壳聚糖-半胱氨酸轭合物对胰岛素酶降解和降血糖作用的影响

目的研究壳聚糖-半胱氨酸轭合物对胰岛素消化酶降解的抑制作用,及其对胰岛素口服降血糖的促进作用.方法合成壳聚糖-半胱氨酸轭合物,体外实验考察其对α-糜蛋白酶和胰蛋白酶降解胰岛素的抑制作用.O1/O2乳化溶剂挥发法制备胰岛素肠溶微球,考察壳聚糖-半胱氨酸轭合物对胰岛素溶液和肠溶微球大鼠灌胃后降血糖作用的影响.结果所合成的壳聚糖-半胱氨酸轭合物巯基含量为200μmol·g-1polymer,体外实验对酶降解胰岛素有明显的保护作用:不加壳聚糖-半胱氨酸轭合物的对照组胰岛素溶液可分别在1 h和5 h内被α-糜蛋白酶和胰蛋白酶完全降解;含4mg·mL-1轭合物的同浓度的胰岛素溶液在相同浓度的酶溶液中经过相同时间,胰岛素剩余量均大于75%.所制备的胰岛素肠溶微球载药量7%,大鼠灌胃给予壳聚糖-半胱氨酸轭合物85 mg·kg-1,可增强胰岛素溶液或肠溶微球的口服降血糖作用.结论壳聚糖-半胱氨酸轭合物对胰岛素口服制剂降血糖作用的增强可能与其酶抑制作用有关.     

胰岛素的种类及特点

胰岛素最初是由加拿大学者班廷(Banting)和拜斯特(Best)在1921年从狗的胰腺中提取出来的,从此开辟了糖尿病治疗的新时代。目前胰岛素制剂的研制开发进展迅速,临床上胰岛素的种类繁多,现结合我院胰岛素品种,按来源,制备工艺,胰岛素作用时间,胰岛素给药装置等几方面对胰岛素进行分类阐述。1根据胰岛素的来源,胰岛素分类为1.1人胰岛素,是一种小分子蛋白质,分子量为5808,是由胰腺胰岛的β细胞分泌的激素物质,由A、B两条氨基酸肽链组成[1]。1.2猪胰岛素,与人胰岛素结构类似,只有一个氨基酸不同,即将人胰岛素的B链30位上的苏氨酸换成了丙氨酸[1]。1.3牛胰岛素,与人胰岛素有3个氨基酸不同,一个是人胰岛     

地特胰岛素的葡萄糖钳夹研究进展

胰岛素在糖尿病治疗中占有极其重要的地位,对其研究的不断深入直接驱动着糖尿病治疗理念的不断更新。近年来,长效胰岛素类似物的问世使基础胰岛素的应用进入一个新的时代。即将进入我国临床使用的地特胰岛素,作为新一代的可溶性长效胰岛素类似物,是将人胰岛素B链上天然排列在第30位的苏氨酸去掉后,通过酰化反应,在第29位赖氨酸上结合1个14碳脂肪酸（肉豆蔻酸）。     

甘精胰岛素的不良反应和合理应用

甘精胰岛素是一种超长效胰岛素类似物，通过DNA重组技术，用大肠埃希杆菌的非病原性菌株生产的。其分子结构是将人体胰岛素的A链上21位上的天冬酰胺用甘氨酸取代，B链30位苏氨酸后增加两个精氨酸，等电点为7．0左右，在酸性注射液中完全溶解，呈无色透明溶液，可用于注射。在生理条件下，其溶解度低，形成细微的胰岛素沉淀物，并缓慢由六聚体分解为二聚体、单聚体后被人体吸收。作为一种基础胰岛素在持续给药后药效能持续24h，     

基因载体PEG化壳聚糖的制备及其表征

目的：用亲水性的聚乙二醇对壳聚糖进行改性，制备适用于基因转染的非病毒类裁体。方法：合成步骤共分三步。首先通过有机合成，将甲氧基聚乙二醇(mPEG)的羟基末端依次活化成为羧基和琥珀酰亚胺端基，形成mPEG-COOH活化物和mPEG-COONSu活化物；然后将亲水性的mPEG-COONSu活化物接枝到壳聚糖的氨基侧链上，得到改性了的壳聚糖-聚乙二醇接枝产物，应用波谱技术FTIR，^1H-NMR，^13C-NMR对中间产物和最终产物进行了表征。结果：在FTIR谱图上基本找到mPEG-COOH，mPEG-COONSu的特征峰；^1H-NMR再一次确认mPEG-COONSu的合成；^13C-NMR确认了壳聚糖-聚乙二醇接枝产物的存在。结论：mPEG-COONSu活化物通过接枝反应对壳聚糖进行改性，得到了PBG化壳聚糖。     

