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不合羟脯氨酸；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。     

不同鲨鱼皮胶原蛋白的分离及其特性研究

目的 探讨不同鲨鱼皮胶原蛋白的分离方法,并对分离胶原蛋白的部分生化特性进行研究.方法 用稀碱溶胀酶解法争缓冲液溶胀分离法分别从水鲨头皮和马鲛鲨皮中分离胶原蛋白,利用聚丙烯酰胺凝胶电泳法(SDS-PAGE)、差示扫描量热仪法(DSC)、园二色谱仪法(CD)对分离胶原蛋白进行纯度及分子大小、变性温度和二级结构的测定.结果 鲨鱼皮胶原蛋白回收率为鲜重的2.9%～4.1%;分离胶原蛋白在SDS-PAGE上呈现清晰的3条谱带,相对分子质量分别为205,134,118 KDa水鲨头皮和马鲛鲨皮胶原蛋白的热变性温度分别为69℃、47℃;两种分离胶原蛋白的二级结构均主要是β-折叠和无规卷曲,不存在α-螺旋.结论 采用稀碱溶胀酶解法和缓冲液溶胀分离法均能制备高纯度的胶原蛋白,从水鲨头皮和马鲛鲨皮分离的胶原蛋白相对分子质量争分子构成一致,但提取条件的不同能影响胶原蛋白的热变性温度和二级结构.     

巴沙鱼皮胶原蛋白的提取、组成及变性温度研究

目的 从巴沙鱼皮中提取胶原蛋白并分析其结构类型及氨基酸组成,同时研究物理、化学交联法对其变性温度的影响。 方法 采用酸提法和酶提法提取巴沙鱼皮中的胶原蛋白,并用SDS-PAGE凝胶电泳、FTIR和HPLC分析胶原蛋白的结构类型和氨基酸组成。通过测定粘度值研究物理、化学交联法对其变性温度的影响。 结果 酸提法提取胶原蛋白的提取率为58.2%,酶提法的提取率为22.0%;巴沙鱼皮鱼胶原蛋白为I型胶原蛋白;经120 ℃热交联后,巴沙鱼皮胶原蛋白的变性温度从34.2 ℃下降至28.0 ℃,经四甲基乙二胺(EDC)交联后,其变性温度上升至35.8℃。 结论 巴沙鱼皮富含I型胶原蛋白,酸提法比酶提法提取效率高,采用化学交联法可提高其变性温度,因而巴沙鱼皮可作为胶原蛋白的重要来源,可用于制备多种生物医用材料,具广泛的应用前景。     

3种不同方式提取鳕鱼鱼鳔胶原蛋白与胶原蛋白肽的基本特性研究

目的 研究三种不同提取方式所制备的大西洋鳕鱼（Gadus macrocephalus）鱼鳔胶原蛋白的基本特性。方法 采用低温酸溶提取酸溶性胶原蛋白(acid-soluble collagen, ASC),热水抽提明胶(gelatin, GEL),复合蛋白酶酶解制备鳕鱼鱼鳔肽(swim bladder peptides, SWP)对鳕鱼鱼鳔中胶原蛋白的特性进行研究。对提取的胶原蛋白进行感官评定、SDS-PAGE电泳分析、紫外光谱分析、傅里叶红外光谱分析、氨基酸组成分析与扫描电镜分析。结果 ASC、GEL与SWP三种胶原蛋白的提取率分别为42.15%、72.20%与89.11%,且羟脯氨酸的含量为8.73%、8.97%与7.94%;SDS-PAGE图谱显示ASC至少由两条α链与一条β链组成,GEL中存在少量的α链与β链,SWP种α链与β链被降解。紫外光谱结果显示三种蛋白在230 nm波长处均有最大吸收;扫描电镜结果显示ASC与GEL具有一定的交联结构,SWP中无交联结构存在。结论 三种不同提取方式制备的胶原蛋白具有不同的特性,为鳕鱼鱼鳔胶原蛋白的开发与应用拓宽了领域。     

水母胶原蛋白的提取及性能研究

目的 优化越前水母(Nemopilema nomurai)胶原蛋白提取工艺,并探究其性能。方法 利用乙酸和胃蛋白酶提取胶原蛋白,通过盐析和超滤纯化水母胶原蛋白粗提物,并对水母胶原蛋白进行表征和性能分析。结果 水母胶原蛋白纯度97%,得率33%（干重）;X射线衍射、紫外光谱和傅里叶变换红外光谱检测结果表明,水母胶原蛋白在提取和纯化过程中保持了胶原蛋白三螺旋结构;氨基酸组成和凝胶电泳分析提示,水母胶原蛋白符合Ⅰ型胶原蛋白特征。溶解度实验表明,水母胶原蛋白在pH 3～5,NaCl浓度小于0.9 mol/L的条件下都具有良好的溶解性。结论 所得水母胶原蛋白与牛Ⅰ型胶原蛋白特征相似,有望成为新的胶原蛋白资源。     

