重组类人胶原蛋白与牛源Ⅰ型胶原蛋白的比较研究

目的比较重组类人胶原蛋白(RHCg)和牛源Ⅰ型胶原蛋白(BTCg)的异同。方法用有限胃蛋白酶水解和盐析沉淀法从牛肌腱中提取纯化BTCg。通过氨基酸分析测定BTCg和RHCg的氨基酸组成;用园二色谱表征BTCg和RHCg在溶液中变性前后的二级结构;用差示扫描量热仪(DSC)、调制差示扫描量热仪(MDSC)以及热重仪(TG)分析BTCg和RHCg的热学性质。结果RHCg和BTCg的氨基酸组成有显著差异。特别是RHCg不含羟脯氨酸;亚氨基酸含量为17.83%(残基数),低于BTCg的22.03%(残基数);赖氨酸含量为0.91%低于BTCg的2.37%;谷氨酸含量为8.13%低于BTCg的12.91%;精氨酸为1.01%低于BTCg的5.33%。在溶液中,温度为20℃时,BTCg和RHCg分子的二级结构均为左旋聚脯氨酸(P-Ⅱ)构型。BTCg和RHCg在140~280℃区间的热学性能显著不同。结论BTCg和RHCg分子的二级结构相似,但氨基酸组成和热学性质存在显著差异。充分认识和理解不同来源胶原蛋白质的特性对于合理设计和使用胶原基生物材料具有重要意义。     

重组类人胶原蛋白Ⅱ的表达与纯化

研究了类人胶原蛋白Ⅱ在基因工程菌E.coli BL213．7中的表达与纯化。经高密度发酵及代谢调控，重组菌在12．8L发酵罐中的最终菌体密度约为150。菌体经高压匀浆破碎、沉淀、超滤、阳离子交换色谱纯化后，表达产物纯度达96．4％，总回收率71．6％。     

2种海参胶原蛋白多肽对血管内皮细胞保护作用的比较研究

目的 观察革皮氏海参和北极刺参胶原蛋白多肽对氧化型低密度脂蛋白(ox-LDL)损伤的血管内皮细胞的保护作用,探讨其保护内皮细胞的作用机制。方法 采用ox-LDL处理血管内皮细胞(ECV304)建立氧化应激损伤模型,以MTT法测定ECV304的增殖活性,硫代巴比妥酸法测定细胞内的丙二醛(MDA)含量,比色法测定一氧化氮合酶(NOS)活力和一氧化氮(NO)释放量,Hoechst33258染色法检测细胞的凋亡,Western blotting法检测caspase-3蛋白的表达。结果 经2种海参胶原蛋白多肽预处理后,ECV304细胞的增殖率显著升高 (P<0.05,P<0.01),细胞凋亡比率和MDA含量显著降低(P<0.05,P<0.01),细胞NOS活力和NO释放量均显著提高 (P<0.05,P<0.01),凋亡蛋白caspase-3的表达量显著降低。结论 2种海参胶原蛋白多肽均能有效保护脂质过氧化物损伤的血管内皮细胞,其中北极刺参胶原蛋白多肽在抑制细胞凋亡和提高NOS活性方面效果更突出,可能与其氨基酸组成有关。     

I型胶原蛋白对斑马鱼模型促组织再生及抗炎作用研究

目的 观察I型胶原蛋白对斑马鱼模型的促组织再生及抗炎作用。方法 通过检测斑马鱼尾鳍再生面积和col1a1b基因的相对表达量,表征I型胶原蛋白促组织再生作用;通过测量斑马鱼皮肤中性粒细胞数量变化表征I型胶原蛋白的抗炎作用。结果 与模型对照组相比,I型胶原蛋白溶液处理斑马鱼尾鳍面积明显增大,中性粒细胞数量明显减少。I型胶原蛋白溶液处理后的斑马鱼col1a1b基因的相对表达量明显高于正常对照组。结论I型胶原蛋白对斑马鱼模型具有促组织再生及抗炎作用。     

