聚乙二醇(PEG-5000)对胰激肽释放酶化学修饰的初步研究

以自制单甲氧基聚乙二醇琥珀酰亚胺碳酸酯对猪胰激肽释放酶进行化学修饰，修饰产物以superdex 675凝胶色谱柱进行分离纯化。结果表明，修饰PPK分子质量增加，保留活性降低，对温度的耐受性加强，稳定性增加。     

白眉蝮蛇蛇毒纤溶酶的聚乙二醇修饰

为提高纤溶酶的临床适用性，采用4种针对氨基修饰的mPEG试剂——mPEG—SPA5K、mPEG—SMB5K、mPEG—ButyrALD5K和mPEG2-NHS40K修饰蛇毒纤溶酶，以获得高比例的单修饰产物和较高的酶活性保留率。优化修饰反应条件，用Superdex75凝胶过滤色谱法分离纯化修饰产物。最终确定mPEG—SPA5K为最适宜的修饰剂。产物经sDs—PAGE和MALDI-TOF-MS法检测为单修饰，修饰产物酶活性收率为67．3％，修饰产物的热稳定性提高，免疫原性显著下降。     

聚乙二醇修饰重组人干扰素α-2b修饰产物的初步分析研究

目的 对聚乙二醇(PEG)修饰重组人干扰素α-2b(rhIFNa-2b)的修饰产物进行了初步分析研究。方法以单甲氧基聚乙二醇甲酸琥珀酰亚胺酯对rhIFNα-2b进行修饰,修饰产物以Superdex 75 Highload制备型凝胶色谱柱进行分离、纯化,SDS-PAGE鉴定各组分,采用Mariner电喷雾飞行时间质谱对单修饰IFNα-2b(Inono-PEG-IFNα-2b)进行分析。结果 分别获得了纯化的mono-PEG-IFNα-2b和多修饰IFNα-2b(poly-PEG-IFNα-2b)。ESI-TOF质谱也证明mono-PEG-IFNα-2b分子量比rhIFNα-2b多5 000左右;紫外光谱分析表明,PEG修饰产物的结构没有发生改变。结论获得了纯化的PEG修饰产物。     

降纤酶的聚乙二醇修饰及产物的纯化研究

研究了降纤酶的聚乙二醇修饰及纯化方法。采用正交法考察影响修饰的4个因素，确定了最佳条件。利用分子筛柱色谱得到了较高酶活力保留的修饰产物，SDS-PAGE检测为单一条带。     

聚乙二醇单修饰的重组人粒细胞集落刺激因子突变体的纯化与分析

目的 获得高活性的聚乙二醇定点单修饰的产物PEG-rmhG-CSF-Cys176.方法 用聚乙二醇马来酰亚胺基和高活性的重组人粒细胞集落刺激因子突变体(rmhG-CSF)的半胱氨酸(Cys)基团特异性结合,并用离子交换层析分离纯化修饰产物,通过高效凝胶过滤色谱检测纯度,MTT法检测体外的生物活性.结果 蛋白纯度为97%,体外生物活性为0.8×10~8 IU·mg~(-1).结论 半胱氨酸反应性PEG和游离Cys的结合是在rmhG-CSF活性区以外的位置C末端,修饰后的蛋白活性基本未受影响.     

