urea. It was not denatured completely even in 10.0 mol/L urea. 2-ME can destroy the intramolecular disulfide bond in Cygb and decrease the stability of Cygb. The unfolding of Cygb in urea is a three-state model, that is, N→Iurea→U. But in the presence of 2-ME, the case is different. The unfolding process became the complex four-state model, that is, N→I1urea→I2urea→U. GdnHCl is a kind of intensive denaturant, the unfolding process of Cygb in GdnHCl is relatively complete. The results indicated that, in the absence of 2-ME, the protein has denatured completely when GdnHCl concentration reached 4.5 mol/L, the denaturation midpoint concentration is at 3.5 mol/L. Whether 2-ME exists in the denaturant solution or not, the unfolding process of Cygb obey a complicated three-state model, that is, N→IGdmCl→U.The unfolding and refolding of Cygb induced by methanol and ethanol were investigated by means of the UV-visible spectra, circular dichroism spectra and the fluorescence spectra. The results showed that a blue shift of the Soret band in UV-Vis spectra and a red shift of the maximal emission wavelength. By comparison, the unfolding of Cygb induced by ethanol is stronger than that by methanol. All the results suggested that the pathway of the refolding process is almost the same with that of the unfolding process of Cygb in methanol and ethanol. Methanol and ethanol can destroy the protein structure to some extent, while they can induce theα-helix content to increase apparently.4. The investigation of Cygb on the potential biological functions. The peroxidase activity of Cygb for the catalytic oxidation of o-methoxyphenol by H2O2 was investigated, the maximal rate, Km and Kcat of the reaction are 54.9μmol?L-1?min-1, 5.11×10-3 mol?L-1 and 11.0 min-1, respectively. Under high pH and high temperature conditions, the reaction rate of this reaction increases obviously.The interaction of Cygb with H2S was investigated by UV-visible spectra. The results showed that addition of excessive H2S to ferric Cygb leads to a reduction in the intensity of the Soret band and a shift in the wavelength of maximum absorption from 416 nm to 428 nm, and two new peaks appear at 531 nm and 560 nm, which are the characteristic absorption of ferrous heme. In addition, a new peak appears at 606 nm by adding the amount of H2S. The reaction rate of Cygb with H2S gradually increases with the increase of temperature. The effect of pH on this reaction indicated that it has the maximal rate constant at pH 6.0.5. Studies on the recombination of Apocygb with metalloprotoporphyrin and the stability of Apocygb. When the Fe(III)- porphyrin was added to the Apocygb, the UV-Vis spectra indicated that the new strong peak at 412 nm appears at once. Then this peak shifts slowly to 416 nm after 12 h which is the same as the spectra of ferric Cygb. The recombination of Apocygb with Fe(III)-porphyrin reduced by addition of disodium dithionite has a strong absorption peak at 428 nm. The results of Stopped Flow Kinetics showed that the recombination of Apocygb with Fe(III)-porphyrin contains two portions. One is a fast reaction which is less than 0.2 s, the other is a slow reaction which is more than 5 min.The recombinations of Apocygb with Co(III)- porphyrin or Mn(III)- porphyrin have the same process.The effects of acid, urea and GdnHCl on Apocygb stability had been investigated by fluorescence and CD spectra. The results showed that the native state prevails between pH 7.5 and 6.0. A second conformational state is observed from the decreased fluorescence emission between pH 5.5 and 4.0. When bound to Apocygb between pH 6.0 and 2.5, ANS exhibits a large increase of fluorescence yield and a blue shift, i.e., from 478 to 466 nm. With the increase of denaturant concentration, the protein's fluorophores exposed to more polar environment, leading to its unfolding. In the presence of 2-ME no metter the effects of urea or GdnHCl, the case is the same as in the absence of 2-ME except that the unfolding midpoint concentration decreases by 1.0 mol/L.