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。     

大豆磷脂对小鼠皮肤胶原蛋白含量的影响

目的 研究大豆磷脂对小鼠皮肤胶原蛋白含量的影响。方法 小鼠用不同剂量的大豆磷脂2.5g/(kg.d)(低剂量组)、5.0 g/(kg.d)(中剂量组)和10.0 g/(kg.d)(高剂量组)灌胃30d后,取背部皮肤,用分光光度法测定小鼠皮肤中胶原蛋白的含量。结果 与对照组小鼠皮肤胶原蛋白含量[(31.70±5.00)mg/g]比较,中剂量组小鼠皮肤胶原蛋白含量[(36.80±5.50)mg/g]显著增加(P<0.05),高剂量组小鼠皮肤胶原蛋白含量[(41.23±7.54)mg/g]极显著增加(P<0.01)。结论 大豆磷脂可提高皮肤中胶原蛋白含量,具有延缓皮肤衰老作用。     

高效液相色谱法测定星虫体壁胶原蛋白

目的建立快速检测胶原蛋白的高效液相色谱法。方法采用异硫氰酸苯酯柱前衍生反相高效液相色谱,以乙晴-醋酸盐缓冲系统梯度洗脱,于254 nm波长处根据羟脯氨酸的含量来推算星虫体壁胶原蛋白。结果羟脯氨酸衍生物与其它氨基酸较好分离,在5~200μg/mL浓度范围内有良好的线性关系(r=0.999 9),回收率为102.6%,RSD为1.58%。结论此法快速、灵敏、分辨率高,适用于动物组织中胶原蛋白含量的测定。     

离子色谱法测定鱼鳞胶原蛋白中羟脯氨酸含量方法的研究

目的建立离子色谱法测定鱼鳞胶原蛋白中羟脯氨酸的含量,为工艺研究提供检测依据。方法采用Aminopac PA-10氨基酸分析柱,用积分安培离子色谱法测定鱼鳞中羟脯氨酸的含量。结果羟脯氨酸在0.32~32μmol.L-1(进样量25μL)的范围内呈良好线性关系(y=3.341 1x+1.0087,r=0.999,平均回收率98.25%,RSD=2.04% n=5)。结论所建立的方法稳定可行,可作为判断鱼鳞胶原蛋白含量的一个定量指标。     

酶解鱼可溶性肽分子组成结构及营养评价

本文叙述了用胃蛋白酶、胰蛋白酶水解的鱼可溶性肤类水解物,经凝胶高效液相色谱和氨基酸分析仪分析测定其肤类分子组成结构和氨基酸组成成分.测得水解产物中肽类相对分子质量在7400以下,其中相对分子质量6600～7400、由52～58个氨基酸组成的较长肽链占1.74%;相对分子质量2500～5300、由20～41个氨基酸组成的中长肽链占29.75%;相对分子质量在1000以下的由2～10个氨基酸组成的寡肽占50%.水解物的总氮与氨基酸态氮比为25.91,约有96%的氨基酸以肽类形式存在,4%为游离氨基酸.测得可溶性肽的总氨基酸含量为73.98%,必需氨基酸为32.39%,占总氮基酸的43.78%,与FAO/WHO相比,苯丙氨酸为第一限制性氨基酸,氨基酸分值为61.根据分析结果,深入探讨了鱼可溶性肽氨基酸组成的平衡性及其有关寡肽在动物机体的生理功能作用.     