鸡Ⅱ型胶原蛋白的药物动力学研究

目的研究了鸡Ⅱ型胶原（chicken typeⅡcollagen, CCⅡ）在大鼠体内的药物动力学。方法采用氯胺T法进行125I-CCⅡ标记，放化纯度>96.26%。给大鼠灌胃7.4×105、1.48×106 及2.96×106 Bq*kg－1 的125I-CCⅡ，然后测定9个时间点的血药浓度，用一室模型进行参数计算。大鼠灌胃2.96×106 Bq*kg－1后，测定1 、3 、6 h的分布及排泄情况。结果125I-CCⅡ的吸收相较短，t1/2Ka约为0.56～0.74 h。消除较快，t1/2Ke约为3.60～4.32 h。该药分布较快，以肾、胃、肠、肺、肝、脾的分布量较高。在72 h 内从尿及粪中分别排出27.9%和4.8%，26 h 内从胆汁中排出6.7%。结论CCⅡ口服给药后，吸收较快，消除也较迅速，并且分布广泛。     

Ⅱ型胶原蛋白的热稳定性、圆二色性和红外光谱研究

目的以蓝鲨Ⅱ型胶原蛋白为材料,分析Ⅱ型胶原蛋白的热稳定性、圆二色性和红外光谱特性。方法采用限制性酶解法分离蓝鲨软骨Ⅱ型胶原蛋白,通过SDS-PAGE、DSC、CD、IR分析Ⅱ型胶原蛋白的相对分子质量、热变性温度、二级结构特征和红外光谱特征。结果蓝鲨Ⅱ型胶原蛋白是由相对分子质量为130kDa的α1链构成;蓝鲨Ⅱ型胶原蛋白的热变性温度为40.5℃,[Na+]和[H+]改变了胶原蛋白的热稳定性;蓝鲨Ⅱ型胶原蛋白在3411、3008、1648、1549cm-1有红外特征性吸收峰;蓝鲨Ⅱ型胶原蛋白以β-折叠为主,占主链的36.9%,接近于全β型三级结构,[H+]将改变Ⅱ型胶原蛋白的二级结构元件构成,使其β-折叠消失,出现少量的α-螺旋,Ⅱ型胶原蛋白肽链转变成无规卷曲。结论蓝鲨Ⅱ型胶原蛋白是典型的纤维状蛋白质,具有作为生物功能物质的优良生物化学特性。     

胶原蛋白敷料与常规敷料治疗糖尿病足溃疡的比较

目的:将胶原蛋白敷料应用于糖尿病足溃疡治疗中,探讨其临床治疗效果和安全性.方法:选择2019年1月～2020年12月某院住院治疗的糖尿病足溃疡94例,随机分为对照组(常规敷料治疗)和观察组(胶原蛋白敷料治疗)各47例,比较两组治疗前和治疗第1周、2周、4周、8周足部溃疡面积、愈合率,统计愈合时间和不良事件发生率.结果:...     

一些紫草素衍生物的园二色谱研究

从四川密花滇紫草(Onosma confertum W.W.Smith)和新疆软紫草[Arnebia euchroma (Royle) Johust]的根中分到九个紫草素衍生物,其中七个为手性化合物。测定了它们的CD谱和UV谱,结果得到三对几乎完全对称的CD谱图和完全相同的UV谱图、确定了四川密花滇紫草成分均为R构型;新疆软紫草成分均为S构型。     

罗非鱼鱼鳞胶原蛋白多肽对小鼠成骨细胞增殖与Ⅰ型胶原蛋白分泌的影响

目的 研究不同分子量的罗非鱼鱼鳞胶原蛋白多肽对体外培养新生小鼠颅骨MC3T3-E1细胞增殖与I型胶原蛋白分泌及I型胶原蛋白mRNA表达的影响.方法 采用WST-8法测定胶原蛋白多肽对小鼠成骨细胞株MC3T3-E1细胞增殖的影响,ELISA法检测胶原蛋白多肽对MC3T3-E1细胞I型胶原蛋白分泌的影响,采用real-ti...     

