用亲和层析法分离纯化降纤酶

目的用亲和层析法从尖吻蝮蛇蛇毒中分离纯化降纤酶。方法用离子交换色谱、单克隆抗体亲和色谱技术进行分离纯化。结果得到电泳纯的降纤酶，其分子量为36000。结论用单克隆抗体亲和色谱法纯化降纤酶是切实可行的。     

黑斑双鳍电鳐电器官烟碱样乙酰胆碱受体的纯化及特性

用亲和层析法自黑斑双鳍电鳐电器官提纯出烟碱样乙酰胆碱受体,产率41.1％,比结合力7.3nmol/mg protein。饱和结合分析获得的Scatch-ard曲线呈上凸的抛物线形状,K_(D1),K_(D2)分别为9.63nmol和2.74nmol,Hill系数为1.3。根据氨基酸定量测定值推算,该受体的最小分子量约为220000～230 000;测得其等电点为4.75。在SDS电泳上,纯化受体解离成表观分子量分别为 39000,43000,47000及53000的四条区带,提示它是由多个亚基组成的大分子蛋白。     

长白山白眉蝮蛇血凝酶的分离纯化研究

摘 要 目的：建立一种分离纯化长白山白眉蝮蛇血凝酶的方法。方法: 先后采用分子筛层析法、离子交换色谱法、肝素 Sepharose亲和层析法从长白山白眉蝮蛇蛇毒中分离纯化得到一种具有有凝血活性的酶成分,并采用SDS-Page法、RP-HPLC法测定其纯度,凝胶电泳法(SDS-Page)考察其对牛纤维蛋白原的作用方式、高效空间排阻色谱法（HPSEC）测定其相对分子质量,等电聚焦电泳分析法（IEF）测定其等电点,Lowry法测定其蛋白浓度。结果: 从长白山白眉蝮蛇蛇毒中分离纯化了一种凝血酶成分,SDS-Page显示为一条带,HPLC得到单一的色谱峰,该酶只作用于纤维蛋白原的α链,其相对分子质量为32.2 kD,等电点为5.21,此酶具有体外凝血活性。结论:该方法可用于长白山白眉蝮蛇血凝酶的分离纯化。     

肿瘤源性早孕因子的筛检、纯化及鉴定

目的筛检出具有t EPF样活性的肿瘤样本 ,并对其进行分离纯化。方法用活性玫瑰花结抑制实验进行筛检 ,采用DEAE纤维素离子交换色谱、S SepharoseF .F离子交换色谱、ConA SepharoseCl 4B亲和色谱、Heparin SepharoseCl 6B亲和色谱等方法进行纯化 ,用SDS PAGE测定其相对分子量 ,以等电聚焦电泳法检测其等电点。结果膀胱癌、黑色素瘤、绒癌、恶性葡萄胎 4种肿瘤血清和恶性葡萄胎清宫人流血及黑色素瘤标本组织液具有t EPF活性。蛋白含量为 0 .49mg/ml,相对分子量为 134 2 4、2 32 19、382 73,等电点为 6 .5 3。结论 4种肿瘤血清和 2种肿瘤组织液中纯化获得的t EPF在相对分子量、等电点等生化指标上完全一致 ,且与胎源性EPF无本质区别     

中草药桃仁纤溶酶的亲和层析分离

目的分离出高效、特异、安全、不良反应较小的纤溶酶,为溶栓药物的研究奠定基础。方法利用硫铵沉淀法提取出桃仁纤溶酶,测定其比活力,并比较不同蛋白酶抑制剂对桃仁纤溶酶活性的抑制作用,采用亲和层析法对桃仁纤溶酶进行分离纯化。结果亲和层析法分离得到了纤溶酶活性单一组分,比活力为89.08 U.mg？1,比纯化前提高了7.37倍,该组分属于丝氨酸蛋白酶家族。结论利用大豆胰蛋白酶抑制剂作为配基,采用亲和层析法分离桃仁纤溶酶可以得到单一活性成分,且比活力相对提高。     