Extraction,characterization and biocompatibility evaluation of silver carp (Hypophthalmichthys molitrix) skin collagen

Collagen as a biomaterial is commonly obtained from terrestrial animals. However, nowadays, the use of collagen with terrestrial animals' sources due to possible transmission of infectious diseases and religious beliefs is restricted. This study was conducted to evaluate the physicochemical characterization, morphology, and biocompatibility of extracted collagen from silver carp fish skin by-product. Type I collagen was extracted from the silver carp fish skin by-product using acetic acid and pepsin enzyme. The results showed that the extraction yield of ASC and PSC was 43% and 59% (on a dry weight basis), respectively. The presence of two different α-chains confirmed the type I collagen structure for both collagens. Moreover, FTIR spectra investigation demonstrated the triple helical in ASC and PSC collagens. The PSC showed higher imino acids content than ASC. Hence, the fractional viscosity and DSC curves revealed higher denaturation temperature (30 °C), shrinkage temperature (81 °C), and melting temperature (209 °C) for PSC than ASC (29 °C, 77 °C, and 187 °C, respectively). Finally, observations of the microscopic and the cell viability evaluation confirmed biocompatibility and suitable structure to cell growth. Accordingly, the obtained collagen from silver carp fish skin can be a proper alternative for terrestrial animals’ collagen and a safe biomaterial for biomedical use.     

人肌腱胶原蛋白的提取及凝胶制备

目的探索人肌腱胶原蛋白在组织工程皮肤及整形外科中的应用。方法从人肌腱提取酸溶性胶原,制备人肌腱胶原蛋白。结果所提取的胶原在pH5.8左右迅速形成凝胶。光镜下可见酸溶性胶原纤维呈长细丝状,交织成网状。纯度以羟脯氨酸质量分数为基准,分光光度法测定为95%。氨基酸成分主要为甘氨酸占35%,脯氨酸占10%。结论氨基酸分析表明,所提取的胶原为典型的Ⅰ型胶原蛋白,热稳定性测定Td为39～41℃。     

Granulocyte-colony stimulating factor maintains a thermally stable,compact, partially folded structure at pH 2

At acidic pH many proteins exist in a partially unfolded form, called the “A” state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.O. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stake's radius even at pH 2.0.The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some α-helical structure persists even at 86°C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the “A” state.     

Species Differences of Serum Albumins: II. Chemical and Thermal Stability

Purpose. The chemical and thermal stability of five species of mammalian serum albumins (human, bovine, dog, rabbit, and rat) were investigated, and conformational stabilities were compared to obtain structural information about the different albumins. Methods. The chemical stability was estimated by using guanidine hydrochloride (GdnCl), and monitored by fluorometry and circular dichroism (CD). Thermal stability was evaluated by differential scanning calorimetry (DSC). Results. In human, bovine, and rat albumin, two transitions were observed when GdnCl-induced denaturation was monitored fluorometrically, indicating at least one stable intermediate, although, in dog and rabbit albumin, only one transition was observed. However, GdnCl denaturation, as monitored by the ellipticity, showed a two-state transition in all species used in this study. Since these proteins, showing two transitions, contained a conserved tryptophan residue within domain II, these structural changes might have occurred in domain II during intermediate formation. DSC measurements showed that human, bovine, and rat albumin exhibited single sharp endotherms and these were clearly consistent with a two-state transition, while the deconvolution analysis of broad thermograms observed for dog and rabbit albumin showed that the absorption peaks could be approximated by a two-component composition, and were consistent with independent transitions of two different cooperative blocks. Conclusions. These experimental results demonstrate that species differences exist with respect to the conformational stability and the mechanism of the unfolding pathway for mammalian albumin.     

Synthesis of polypeptide models of collagen

It is well known that the amino acid sequence of collagen contains a large number of tripeptide units such as Y-X-Gly, where Y and X can be any amino acid residue but most frequently are proline or hydroxyproline. Accordingly, several sequential polytripeptides, including repeating sequences of that type containing one or two imino acid residues, have been synthesized and studied in solution and/or in the solid state (1–14). In this paper we describe in detail the syntheses via active esters of four polytripeptides of the type poly(Pro-X-Gly) and poly(X-Pro-Gly), where X is isoleucine or norleucine. The general objective was to study the influence of hydrophobic residues on the stability of collagen three-dimensional structure; in particular we were interested in the introduction of branching at C^β in view of the recent calculations of Némethy & Scheraga (15). The Nle residue was introduced in order to obtain reference polymers without any branching in the side chain. By way of comparison, we have shown earlier that polymeric sequences where X is leucine (branching at the C^γ) possess collagen-like structures in an appropriate milieu (10, 11). The results of both theoretical and experimental conformational studies on the polymers will be published in a forthcoming paper. Syntheses via p-nitrophenyl esters are described for poly(Pro-Ile-Gly), poly(Ile-Pro-Gly), poly(Pro-Nle-Gly) and poly(Nle-Pro-Gly), four synthetic polytripeptide analogues of the non-polar regions of collagens. The obtained polymers exhibit molecular weights in the range 5000–10000 as judged from viscosity measurements.     