HPLC-ESI-ITMS在药品质量控制中确证胰岛素和胰岛素B链C端氨基酸序列的应用研究

在药品质量控制中应用HPLC-ESI-IT (Ion Trap) MS分别确证胰岛素和胰岛素B链的C端氨基酸序列。取胰岛素标准品溶液或者胰岛素B链溶液适量，加入羧肽酶P及羧肽酶Y溶液适量降解胰岛素或者胰岛素B链C端，在预定的时间点取出酶降解反应液适量，加入等体积1%甲酸停止反应，混旋均匀后制成供试液，供HPLC-ESI-IT MS分析测定。采用Zorbax Prosphere C₁₈柱(2.1 mm×150 mm，300A，5 μm)分离，以流动相进行梯度洗脱［A相：水-乙腈-三氟乙酸(98∶2∶0.02)，B相：乙腈-水-三氟乙酸(98∶2∶0.02)］。柱后修饰“TFAfix”溶液为丙酸-异丙醇(2∶8)的混合溶液。ESI-ITMS测定供试液中梯度多肽系列(彼此相差1个C端氨基酸残基)的分子质量，计算出分子质量最相近的相邻两肽段的系列分子质量差值，即可得到待测样品C端系列氨基酸残基质量的实验值。胰岛素的B链C端的第1个氨基酸残基丙氨酸残基可以被准确地确证；同样还原修饰的胰岛素B链C端的第1个氨基酸残基丙氨酸残基也能被准确地确证。本研究在nmol样品水平准确地确证胰岛素和胰岛素B链各一个C端氨基酸，符合重组DNA制品中试产品的质量控制要求。本实验方法不必将胰岛素经还原修饰将A链从B链裂解，直接确证胰岛素的C端氨基酸残基，避免还原修饰和纯化的烦琐操作，方法简便。     

聚乙二醇——酶轭合物研究进展

<正> 用水溶性多聚物对酶蛋白进行化学修饰,改变酶分子的性质、探索解决药用酶中存在问题的途径是酶工程的研究方向之一。近年来得以广泛研究和利用的便是聚乙二醇(PEG)与酶的轭合物。PEG是无毒的线性分子,易于广泛收集从二聚体到分子量达10~6多聚体的不同组分,具有良好的生物相容性。美国FDA已批准 PEG用于药物(肠外、局部、栓剂、鼻喷雾剂)、食物和化妆品。酶经PEG化学修饰后改变了酶的理化性质,使之在有机溶剂中也呈现活性,增加了稳定性和体内半衰期,降低或消除了免疫原性;因PEG轭合而增加了酶的分子量,导致药代动力学性质的改变。酶经PEG化学修饰称为PEG-酶轭合物,作为药物和生物反应器的催化剂有巨大的实用意义。     

小C肽人胰岛素原类似物(B-Arg-Arg-A)和人胰岛素原(B-C-A)A、B链重组条件的研究

目的 :获得更佳的变复性条件和提高人胰岛素原的重组率。方法 :采用特有的重组方法 ,将小C肽人胰岛素原类似物 (B Arg Arg A)与人胰岛素原 (B C A)进行重组条件的比较。 结果 :发现二者在 pH10 .6左右处重组率达到最大 ,且在相同的条件下B Arg Arg A蛋白质的重组率 (94% )较B C A蛋白质 (82 % )的高。结论 :进一步验证了“胰岛素的A、B链含有足够使二硫键正确配对的结构信息”的论点。     

赖脯胰岛素和门冬胰岛素各自的优缺点是什么？

赖脯胰岛素和门冬胰岛素分别是美国礼来公司和丹麦诺和诺德公司研制并生产的超短效或称速效胰岛素类似物。人胰岛素由两个多肽链（A链B链）通过2个二硫键连接而成，A链有21氨基酸，B链有30个氨基酸。     

肽和蛋白质类药物的给药系统(Ⅱ)

<正>3 蛋白质─聚合物共轭体 重组蛋白因缺乏糖基而使其溶解性和稳定性较差,并且有抗原性,与以聚乙二醇（PEG）为代表的水溶性聚合物通过共价键形成轭合物后,轭合物的性质较之未经修饰的原物质有很大改善,溶解度提高,稳定性增强,生物半衰期延长,毒副作用（免疫原性和过敏反应）降低甚至消除,疗效提高,因此在临床上得到应用。 白细胞介素-2经PEG修饰后的轭合物以适当的剂量和方式给药,可以延长血液中IL-2活性达到最大值的时间,延长循环寿命,减轻免疫原性,提高治疗指数和疗效。PEG-天冬酰胺酶轭合物的半衰期较之天然酶延长了17倍,免疫原性也有所降低,治疗指数得以提高。用PEG修饰,在临床上得到应用的酶还有:SOD（超氧化物歧化酶）、ADA（腺苷脱氨酶）、尿酸酶等,都表现出相似的特性。     

Facile synthesis of insulin fusion derivatives through sortase A ligation

Insulin derivatives such as insulin detemir and insulin degludec are U.S. Food and Drug Administration (FDA)-approved long-acting insulin currently used by millions of people with diabetes. These derivatives are modified in C-terminal B29 lysine to retain insulin bioactivity. New and efficient methods for facile synthesis of insulin derivatives may lead to new discovery of therapeutic insulin. Herein, we report a new method using sortase A (SrtA)-mediated ligation for the synthesis of insulin derivatives with high efficiency and functional group tolerance in the C-terminal B chain. This new insulin molecule (Ins-SA) with an SrtA-recognizing motif can be conjugated to diverse groups with N-terminal oligoglycines to generate new insulin derivatives. We further demonstrated that a new insulin derivative synthesized by this SrtA-mediated ligation shows strong cellular and in vivo bioactivity. This enzymatic method can therefore be used for future insulin design and development.     