新型定点修饰的聚乙二醇化重组人生长激素修饰位点研究

目的:建立新型定点修饰的聚乙二醇化重组人生长激素(PEG-rhGH)修饰位点的研究方法,为该类新型修饰技术产物的质量评价提供依据。方法:采用MALDI-TOF质谱分析修饰产物中聚乙二醇(PEG)的修饰个数及修饰肽段的质量数;采用LC-Q-TOF质谱分析修饰位点,分析色谱柱为UPLC色谱柱(Protein ACQUITY BEH C₄ Column, 150 mm×2.1 mm, 1.7μm, 300?),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,进行梯度洗脱,柱温为40℃。质谱数据采集条件为MS~E模式,一级质谱能量4 eV,二级碎裂电压30～55 eV。结果:修饰蛋白中PEG的平均相对分子质量为30 000,并且每个PEG-rhGH分子中仅存在1个PEG修饰;原型蛋白重组人生长激素中第134位精氨酸被替换为赖氨酸,且该赖氨酸的ε-氨基与连接子HOOC-O-CH₂-CH₂-N₃中的羧基端共价结合,连接子另一端的叠氮基团与活化后的PEG偶联后生成修饰产物。结论:联合运用多种质谱技术,通过比对...     

聚乙二醇修饰牛血红蛋白产物的分离纯化和检测

目的:研究聚乙二醇修饰剂单甲氧基聚乙二醇琥珀酰亚胺酯(SC-mPEG)对牛血红蛋白(BHB)修饰产物的合适的分离纯化、检测方法。方法:采用阳离子交换层析以及有机溶剂沉淀法进行分离,并采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和三硝基苯磺酸(TNBS)法进行检测。结果:阳离子交换层析法分离出修饰与低修饰产物,TNBS法检测阳离子交换层析分离所得2个峰值产物的平均修饰率分别为52.95%和14.19%。结论:阳离子交换层析法可作为分离纯化SC-mPEG-BHB的方法;TNBS方法可作为检测SC-mPEG-BHB平均修饰率的方法。     

聚乙二醇修饰超氧化物歧化酶的研究

以氰尿酰氯活化单甲氧基聚乙二醇（ＭＰＥＧ Ｍｒ５０００）对猪血Ｃｕ，Ｚｎ－ＳＯＤ进行修饰，制得ＭＰＥＧ２－ＳＯＤ。产物的酶活性保持８２．０％，期半衰期比修饰前延长至７．６倍，在动物脑中的ＳＯＤ活性较高。修饰ＳＯＤ抗炎活性增强，抗原性降低，稳定性提高。     

绞股蓝皂甙对正常及利血平化小鼠脑单胺递质量和体征的影响

在正常及利血平化小鼠上用反相离子对高效液相色谱电化学检测法研究了绞股蓝皂甙对小鼠中枢神经系统单胺递质及其代谢产物的影响，发现绞股蓝皂甙可以改善利血平对中枢单胺递质的耗竭作用．同时改善利血平引起的一系列体征改变，而绞股蓝本身对正常小鼠脑内单胺递质及部分代谢产物影响不明显，也不引起明显的体征改变．     

云南红豆杉的化学成分研究及1-去羟基巴卡亭VI的结构修饰

目的 研究云南红豆杉(Taxus yunnanensis)心木的化学成分并对1-去羟基巴卡亭VI进行了结构修饰.方法 采用各种现代色谱技术进行分离纯化,根据化合物的理化性质和波谱数据进行结构鉴定.并对1-去羟基巴卡亭VI进行了选择性的去乙酰化反应.结果 从云南红豆杉心木的95%乙醇提取物中分离并鉴定了7个紫杉烷类化合物.对其中一个含量较高的化合物进行了结构修饰,得到两个去乙酰化的产物,并鉴定了其结构.结论 结构修饰所得到的13位去乙酰产物是我们所需要的,它为今后进一步合成符合紫杉醇构效关系的产物提供了物质基础.     

