用MALDI-TOF质谱和电子光谱研究纳米胰岛素核-铁蛋白的构建技术和特性

小批量制备了电泳纯鲨鱼肝铁蛋白(Liver ferritin of Sphyma zygaena, SZLF). 用透射电子显微镜技术研究SZLF的铁核和蛋白壳亚基解离和重组过程. 用盐酸解离(pH=1.5)和分离膜透析技术制备铁蛋白亚基, 并用酸碱中和方法重组亚基成为脱铁核铁蛋白(apoSZLF), 同时将胰岛素(Insulin, INS)包裹于apoSZLF蛋白壳内, 构建纳米INS核-SZLF. 用电子光谱、MALDI-TOF质谱和SDS-PAGE技术分别揭示了纳米INS核-SZLF分子结构的真实性, 提出铁蛋白亚基包裹INS构建为纳米INS核-铁蛋白的途径.     

电泳/质谱技术研究鲨鱼肝铁蛋白及亚基多聚体特性

改良鲨鱼肝铁蛋白(liver ferritin ofsphyrna zygaena,SZLF)分离技术,并结合非变性梯度聚丙烯酰胺凝胶电泳(NGPAGE)制备质谱纯SZLF,以维系铁蛋白分子和它的亚基之间的作用类型,供研究铁蛋白结构与功能。实验结果表明,SZLF由分子量约为20kDa的单类型亚基组成。SZLF和它的亚基均组成不同的聚合体。聚合体类型和聚合数目与铁蛋白亚基和分离介质有关。MALDI-TO F质谱仪的激光和基质协同能有效解吸SZLF中的亚基成为准分子离子,并供质量分析,其亚基特征质谱峰m/z值分别为10889.35和22030.45,确定为带双电荷[M 2H]2 和单电荷[M H] 的SZLF亚基分子量。SDS-PAGE和变性IEF技术研究指出,形成不同聚合态的SZLF,其分子之间的相互作用强度高于SZLF亚基自身,难以通过MALDI-TOF质谱技术测定其亚基的结构信息。尽管SZLF由单类型亚基组成,但它能通过聚合体和自身亚基之间的相互作用差异性发挥极其重要的生理功能。     

用圆二色性和荧光光谱技术研究纳米吡啰红G核-铁蛋白的构建机理

小批量制备了电泳纯鲨鱼肝铁蛋白(Liver ferritin ofSphyrna zygaena,SZLF),对SZLF的结构与功能进行了研究.在pH=2.0~10.0条件下,选用圆二色性(Circular dichroism,CD)光谱技术研究了SZLF和脱铁核SZLF(apoSZLF)二级结构转换的基本特征和变化趋势,揭示了SZLF蛋白壳的亚基稳定性、相互作用强度和去折叠现象.利用酸碱中和方法,解离SZLF蛋白壳亚基,并再次构建成完整SZLF.用紫外-可见分光光度法和荧光光度法研究了构建纳米吡啰红G核-铁蛋白的途径.定量分析结果表明,每分子apoSZLF可捕获12分子吡啰红G于蛋白壳内,构建纳米吡啰红G核-铁蛋白.分析了apoSZLF直接捕获和释放吡啰红G的途径与速率,为后续构建高存量纳米顺铂核-铁蛋白药物载体提供了可行性技术.     

蛋白质辅助基质提高激光解吸/电离多肽离子化率和绝对强度的研究

人血清转铁蛋白(Human serum transferrin, HTF)、牛血清白蛋白(Bovine serum albumin, BSA)、鲨鱼肝铁蛋白(Sphyrna zygaena liver ferritin, SZLF)、马脾铁蛋白(Horse spleen ferritin, HSF)均能辅助基质提高激光解吸/电离胰岛素(Insulin, INS)和海兔酸性多肽(Aplysia acidic peptide, AP)离子化率和质谱峰的绝对强度(简称为绝对强度), 其中绝对强度分别提高10和4倍, 这一现象不依赖于INS浓度, 而与蛋白质类型和结构有关. SZLF和脱铁核SZLF(apoSZLF)辅助基质提高INS绝对强度的能力几乎相同, 蛋白质中的金属离子含量对这种效应无明显影响, 主要取决于蛋白质的氨基酸组成与结构. 采用胶内酶解和肽指纹技术(PMF)鉴定HTF过程中, 发现基质中分别添加SZLF, apoSZLF和HSF后, HTF肽片段质谱检测的质谱峰数目及绝对强度均有明显增加, 进一步提高了数据库鉴定HTF的可信度.     

