【作者】 田素燕；

【导师】 李连之；

【作者基本信息】 聊城大学 ， 分析化学， 2009， 硕士

【摘要】 细胞红蛋白(Cytoglobin, Cygb)是近期在脊椎动物组织中发现的第四种珠蛋白,它虽具有典型珠蛋白“three-over-three”类型的α-螺旋三明治折叠结构,但其蛋白肽链E-螺旋中的His81却与血红素配位,形成与神经红蛋白(Neuroglobin, Ngb)相似的六配位结构。在Cygb的血红素附近有一个与外部相连的大的疏水性蛋白基质空腔,可能用于提供配体进出的路径。Cygb几乎在人体各类组织中都有表达,可能具有携氧、储氧、氧感受器以及过氧化物酶等功能,此外它也可能与胶原形成有关,但其具体的生物功能仍未完全明确。因此本文对细胞红蛋白进行了基因表达和分离纯化,并研究了其基本的化学生物学性质以及可能的生物功能。主要的工作有:一、细胞红蛋白的基因表达、分离纯化与谱学表征。将带有人细胞红蛋白基因pET3a质粒转化至E. Coli BL21(DE3)plys中进行发酵表达,细胞红蛋白以可溶性和包涵体两种形式同时表达。可溶性蛋白依次经硫酸铵分级沉淀,Hiprep 16/10 Q FF阴离子交换柱,Hiload16/60 Superdex 75凝胶过滤柱和CM Sepharose FF阳离子交换柱纯化,得到电泳纯的可溶性蛋白(Scygb);包涵体蛋白经盐酸胍变性溶解、外加血红素重组和柱层析得到了电泳纯的可溶性蛋白(Icygb)。电喷雾质谱测得Scygb的分子量为21403.8 Da,Icygb的分子量则为21556.8 Da。紫外可见光谱表明,Scygb和Icygb的氧化态都在416 nm处有强的吸收峰,500 nm～600 nm间有宽的弱吸收峰,还原态都在428 nm,531 nm和561nm有吸收峰,但两者的A428(R)与A416(O)峰值比有所差别。荧光光谱表明,Scygb和Icygb的发射光谱的最大波长都为340 nm。但相同浓度时,Scygb的荧光强度要比Icygb的荧光强度高一倍左右。圆二色光谱表明,Scygb和Icygb的α-螺旋相对含量分别为64.4%和62.0%。还原态均在424 nm和555 nm处有正峰,氧化态都在257 nm和412 nm处有正峰,在285 nm处有负峰。但与Icygb相比,Scygb的还原态在425 nm处呈现一负峰,氧化态在453 nm处呈现一负峰。两者的热稳定性和酸碱稳定性的变化情况也不相同。通过以上对比,得出由可溶性形式纯化出的Cygb应该更接近于天然态,因此主要对这种形式的蛋白进行了深入研究。二、细胞红蛋白的荧光光谱和圆二色光谱研究。利用同步荧光光谱、荧光探针和荧光猝灭研究了Cygb的荧光特性以及溶液pH和巯基乙醇(2-ME)对Cygb构象的影响。波长差为20 nm和80 nm时Cygb在不同浓度下的同步荧光光谱结果表明,蛋白浓度对肽链中色氨酸和酪氨酸的荧光强度的影响不相同。波长差为20 nm和80 nm时Cygb在不同pH值溶液中的同步荧光光谱结果表明,在酸性环境中色氨酸和酪氨酸荧光峰的强度迅速增大,在碱性环境中两者荧光峰的强度明显降低。ANS荧光探针表明,在酸性环境中蛋白分子的构象发生了改变,出现能够结合ANS的疏水区。利用丙烯酰胺和I-、Cs+离子在有或无2-ME存在下对Cygb内源荧光进行猝灭作用的研究表明,三种猝灭剂对Cygb的荧光猝灭遵循Stern-Volmer方程,但它们的猝灭效率明显不同。当向蛋白溶液中加入2-ME后,三种猝灭剂对Cygb荧光基团的猝灭常数均有所增加。利用圆二色光谱研究了环境因素对Cygb二级结构的影响。结果表明,在蛋白浓度较低时,蛋白含有较高的α-螺旋。随着温度的升高,Cygb的α-螺旋含量逐渐减小。即使温度达到95℃,它仍保持有20%的α-螺旋结构,说明Cygb具有较高的热稳定性。在弱酸性和弱碱性溶液中,Cygb的二级结构都会有不同程度的破坏。