Use of Biophysical Characterization in Preformulation Development of a Heavy-Chain Fragment of Botulinum Serotype B: Evaluation of Suitable Purification Process Conditions

PURPOSE: The purpose of this study was to investigate the physicochemical and structural characteristics of recombinant botulinum serotype B (rBoNTB(Hc)) under various conditions and to use the information in evaluating suitable purification process conditions. METHODS: The solubility of rBoNTB(Hc) was evaluated at pH 4, 5, 6 7.5, 8, and 9. Secondary structure was evaluated using circular dichroism, and conformational stability was monitored using highsensitivity differential scanning calorimetry. Hydrophobic interaction chromatography, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), peptide mapping, and UV spectroscopy were used to monitor stability under the various conditions. RESULTS: The secondary structure of rBoNTB(Hc) consists predominantly of beta-sheets. Solubility of rBoNTB(Hc) was lowest at its pI and highest at low and high pH. In the presence of NaCl, however, solubility decreased with increase in pH. Conformational and chemical stability are improved below pH 7.5. In the presence of 150 mM NaCl at high pH, conformational and chemical stability of rBoNTB(Hc) are further decreased. The study suggests that the purification process should minimize exposure of rBoNTB(Hc) to high pH and salt conditions. CONCLUSIONS: Optimal stability of rBoNTB(Hc) is achieved at low pH. The biophysical and analytical studies provide us with an understanding of rBoNTB(Hc) stability behavior in solution and assists in developing efficient purification conditions.     

Conformational and biological properties of partial sequences of salmon calcitonin

According to the Chou-Fasman rules for predicting the secondary structures of proteins, the 12–20 portion of salmon calcitonin should adopt an alpha helical conformation. These residues would form an amphipathic helix and contribute to the solubilization of certain phospholipids by the peptide. Circular dichroism was used to assess the extent that peptide segments of salmon calcitonin fold into structures of higher helical content in the presence of dimyristoylphospha-tidylglycerol, lysolecithin or sodium dodecyl sulfate. All of the segments studied are carboxyl terminal amides as is the native, intact, salmon calcitonin. Salmon calcitonin segments 11–23 or 12–23 form no more helical structure in the presence of lipids or detergents than does a segment comprising the hydro-philic carboxyl terminal residues 22–32 which is not predicted to adopt a helical conformation. Even a larger segment containing residues 12–32 does not exhibit a great increase in helical content in the presence of lipids or detergents, and it causes only a small broadening of the phase transition of dimyristoyl-phosphatidylglycerol. In contrast, a preparation with an equivalent molar ratio of dimyristoylphosphatidylglycerol to the salmon calcitonin segment 1–23 exhibits a very marked broadening of the phase transition, similar to what is found with the 32 amino acid native hormone. This amino terminal segment also adopts a conformation of higher helical content than even the intact hormone. This 1–23 segment is the only one studied that showed significant interaction with lipids, and it is also the only one which exhibited any hypocalcemic activity. The association constant for the binding of the 1–23 segment to dimyristoylphosphatidylglycerol is 2.4 × 10⁴ M^-1 compared with 1.0 × 10⁵ M^-1 for salmon calcitonin. The reduction in the affinity for lipid which occurs by removing residues 24–32 is much less than the reduction in biological activity. These and previous results indicate that the properties which give rise to lipid binding contribute to receptor affinity but are not the sole determining factor.     

Interaction of atriopeptin III with lipids and detergents

Atriopeptin III, a potent natural hypotensive agent, contains little α-helical structure but substantial amounts of β-structure. The peptide can self-associate at millimolar concentrations or can associate with the anionic phospholipid, dimyristoylphosphatidylglycerol. Both of these processes are accompanied by a conformational change suggesting the formation of an increased amount of β-structure. The peptide can broaden the transition and lower the transition enthalpy of dimyristoylphosphatidylglycerol. The results demonstrate that a peptide hormone can associate with lipid largely in the form of a β-structure.     

Lipophilic derivatization of synthetic peptides belonging to NS3 and E2 proteins of GB virus‐C (hepatitis G virus) and its effect on the interaction with model lipid membranes

Abstract: The synthesis by solid‐phase methodologies of peptides belonging to structural and non‐structural proteins of GB virus C as well as its N‐α‐acylation with myristate and palmitate fatty acids is described. To explore the peptide–lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. Our results show that structural and more clearly the structural lipophilic peptide sequences incorporated into lipid bilayers perturb the packing of lipids and affect their thermotropic properties, more than the non‐structural selected sequence. However, the binding of the synthetic sequences to lipid membranes occurred without any restructuration of the peptides.     