壳聚糖固定化酵母蔗糖酶的研究

用壳聚糖作吸附剂、戊二醛作交联剂,对酵母蔗糖酶进行固定化研究,同时用琼脂糖固定化的蔗糖酶和游离酶与其进行比较。结果表明,壳聚糖固定化蔗糖酶贮藏稳定性、对变性剂以及对温度的耐受性均明显优越,而Km值与琼脂糖固定化酶相当,但比游离酶明显增高,酶的最适pH三者相近。研究表明,壳聚糖固定化酶所需的戊二醛浓度为0.6%,交联时间不低于6 h。固定化蔗糖酶对变性剂(乙醇、脲)的耐受力明显高于游离酶。     

毛蚶超氧化物歧化酶的纯化、部分性质与修饰

经热变性 硫酸铵分级沉淀,微量铜离子溶液秀析,Sephadex G-100凝胶过滤和DE－52柱层析,从毛蚶中分离纯化铜锌超氧化物歧化酶（Cu,Zn-SOD),并对其是化性质进行分析鉴定,实验结果获得该 比活力为5294．1U／mg,提纯倍数为885．9,该酶对KCNM和H2O2敏感,而suchihashi液对酶活性没影响,对热较稳定,紫外吸收峰城260nm处,聚丙烯酰凝胶电泳蛋白带与活性带相对应,该酶是由2个相同亚基组成的二聚体,分子量为28．8kD,每个亚其含有一原子铜和一原子锌,经右旋糖甙修饰后抗胃蛋白酶水解的能力增强。     

蝮蛇毒纤溶酶的分离纯化及性质研究

目的 :寻找一种分离纯化蝮蛇毒纤溶酶的工艺并研究其理化性质。方法 :采用DEAE SepharoseCL 6B和HeparinCL 6B层析方法 ,从蝮蛇毒中分离纯化纤溶酶。结果 :蝮蛇毒纤溶酶经HPLC为单一峰 ,等电聚焦电泳为一条带 ,其等电点为 4.5 5 ,经SDS 聚丙烯酰胺凝胶电泳测得分子量为 2 9.4kD。该酶对热不稳定 ,在 pH6～ 9时稳定 ,氨基酸组成分析表明含酸性氨基酸较多。结论 :用此工艺可制得高纯度的蝮蛇毒纤溶酶。     

Purification and characterization of a thrombin like enzyme, elegaxobin II, with lys-bradykinin releasing activity from the venom of Trimeresurus elegans (Sakishima-Habu).

A thrombin like enzyme, named elegaxobin II, with Lys-bradykinin releasing activity was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel-filtration on Sephadex G-100, and ion-exchange chromatography on the Q-Sepharose Fast Flow. By this procedure, about 9mg of purified enzyme was obtained from 1.1g of the venom. The purified enzyme showed a single protein band, the molecular weight of which was estimated to be about 35,000Da by sodium dodecyl sulfate-PAGE) under reducing condition, and this enzyme was found to contain a carbohydrate moiety. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 250 TAME units/mg of protein. This enzyme clotted only rabbit fibrinogen, whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin conversion, this enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas it did not release fibrinopeptide B. Furthermore, elegaxobin II released Lys-bradykinin when the enzyme was incubated with bovine plasma. The esterase activity was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), suggesting that this enzyme is a serine protease. The N-terminal sequence (Val-Ile-Gly-Gly) of this enzyme was identical to the typical sequence of serine proteinases.     

Properties of partially purified beta-N-acetylglucosaminidase from bovine crystalline lens.

This paper reports that beta-N-acetylglucosaminidase from bovine lens has potent enzyme activity compared with other glycosidases in the lens. The partially purified enzyme was used to determine its physiological properties. The optimal pH and optimal temperature of this enzyme was approximately 6.3 and 40 degrees C, respectively. The apparent native molecular weight of this enzyme obtained by gel filtration chromatography was 540 kDa. Furthermore, the enzyme fraction contained 3 polypeptides with molecular weights of 28.8, 28.0 and 26.0 kDa, although it is not certain if they were one of the components of this enzyme in the current study. The Km value of this enzyme was 92.3 microM and it was inhibited strongly by HgCl2 and sodium dodecyl sulfate (SDS).     