Bone Regeneration with Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Using Absorbable Collagen Sponges (ACS): Influence of Processing on ACS Characteristics and Formulation

The effects of variability in three parameters (mass, cross-linking with CH₂O, and EtO sterilization) of three surgically implantable absorbable collagen sponges (ACS) were studied. Sponges soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) solution were analyzed for pH, conductivity, and rhBMP-2 precipitation. A method using trinitrobenzenesulfonic acid was developed to quantify the free amino groups of the collagen sponge. With up to 240 min exposure to CH₂O, the amount of free amino groups was reduced to 80%. In comparison, the denaturation temperature as determined by differential scanning calorimetry (DSC) after the sponges were soaked with phosphate-buffered saline, increased from 48 to 55°C, indicating stronger interactions due to cross-linking. Subsequent sterilization with EtO caused a marked decrease in the amount of free amino groups (approximately 33% of nonsterilized controls) independent of previous CH₂O treatment. However, the denaturation temperature was on average 5°C lower in sterilized sponges than in nonsterilized material. In contrast to CH₂O, exposure, the strong reaction with EtO appeared to weaken the collagen structure. Resistance of the sponge to collagenase correlated with the degree of collagen cross-linking but was slightly reduced by sterilization. In addition, the pH of ACS soaked with water was substantially increased by sterilization. Protein precipitation was a function of pH and salt concentration but there was no effect due to collagen alone. Results indicated that ACS weight has to be limited to avoid rhBMP-2 precipitation.     

Effect of irradiation on collagen solutions in relation to biomedical applications

The aim of this work was to investigate physico-chemical properties of collagen solutions and evaluate the influence of ionizing radiation on: formation of collagen gels, thermal stability of protein, amino acid composition and cross-linking of collagen. Collagen soluble in acetic acid was prepared from bovine Achilles tendons by the enzymatic (pepsin) method. The results obtained in this study are discussed in view of the possible use of radiation cross-linked collagen for the biomaterials.     

Solid state stability of proteins III: calorimetric (DSC) and spectroscopic (FTIR) characterization of thermal denaturation in freeze dried human growth hormone (hGH)

This research is a study of the changes in secondary structure (Fourier transform infrared spectroscopy, FTIR), aggregation, and loss of the magnitude of the heat of denaturation upon scanning to and partially through the temperature range of the thermal denaturation peak of a model protein, human growth hormone (hGH). We study two formulations, a system of essentially pure protein (with a trace of phosphate buffer) and a system formulated with trehalose in a 3:1 trehalose:hGH weight ratio. The extent of denaturation is measured by loss of secondary structure by FTIR, the loss of heat of denaturation by differential scanning calorimetry (DSC), and the fraction of protein aggregated by HPLC. We examine loss of structure on heating to the DSC onset of thermal denaturation and restoration of structure by cooling below the denaturation temperature and holding to (nominally) allow time for refolding, and we also examine restoration of structure upon dissolving and refreeze drying samples heated to selected temperatures in the denaturation range. We find that denaturation occurs only above the glass transition temperature, is highly cooperative, and is only reversible by redissolving the "denatured" formulated (trehalose) solid. Further, all measures of the extent of denaturation are in essential agreement.     

The effect of PEGylation on the stability of small therapeutic proteins

The influence of PEGylation on the thermal stability of small therapeutic proteins was evaluated using two model proteins. Changes in the midpoint of thermal unfolding and the ability to properly refold after thermal denaturation were monitored by differential scanning calorimetry (DSC) as a function of PEGylation and pH. The results showed that PEGylation increased the thermal stability of both model proteins as well as their ability to refold properly after thermal denaturation. The DSC results were compared to traditional accelerated stability data that were collected using size exclusion high performance liquid chromatography (SE-HPLC). The DSC data agreed reasonably well with those from SE-HPLC indicating that microcalorimetry can be an efficient screening tool for PEGylated proteins.     

Isolation and characterization of a genetic variant of bovine proinsulin

A genetic variant of bovine proinsulin has been isolated using preparative reverse-phase HPLC. The new proinsulin (bovine proinsulin II) differs from the known proinsulin (bovine proinsulin I) by a single amino acid residue at position C-48 in the connecting peptide. The amino acid replacement is a leucine substitution for proline. The two proinsulins were found in a ratio of approximately 9:1, proinsulin I: proinsulin II. No chemical or biological differences were observed for the two proinsulins other than their different elution times on reverse-phase HPLC.