中草药桃仁纤溶酶的亲和层析分离

目的 分离出高效、特异、安全、不良反应较小的纤溶酶,为溶栓药物的研究奠定基础。方法 利用硫铵沉淀法提取出桃仁纤溶酶,测定其比活力,并比较不同蛋白酶抑制剂对桃仁纤溶酶活性的抑制作用,采用亲和层析法对桃仁纤溶酶进行分离纯化。结果 亲和层析法分离得到了纤溶酶活性单一组分,比活力为89.08 U·mg^-1,比纯化前提高了7.37倍,该组分属于丝氨酸蛋白酶家族。结论 利用大豆胰蛋白酶抑制剂作为配基,采用亲和层析法分离桃仁纤溶酶可以得到单一活性成分,且比活力相对提高。     

人胎盘抗凝蛋白的分离纯化及鉴定

目的　从人胎盘中分离纯化一种胎盘抗凝蛋白 (theanticoagulationprotein ,PAP) ,并对其生化性质进行研究。方法　用饱和硫酸铵分步盐析、阴离子交换、凝胶过滤和亲和层析方法分离纯化人胎盘抗凝蛋白 ,用活化部分凝血活酶时间 (APTT)测定其抗凝活性 ,用SDS 聚丙烯酰胺凝胶电泳(SDS PAGE)测定其蛋白分子量 ,用等电聚焦法测定蛋白的等电点。结果　从人胎盘中分离纯化出相对分子质量约为3 4 9× 1 0 4,等电点约为pH4 9的单聚体抗凝蛋白。该蛋白能延长APTT时间 ,且延长的时间与剂量成线性关系 ,得率占胎盘总重量的 0 0 1‰。结论　此方法成功地从人胎盘中纯化出一种抗凝蛋白。该蛋白的性质与膜联蛋白Ⅴ比较相似 ,可能是属于膜联蛋白家族     

东亚钳蝎(ButhusmarthensiiKarsch)毒镇痛活性肽的分离纯化及其镇痛作用研究

本文采用凝胶过滤及离子交换层析法从东亚钳蝎毒中分离纯化出一种蝎毒镇痛活性肽（ＳｃｏｒｐｉｏｎＡｎａｌｇｅｓｉｃＰｅｐｔｉｄｅ简称ＳＡＰ）．ＳＡＰ经ＰＡＧＥ得单一条带，ＨＰＬＣ层析为单一峰；以ＳＤＳ－ｐＡＧＥ法测定其分子量为９１２０，等电聚焦法测定ＳＡＰ的等电点为７．８５，ＳＡＰ对热比较稳定．用小鼠醋酸扭体法等３种动物实验模型测定ＳＡＰ的镇痛作用，结果表明均有较强的镇痛活性．     

石杉碱甲多克隆抗体纯化方法的研究

目的：根据石杉碱甲多克隆抗体的性质,采用不同的方法对其进行纯化及性质鉴定。方法本实验主要采用硫酸铵沉淀法、辛酸-硫酸铵沉淀法和Protein A亲和层析法分别对石杉碱甲多克隆抗体进行纯化,并分别对纯化后的抗体纯度、蛋白量和效价进行检测。结果电泳结果示Protein A亲和层析法纯化后仅出现两条色带,无明显杂带,纯度高。饱和硫酸铵沉淀法、辛酸-硫酸铵沉淀法和Protein A亲和层析法纯化后的抗体蛋白含量分别为4.67mg· mL －1、6.89mg· mL－1和13.44mg· mL －1。 Protein A亲和层析法纯化后抗体的效价大于1∶256000,其性质未发生变化。结论相较于硫酸铵沉淀法、辛酸-硫酸铵沉淀法,Protein A亲和层析法纯化石杉碱甲多克隆抗体的纯度、蛋白含量、抗体效价更高,纯化效果更优。     

双脱甲氧基姜黄素的提取、纯化及鉴定

目的 由中药姜黄中提取双脱甲氧基姜黄素。方法 采用柱层析法由姜黄醇提物中分离双脱甲氧基姜黄素，然后采用重结晶法对其进行纯化；分别采用薄层层析法和高效液相色谱法(HPIC)对分离产物进行纯度鉴定，最后采用质谱法测定其分子量。结果 (1)薄层层析法检测结果表明，经柱层析法获取的双脱甲氧基姜黄素中无其他色素成分；但HPLC法的检测结果则表明，分离产物中的杂质峰总面积约占8．2％。采用结晶法纯化后，其中的杂质峰总面积降至3．4％。(2)质谱法鉴定结果表明，分离产物的分子量为308，与双脱甲氧基姜黄素的分子量完全一致，证明所获取的产物即双脱甲氧基姜黄素。结论 柱层析法与结晶纯化法相结合是分离、纯化双脱甲氧基姜黄素的有效方法。     