在甲醇和乙醇中,Cygb的α-螺旋含量明显增加,这说明醇类可以诱导其α-螺旋的生成。三、细胞红蛋白的去折叠研究。利用紫外可见光谱、荧光光谱和圆二色光谱研究了酸诱导细胞红蛋白的去折叠过程。结果表明,随着酸度的增加,Cygb分子中血红素逐渐脱离蛋白链;蛋白的荧光强度显著增加;二级结构中α-螺旋含量逐渐降低。但酸诱导细胞红蛋白的去折叠并不彻底。利用紫外可见光谱、荧光光谱、荧光相图法和圆二色光谱研究了在有或无2-ME存在时,尿素和盐酸胍诱导细胞红蛋白的去折叠过程。结果表明:Cygb具有较强的抗尿素变性作用,即使尿素浓度达到10.0 mol/L时,变性也不完全;二硫键的破坏,降低了蛋白分子的稳定性。Cygb在尿素中的变性为“三态模型”,N→Iurea→U。在2-ME存在的尿素溶液中,其变性过程变为复杂的“四态模型”。Cygb在盐酸胍中的变性较为彻底,其血红素明显脱落。无2-ME存在时,当盐酸胍浓度达到4.5 mol/L时,蛋白变性完全,其变性中点Tm为3.5 mol/L。Cygb在盐酸胍中的去折叠过程为的“三态模型”,N→U→R。2-ME存在下蛋白从0.5 mol/L盐酸胍即开始变性,变性中点Tm降为2.0 mol/L,仍为“三态模型”。利用紫外可见光谱、荧光光谱和圆二色光谱研究了细胞红蛋白在甲醇和乙醇溶液中的去折叠和再折叠过程。去折叠过程的研究结果表明,随着醇浓度的增加,紫外可见吸收光谱中Soret带的最大吸收峰发生蓝移;荧光发射光谱的最大峰位发生红移;二级结构中α-螺旋含量明显升高。通过比较,乙醇的变性能力大于甲醇。再折叠过程的研究结果表明,再折叠过程与去折叠过程是可逆的,说明Cygb中肽链与血红素重组的路径和解离过程基本相同。四、细胞红蛋白的性能研究。利用紫外可见吸收光谱和动力学过程研究了细胞红蛋白的过氧化物酶催化活性。研究结果表明,Cygb具有一定的类过氧化物酶的酶活性。Cygb催化H2O2氧化邻甲氧基苯酚的最大反应速率Vm为54.9μmol?L-1?min-1,米氏常数Km为5.11×10-3 mol?L-1,转化数为Kcat = 11.0 min-1。在碱性环境和较高温度下,酶促反应的初速度明显增大。利用紫外可见吸收光谱研究了细胞红蛋白与硫化氢的相互作用。随着H2S浓度的增大,细胞红蛋白416 nm处的吸收峰逐渐降低并红移至428 nm,之后再逐渐增大,在531 nm、560 nm和606 nm处形成较明显的吸收峰,这说明H2S可以还原氧化态的Cygb。当温度高于45℃时,Cygb与H2S的反应速率明显增大。在pH 6.0时Cygb与H2S的反应速率最大。五、脱辅基细胞红蛋白(Apocygb)与金属卟啉的重组及脱辅基蛋白的稳定性研究。紫外可见光谱结果表明,Apocygb与Fe(III)PPIX重组后,立即在412 nm处形成新吸收峰,后移至416 nm处,还原态在428 nm处形成强吸收峰。Apocygb与Fe(III)PPIX的重组动力学过程包括小于0.2 s的快反应阶段和直到5 min还未完全平衡的慢反应阶段。Apocygb与Co(III)PPIX和Mn(III)PPIX的重组具有相似的过程。利用内源性荧光、外源性荧光探针ANS的荧光光谱和圆二色光谱研究了脱辅基细胞红蛋白在酸、尿素和盐酸胍中的去折叠过程。结果表明,Apocygb在pH7.5～6.0范围内以天然态存在,在pH5.5～4.0之间出现了一个较稳定的中间态。在pH6.0～2.5范围内,ANS逐渐与Apocygb结合,其荧光强度迅速增大,最大峰位由478 nm蓝移至466 nm。尿素和盐酸胍诱导脱辅基细胞红蛋白的去折叠过程表明,随着变性剂浓度的增加,肽链中的荧光基团逐渐暴露于极性环境中,蛋白肽链逐渐去折叠。变性剂不论采用尿素还是盐酸胍,加入2-ME后,变性中间点都提前1.0 mol/L,但对变性过程态没有较大影响。更多还原

【Abstract】 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.更多还原