Stabilization of Lyophilized Porcine Pancreatic Elastase

Porcine pancreatic elastase, a well-characterized serine protease, has been used as a model to assess the effects of excessive humidity on solid-state stability of the lyophilized protein. Elastase lyophilized without excipients retained full activity immediately after freeze-drying but became denatured upon continued storage at 40°C, 75% relative humidity. The extent of inactivation could be monitored through assays of amidolytic activity, as well as through changes in the circular dichroism (CD) and fluorescence spectra. Differential scanning calorimetry (DSC) was employed as a means of screening potential stabilizing additives; based on the results, sucrose and dextran 40 were selected for further evaluation. Both additives were effective in preventing denaturation. Possible mechanisms for the denaturation and stabilization of elastase are discussed.     

Isolation and characterization of a novel superoxide dismutase from fungal strain Humicola lutea 110

Abstract: A novel thermostable MnSOD was purified to electrophoretic homogeneity from the fungal strain Humicola lutea 110. The preparation of the pure metalloenzyme was performed using treatment with acetone followed by ion exchange and gel permeation chromatography. We found that the activity of this enzyme comprises about 80% of the total superoxide dismutase activity in the crude extract, containing two proteins: MnSOD and Cu/ZnSOD. The MnSOD has a molecular mass of ≈ 76 kDa and 7200 U/mg protein specific activity. It is a tetrameric enzyme with four identical subunits of 18 860 Da each as indicated by SDS-PAGE, amino acid analysis and mass spectrometry. N-terminal sequence analysis of MnSOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Physico-chemical properties were determined by absorption spectroscopy and circular dichroism measurements. The UV absorption spectrum was typical for an MnSOD enzyme, but displayed an increased absorption in the 280 nm region (ε₂₈₀ = 10.4 mM^?1. cm^?1), attributed to aromatic amino acid residues. The CD data show that MnSOD has two negative Cotton effects at 208 and 222 nm allowing the calculation of its helical content. The ellipticity at 222 nm is 6800 deg.cm².dmol^?1 and thus similar to the values reported for other MnSODs. The MnSOD from H. lutea 110 is stable over a wide range of pH (4.5–8), even in the presence of EDTA. The enzyme is thermostable at 70–75°C, and more stable than MnSODs from other sources.     

Biochemical characterization of crystallins from frog lenses

Lens crystallins were isolated from the homogenate of frog (Rana catesbeiana) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, amino acid analysis and circular dichroism. Four well-defined fractions corresponding to α/β-, β-, frog 39.5 kDa and γ-crystallins comprising the relative weight percentages in the total soluble cytoplasmic proteins of 18%, 15%, 14% and 48% respectively were obtained. The native molecular masses for each purified fraction were determined to be 432, 207, 40 and 23 kDa, respectively. The polypeptide compositions as determined by SDS-gel electrophoresis revealed the typical subunit structures of mammalian crystallins with the exception of 39.5 kDa monomeric crystallin, which has not been shown in other classes of vertebrate lenses. The spectra of circular dichroism indicate a predominant β-sheet structure in all four fractions, which also bears a resemblance to the secondary structure of mammalian crystallins. Comparison of the amino acid compositions of frog crystallins with those of mammalian and fish crystallins suggests that γ-crystallin from the frog is more closely related to that of porcine than fish crystallins, and the frog 39.5 kDa, frog β- and lamprey 48 kDa crystallins are probably mutually interrelated.     

Trypsin-pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type)

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.     

Structure analysis of trypsin inhibitors isolated from Cucurbitacae seeds. Circular dichroism studies

Structure analysis jointly based on circular dichroism and Chou and Fasman analysis has been performed on a homologous group of 12 squash proteinase inhibitors. The analysis favours a structure incorporating 40–45% helix which is tightly crosslinked by three disulphide bonds. The proposed structure is consistent with the previously published CD and ¹H n.m.r. spectra. The amino acid which forms the centre of the interaction between trypsin and the inhibitor, arginine⁵ or lysine⁵, is located on the N-terminus of one of the helical segments. The basic residue is orientated away from the rest of the structure and thus is ideally situated for interaction with the confined space of the trypsin active site.