短尾蝮蛇毒中磷脂结合抗凝蛋白的分离纯化及鉴定

目的从短尾蝮蛇毒中分离纯化一种抗凝蛋白,并对其生化性质进行研究。方法利用阳、阴离子交换、凝胶过滤的方法分离纯化这种抗凝蛋白,用活化部分凝血活酶时间(APTT)测定其抗凝活性,用SDS-PAGE测定其蛋白相对分子量,用等电聚焦法测定蛋白的等电点.用薄层析方法确定抗凝蛋白与磷脂酰胆碱结合。结果从短尾蝮蛇中分离纯化出的抗凝蛋白是二聚体,相对分子量为24.0×103(非还原)和14.6×103(还原)。等电点为pH 5.2。该蛋白具有精氨酸酯酶活性,能明显地延长活化的部分凝血活酶时间(APTT),其抗凝活性与磷脂结合有关。结论此方法成功地从短尾蝮蛇毒中纯化出一种抗凝蛋白。因其能够与磷脂结合,又具有明显的抗凝活性,因此把该蛋白称为磷脂结合抗凝蛋白(phospholip id-b ind ing anticoagu lation prote in,PBAP)。     

Hydrolysis of ester-type drugs by the purified esterase from human intestinal mucosa.

Esterase from human intestinal mucosa was purified 210 fold by solubilization with Triton X-100, chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite, and isoelectric focusing. The purified esterase showed a single band by polyacrylamide gel electrophoresis. The molecular weight of the purified esterase was estimated to be about 55,000 by gel filtration on Sephadex G-150, and the isoelectric point was 5.02. The purified esterase was strongly inhibited by diethyl p-nitrophenyl phosphate (E-600) and diisopropyl fluorophosphate (DFP), and was not inhibited by eserine sulfate and p-chloromercuribenzoate. The purified esterase from human intestinal mucosa was found to be one of the carboxylesterases. The purified esterase hydrolyzed ester-type drugs, i.e., aspirin, clofibrate, indanyl carbenicillin and procaine, but did not hydrolyze amide-type drugs and choline-type drugs.     

辽宁产东亚钳蝎毒中哺乳动物毒素Ⅲ的分离纯化及部分性质的研究

采用 CM—Sephadex C—50,SP—Sephadex C—25离子交换柱层析和 SephadexG—50凝胶过滤三步分离程序,从东亚钳蝎毒中得到一种新的哺乳动物毒素。经低 pH 系统不连续聚丙烯酰胺凝胶园盘电泳,SDS—不连续聚丙烯酰胺凝胶板电泳及等电聚焦聚丙烯酰胺凝胶园盘电泳鉴定说明该毒素为电泳纯的蛋白质。用 SDS 电泳法测得其分子量为8,750道尔顿,等电聚焦电泳法测定 pH 为8.2。纯化成份的产率为2.5%。     

Purification and characterization of anticoagulant protein from the Tabanus,Tabanus bivittatus

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.     

Characterization of coagulase from Staphylococcus intermedius

A protein coagulase was isolated from Staphylococcus intermedius 6131 using bovine prothrombin-Sepharose 4B and Bio-gel P-4 column chromatographies. Homogeneity was demonstrated by the formation of a single band in polyacrylamide gel electrophoresis and isoelectric focusing. The purified preparation possesses a molecular weight of 64,500, an isoelectric point of 4.1, consists of 615 total amino acid residues and demonstrates coagulase activity for human and rabbit fibrinogen, but does not show the activity for rat or guinea pig fibrinogens. This purified protein contains galactose and fucose, and the amino-terminal amino acid sequence was determined. The coagulase activity is inhibited by N-bromosuccinimide (NBS), suggesting that tryptophan is involved in this activity. The coagulase was heat stable to 80 degrees C and stable to pH over the range of 7-9. This is the first report of coagulase from Staphylococcus intermedius.     