棕色固氮菌细菌铁蛋白捕获能力、稳定性和亚基相互作用的强度

选用柱层析、电泳和反相高效液相色谱(RP-HPLC)技术制备质谱纯棕色固氮菌细菌铁蛋白(Bacteri-al ferritin ofAzotobacter vinelandii,AVBF),并采用释放铁动力学和肽质量指纹图谱(Peptide mass fingerprint-ing,PMF)技术分别鉴定AVBF。基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和电泳技术揭示AVBF亚基之间相互作用强度、稳定性和聚合态。AVBF可直接捕获有机小分子亚甲蓝(MB),其捕获率为15.0±2.0MB/AVBF,认为介于AVBF亚基单体之间的血红素参与捕获MB。较高浓度(40%~50%)的乙腈和丙酮均能使AVBF和鲨鱼肝铁蛋白(Liver ferritin of shark,SLF)释放不稳定亚基,但在较低浓度(20%~30%)的乙腈条件下,却需要借助来源于质谱仪的激光才能使AVBF或SLF释放不稳定亚基,并供质谱分析。AVBF亚基之间的相互作用强度明显低于SLF。铁蛋白亚基之间的相互作用强度高低与铁蛋白执行释放和储存铁的速率有关。     

MALDI-TOF质谱、电泳和透射电子显微镜技术研究魟鱼肝铁蛋白H和L亚基特性

小批量制备质谱纯魟鱼肝铁蛋白(liver ferritin of Dasyatis akajei, DALF),以透射电子显微镜术(TEM)测定DALF、蛋白壳和铁核的分子尺寸.SDS-PAGE技术指出,DALF由H 和L两种不同类型的亚基组成.采用肽质量指纹图谱(PMF)技术进一步佐证H和L亚基类型和同源性.氧化还原剂中性红、劳氏紫、甲基紫精和 pH 1.5酸度均无法削弱DALF的H-L和L-L亚基之间的相互作用强度、并解吸出L亚基离子,供MALDI-TOF质谱仪分析.通过增加激光强度和降低基质pH途径,可直接分析DALF的H和L亚基的分子量,指出DALF中H-L和L-L亚基之间的相互作用强度明显高于H-H亚基类型.     

MALDI-TOF MS和电子光谱技术研究海兔肝铁蛋白亚基电离和释放铁动力学特性

选用肽质量指纹谱(peptide mass fingerprint,PMF)技术鉴定质谱纯海兔肝铁蛋白(liver ferritin ofAplysia,ALF)。来源于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)仪中的激光和基质芥子酸协同解吸海兔肝铁蛋白(ALF)为带双电荷、单电荷[M H] 和二聚体的亚基离子,并可供质谱分析。ALF亚基的质荷比m/z分别为9784.03[M 2H]2 、19678.42[M H] 和39387.80[2M H] ,其中亚基分子量[M H] 略小于鲨鱼肝铁蛋白(liver ferritin of shark,SLF)。在弱碱介质(pH8.0)条件下,电子光谱技术研究指出,抗坏血酸以1/2级反应方式参与ALF释放铁全过程,同时又使ALF以一级反应动力学方式释放铁,呈现两种不同的速率。推测这一异常现象可能与ALF含低铁量、亚基调节能力和海兔的进化地位有关。     

电泳和质谱技术研究4种胰脏铁蛋白的亚基类型和等电点特性

采用电泳和质谱技术对所制备的鸡、鸭、牛和猪胰脏铁蛋白的亚基类型和等电点特性进行了研究。采用天然聚丙烯酰胺凝胶电泳(PAGE)技术研究的结果表明,上述4种铁蛋白呈现不同的迁移率,据此可知鸡胰铁蛋白的相对分子质量(Mr)>鸭胰铁蛋白的Mr>黄牛胰铁蛋白的Mr>猪胰铁蛋白的Mr,而且均大于马脾铁蛋白(HSF)的Mr。采用十二烷基硫酸钠(SDS)-PAGE技术研究的结果表明,上述4种铁蛋白均由H(heavy chain)和L(light chain)类型的亚基组成,但H和L亚基的相对数量（即H/L亚基数量的比值）有差别。采用肽指纹图谱技术分别鉴定各铁蛋白的H和L亚基。选用变性等电聚焦方法研究发现,上述4种铁蛋白分别由3～6种不同等电点的亚基聚合体组成,说明铁蛋白的H和L亚基之间呈现复杂的相互作用和不同的聚合体。不同陆生动物胰脏铁蛋白亚基之间相互作用的强度和聚合态存在着差异,这一差异特性可能与调控铁蛋白释放铁的速率有关,也与动物对铁的需求和铁解毒速率有关。     

铁蛋白纳米蛋白壳结构与功能研究新进展

铁蛋白是广泛存在于生物体内的储存铁的蛋白质,它参与生物体内的铁代谢.本文从铁蛋白的亚基结构、血红素-电子隧道、铁氧化酶位点和捕获重金属离子角度,综述铁蛋白纳米蛋白壳的结构与功能研究的新进展.     

重组人粒—巨噬细胞集落刺激因子去折叠过程的高效液相色谱动态监测

建立了用反相高效液相色谱分析重组人粒－巨噬细胞集落刺激因子（ｈｒＧＭ－ＣＳＦ）在二硫苏糖醇 （ＤＴＴ）作用下去折叠的方法。分离了ｈｒＧＭ－ＣＳＦ去折叠变化过程中的异构体，实现了对这一过程的动态监 测，从色谱分析的角度提供了结构信息，并用二极管阵列检测器和基质辅助激光解吸附飞行时间质谱对结 果进行了验证。     

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.     