超声波对胰蛋白酶活力影响的机理研究

目的探索超声波对胰蛋白酶催化的影响效果和作用方式。方法研究胰蛋白酶经不同频率、不同功率、不同时间的超声波处理后酶活力的变化;用动力学参数变化和光谱学探索其影响催化的机理。结果经超声波处理后酶活力普遍升高。用15 kHz5、0 W超声波处理胰蛋白酶3 min,酶活力可提高45.4%。将粗酶用SephadexG75凝胶色谱,分部液在聚丙烯酰胺凝胶电泳检验显示出一条带,说明酶被纯化到电泳纯程度。对纯酶进行动力学分析,结果表明经超声波处理后Km值变小,Vmax值也降低,说明超声波处理使酶对底物的亲和力增大。超声波处理不会改变胰蛋白酶分子的构型,但会改变酶分子的构象。结论超声波对胰蛋白酶催化活性的提高可能是由于改变酶分子的构象、增强酶分子对底物的亲和力的结果。     

均匀设计在蚯蚓纤溶酶肠溶片研究中的应用

应用均匀设计研究蚯蚓纤港酶肠溶片的处方和工艺，以片剂酶活力不改变为指标观察处方工艺，成功地获得了使蚯蚓纤溶酶活力基本不改变的肠溶衣片剂最佳处方和工艺。     

超声波加酶法提取玉米须黄酮的工艺研究

目的采用正交实验法优化玉米须黄酮的提取工艺条件。方法以加酶量、超声时间、酶解温度和pH值为考察因素,黄酮提取率为考察指标,进行4因素3水平设计,获得最佳提取工艺,并进行方差分析。结果主要影响因素依次为:加酶量、超声时间、pH值、酶解温度。超声加酶法提取玉米须黄酮的最佳提取条件为:加酶量0.030g、超声时间60min、酶解温度30℃、pH值为3,所得最佳黄酮提取率为1.26%。结论 3次平行验证实验得到实际平均提取率为1.26%,优选的工艺稳定可行。     

Characterization of butyrylcholinesterase antagonism of cocaine-induced hyperactivity.

Although there are several published demonstrations that exogenous butyrylcholinesterase (EC 3.1.1.8) works to antagonize cocaine in vivo, a systematic characterization of the enzyme-drug interaction is lacking as is confirmation of the mechanism of effect. This has been addressed using cocaine-induced locomotor activity in mice as a behavioral endpoint. The enzyme was effective, but the enzyme dose-antagonist effect relationship revealed an asymptotic partial maximum effect. This effect was not due to dose-dependent enzyme pharmacokinetics or to a stimulant effect of the cocaine metabolites but rather to partial metabolism of cocaine. Since neither metabolite of cocaine inhibited enzyme activity as potently as cocaine, partial metabolism is not likely due to end-product inhibition. The enzyme reduced the maximum effect of cocaine on locomotor activity. The mechanistic data are generally consistent: the enzyme was inactive against the nonester dopamine/norepinephrine uptake inhibitor, nomifensine, and a paraoxon-inactivated sample of enzyme was ineffective. However, the enzyme was effective against bupropion, a nonester dopamine uptake inhibitor.     

Characterization of equine butyrylcholinesterase disposition in the mouse.

Butyrylcholinesterase administration has been shown to block the effects of cocaine. However, even in model systems, the pharmacokinetics of the enzyme are only partly understood. Measurements of plasma enzyme concentration, antibody titer determinations, and measurement of cocaine-induced locomotor activity in mice were used to describe the disposition of butyrylcholinesterase. Clearance of the enzyme showed biexponential kinetics; the first component was sensitive to asialofetuin, suggesting a role for the asialoglycoprotein receptor. Cocaine did not influence enzyme disposition. An antibody response to enzyme injection was seen; the role of this response is not clear. The antagonist effect of the enzyme was eliminated faster than the enzyme was eliminated from plasma; this may be due to a contribution of tissue esterases to cocaine metabolism. Intraperitoneal enzyme administration was not effective against cocaine, suggesting that the utility of the enzyme is route-dependent.