广西菲牛蛭消化液中抗凝物质的分离纯化

目的从广西菲牛蛭中分离纯化抗凝物质,并对其生化性质进行测定。方法用三氯醋酸沉析、离子交换、凝胶过滤和高效液相色谱法分离纯化广西菲牛蛭消化液中的抗凝物质,用SDS-聚丙烯酰胺凝胶电泳测定其蛋白质的相对分子质量(Mr),用等电聚焦法测定蛋白质的等电点。结果从广西菲牛蛭中分离出2种抗凝物质———菲牛蛭素A(BDA)和菲牛蛭素B(BDB),BDAMr约为15 200,等电点为3.97;BDBMr为14 600,等电点为4.61。每1 000 mL菲牛蛭消化液(含抗凝活性10 ATU/mg)可分离出BDA 630μg,收率约为0.303%,比活为2 777.8 ATU/mg,活性收率约为27.8%;BDB 615μg,收率约为0.300%,比活为2 845.5 ATU/mg,活性收率约为28.4%。结论从广西菲牛蛭消化液中分离出的BDA,BDB对凝血酶具有极强的抑制作用,具有广阔的运用前景。     

Characterization of human erythrocyte aldehyde dehydrogenase

Human erythrocyte aldehyde dehydrogenase was purified to homogeneity. The enzyme exhibited a single band of activity on starch gel electrophoresis and on isoelectric focusing. It was a tetramer with an estimated molecular weight of 230,000 daltons and an isoelectric point of 5.0. Its pH optimum of 8.5, Michaelis-Menten constant for acetaldehyde of 46 microM, and high sensitivity to noncompetitive inhibition by disulfiram resembled human liver cytosolic aldehyde dehydrogenase. Low concentrations of magnesium (5-10 microM) resulted in enhancement of erythrocyte aldehyde dehydrogenase activity, whereas higher physiological concentrations of magnesium resulted in uncompetitive inhibition of enzyme activity. Magnesium inhibited the enzyme activity by increasing the binding of NADH to the enzyme as had been found to be the case for the inhibitory effect of magnesium on the human liver cytosolic enzyme. Erythrocyte aldehyde dehydrogenase may metabolize small amounts of acetaldehyde escaping the liver during ethanol metabolism and protect extrahepatic tissues from acetaldehyde toxicity.     

Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of beta-lactams in Cephalosporium acremonium

The catalytic properties of the partially purified deacetoxycephalosporin C (DAOC)-synthetase and DAOC-hydroxylase from an industrial strain of Cephalosporium acremonium were studied. After mechanical breakage of the cells, purification was achieved by fractional (NH4)2SO4 precipitation, gel chromatography on Sephadex G-75, ion exchange chromatography on DEAE-Trisacryl M and two isoelectric focusing steps. The two enzyme activities could not be separated. Indirect evidence was obtained from SDS-polyacrylamide gel electrophoresis of the purest fractions obtained by isoelectric focusing that the two reactions are catalyzed by a single enzyme with a molecular weight of 33,000 +/- 2,000 and a pI of 4.6 +/- 0.1. Both reactions require alpha-ketoglutarate, FeSO4, ascorbate and O2, whereas additional ATP shows only a slight stimulation.     

Purification of beta-lactamase from Streptomyces cellulosae by affinity chromatography on Blue Sepharose.

A beta-lactamase from culture supernatant of Streptomyces cellulosae was purified about 1,450-fold to apparent homogeneity in polyacrylamide gel electrophoresis and isoelectric focusing on polyacrylamide gel sheet. The methods used were ammonium sulfate precipitation, CM-52 cellulose ion-exchange chromatography and affinity chromatography on Blue Sepharose CL-6B. The molecular weight was determined to be approximately 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was in good agreement with the previous value determined by gel filtration on Sephadex G-75. The isoelectric point was pH 9.5. The enzyme behaved primarily as penicillinase and apparent Km value for benzylpenicillin was 500 microM. The beta-lactamase of S. cellulosae interacted strongly with blue dextran and NADP+-agarose but not with Sepharose. In addition, the presence of NADP+ but not NAD+ and ATP diminished sharply the intrinsic fluorescence intensity of the enzyme and the apparent association constant was calculated to be 1.4 x 10(3) M-1. The beta-lactamase decreases its enzymatic activity against benzylpenicillin in the presence of NADP+. From these results, it is suggested that this beta-lactamase has a dinucleotide binding fold.