Nanoparticles of Prussian blue ferritin: a new route for obtaining nanomaterials

The dissociation of apoferritin into subunits at pH 2 followed by its re-formation at pH 8.5 in the presence of hexacyanoferrate(III) gave rise to a solution containing hexacyanoferrate(III) trapped within the apoferritin and hexacyanoferrate(III) outside it. The addition of Fe(II) to the dialyzed solution resulted in the appearance of the characteristic Prussian blue color. The UV-vis spectrum of this solution showed a broad band centered at 710 nm, and the IR spectrum contained a broad-medium band at 2083 cm(-1). Both features are consistent with the charge-transfer band and the C[bond]N stretching mode in the Fe(II)[bond]CN[bond]Fe(III) fragment of PB. TEM images of the obtained Prussian blue solution showed discrete spherical electron dense iron particles with an average size of about 5 nm. This represents a new route for preparing metallic nanoparticles that offers control over the size and protection against aggregation. Moreover, the fact that the particles are obtained by reaction of hexacyanoferrate(III) and iron(II) building blocks opens up the possibility of obtaining not only homo- but also heterobimetallic nanoparticles.     

Controlled organization of ZnO building blocks into complex nanostructures

ZnO complex nanostructure with special mushroom-like morphology was prepared by hydrolysis of zinc acetate dehydrate (Zn(CH3COO)2 2H2O) in water-methanol mixed solvent at 60 degrees C. The formation mechanism was studied using XRD investigation and FE-SEM observation, which showed that the mushroom-like particles were transformed from cauliflower-like layered basic zinc acetate (LBZA), Zn5(OH)8(CH3COO)22H2O, and composed of ZnO subunits with average size less than 10 nm. The introduction of hexamethylenetetramine (HMTA, C6H12N4) to the solution before deposition led to drastic changes in the morphologies of both aggregation particles and ZnO subunits. The novel ZnO microspheres, which were made of regular hexagonal plate-like ZnO with dimensional size 35 x 10 nm, were formed. These hexagonal plate-like ZnO subunits stacked very compactly and aligned regularly. Kinetic study of this unique complex nanostructure using TEM and FE-SEM observation showed the presence of HMTA played an important role on the formation of hexagonal ZnO subunits through different mechanisms related to the different parts of microspheres.     

环氧树脂复合水分散乳液的制备及其性能研究

通过溶液聚合法制备了丙烯酸单体改性的高相对分子量的环氧树脂复合水分散乳液.利用傅立叶变换红外光谱仪测试了改性聚合物的结构,用ZETA电位分析仪测定了水分散液的粒径及其分布,用透射电子显微镜观察了乳液粒子形态.实验结果表明,改性树脂在水等极性溶剂中有较好的溶解性;随着改性树脂中羧基中和程度的加大,水分散液粒径减小,并成为形状规则的单分散乳液.作者同时进行了改性树脂稀释行为的研究.     

NONCONVENTIONAL EMULSION POLYMERIZATION OF ACRYLONITRILE CATALYZED BY in situ METAL COMPLEX

Nano-sized polyacrylonitrile (PAN) particles were prepared under the catalytic effect of in situ developed CoCl2/EDTA complex with ammonium persulfate as the initiator in the absence of any added emulsifier. The emulsion polymerization was studied at varying concentrations of the initiator, monomer, complex and solvent over a temperature range of 30-70℃. The overall activation energy (Ea, 49.79 kJ/mol), energy of dissociation of initiator (Ed,82.68 kJ/mol), number of micelles (0.163×1018) and the viscosity average molecular weight of the polymer were computed. The distribution of particle sizes was determined by transmission electron microscopy (TEM). It was found that the oil-in-water polymerization was stabilized by the presence of the CoCl2/EDTA in situ complex reducing the particle size into the nano order. The average diameters of PAN nano particles, obtained by TEM, were in the range of 50-150 nm at the maximum conversion. The experimental particle size was mainly dependent on the concentration of the complex and temperature.     

Separation of human glycoprotein hormones and their subunits by reversed-phase liquid chromatography

Separation of human pituitary follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) was effected on a micro-scale by reversed-phase high-performance liquid chromatography (HPLC) on a SynChropak C1 column in series with a Vydac C4 column using a linear gradient of acetonitrile in 0.1 M triethylamine phosphate buffer at pH 6.5. Chromatography on the C4 column alone caused partial dissociation of FSH into its subunits, whereas LH and TSH remained intact. Good yields of the separated subunits were obtained after prior dissociation of each hormone, and the results show that reversed-phase HPLC is useful for the analytical and preparative separation of these structurally related hormones and their subunits.     

Monitoring the dynamics of ligand-receptor complexes on model membranes

Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.