B_(19)-Gly-B_(20)人胰岛素的分离纯化及性质研究

目的研究胰岛素B链羧端的自由度对于胰岛素与受体相互作用的影响。方法在人胰岛素B链羧端 β转角区回折点B1 9与B2 0 之间插入一个Gly。结果B1 9 Gly B2 0 人胰岛素原的表达产物占细胞总蛋白量的 3 0 % ,B1 9 Gly B2 0 人胰岛素的受体活性是标准猪胰岛素的 12 1%。结论以较高的受体结合活性获得了高纯度的B1 9 Gly B2 0 人胰岛素。     

[21 - ARGININE - A] INSULIN

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg²¹] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg²¹-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg²¹-A] sheep insulin shows potencies of 10.5–12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23–25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A²¹ asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg²¹-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the abovementioned interactions come into play, which involve the side chain of the A²¹ amino acid residue and affect the biological activity of the hormone.     

Molecular structure of insulin: the insulin monomer and its assembly

The insulin molecule contains 51 amino acids; it is made up of two peptide chains linked by disulphide bonds. Although it is active as a monomer, during its biosynthesis and storage it assembles to dimers and, in the presence of zinc, to hexamers. X-ray analysis has revealed the 3-dimensional structure of the insulin molecule in its hexameric, dimeric and monomeric states. Two main conformations of insulin which differ in the extent of helix in the B chain (B9-B20 and B1-B20, respectively) have been identified. Other variations are seen in insulin when dimeric or monomeric. Reagents such as chloride and phenol govern the conformations present in the insulin hexamers and this can influence the behaviour and properties of insulin preparations employing them.     

A new B‐chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B‐chain structure

Abstract: The solution structure of a new B‐chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D‐NMR and molecular modeling. This structure is compared with that of the oxidized B‐chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res. 46 , 424–433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B‐chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B‐chain. 2D‐NMR experiments confirmed, among multiple conformations, that the B‐chain mutant presents defined secondary structures such as a α‐helix between residues B9 and B19, and a β‐turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B‐chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B‐chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B‐chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B‐chains.     

A novel protein cross-linking reaction in stressed Neutral Protamine Hagedorn formulations of insulin

The covalent insulin-protamine product molecules formed by heat stress in Neutral Protamine Hagedorn formulations of insulin and the insulin analogue [LysB28,ProB29] were examined by mass spectrometry. The results demonstrated that the covalent cross-link between insulin and protamine was not caused by linkage through the protamine N-terminal amino group, as had been previously thought. Our results indicate that the linkage was formed between the side chain of a protamine arginine and a histidine in the insulin B chain, resulting in a net mass change of -5 Da, compared to the sum of the protamine and insulin molecular masses. A mechanism for this new type of covalent cross-linking reaction is proposed.     

CHEMICAL SYNTHESIS OF [DES(TETRAPEPTIDE B27–30), TYR(NH2)26-B] AND [DES(PENTAPEPTIDE B26–30), PHE(NH2)25-B] BOVINE INSULINS*

Two analogs of bovine insulin, [des(tetrapeptide B^27–30), Tyr(NH₂)²⁶-B] and [des(pentapeptide B^26–30), Phe(NH₂)²⁵-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B^27–30), Tyr(NH₂)²⁶-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B^26–30), Phe(NH₂)²⁵-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.     

速效胰岛素类似物研究进展

速效胰岛素类似物与正规人胰岛素（RHI）相比起效快,常规胰岛素含有引起分子间聚合的氨基酸,通过改变或删除A和B链上部分氨基酸,去除引起聚合的因素,达到形成单体胰岛素的目的,模拟胰岛素的生理性分泌,应用于糖尿病的治疗,且耐受性良好,这样可以消除使用常规胰岛素所引起的并发症,比如：眼病、腿病等。本文综述了速效胰岛素类似物近年来的研究进展,为进一步研究速效胰岛素类似物奠定基础。     

Characterization of mucrotoxin A from the venom of Trimeresurus mucrosquamatus (the Chinese habu snake)

Mucrotoxin A from the venom of Trimeresurus mucrosquamatus was isolated in homogeneous form by a previously published method. Mucrotoxin A did not hydrolyze casein, however, when dimethylcasein was used as a substrate, the toxin cleaved the substrate. This toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The sites of cleavage in the oxidized B chain of insulin were identified as Ser(9)-His(10), His(10)-Leu(11), Ala(14)-Leu(15), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The toxin digested the A alpha chain of fibrinogen first, followed by hydrolysis of the B beta chain. The fact that no fibrin clot formed indicates that the sites of cleavage in the A alpha and B beta chains of fibrinogen by the toxin must be different from those cleaved by thrombin. Mucrotoxin A produced systemic hemorrhage in internal organs such as the heart and stomach.