Large Scale Purification of Alpha-1 Trypsin Inhibitor from Human Plasma

A convenient large scale purification of the alpha-1 trypsin inhibitor from human plasma was achieved by conventional salting out and column chromatography methods. The pure inhibitor complexes 0.68 μgs of bovine trypsin per ug inhibitor, and 0.60 μgs human trypsin per μg inhibitor. The inhibitor is homogeneous by equilibrium chromatography, disc electrophoresis, and ultracentrifugation. Conditions for its storage with minimal loss of activity are described. The advantages of the present preparation are higher potency, long-term stability, and large scale.     

人红细胞NADH－细胞色素b5还原酶的分离纯化与鉴定

 人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶的分离纯化与鉴定黄长晖,朱忠勇,唐玉钗（南京军区福州总院全军医学检验中心实验科,福州３５０００１）ＮＡＤＨ细胞色素ｂ５还原酶（Ｃｙｔｂ５Ｒ,Ｅ．Ｃ．１．６．２．２．）是红细胞内催化高铁血红蛋白还原为血红蛋白的关键酶...     

A Rapid One-Step Extraction Procedure for the Isolation of Ubiquitin from Human Erythrocytes for Antibody Production

Abstract

A procedure is described that employs 5% perchloric acid extraction to isolate ubiquitin from human erythrocytes. The procedure is rapid and economical as it requires no specialized equipment. The extracted protein appeared to be highly purified as judged by electrophoresis and was identified as ubiquitin by immunoblotting and total amino acid analysis. The extraction yields about 78% of the ubiquitin in the hemolysate, which is a higher yield than is obtained with other procedures. The purified ubiquitin was used to make a polyclonal antiserum. As ubiquitin is a small and highly conserved protein, it is necessary to couple it to a larger immunogen to elicit an immune response. This ubiquitin antiserum was produced using an immunogen system that produces an immune response to the ubiquitin, but not to the carrier protein.     

A Rapid Procedure for the Large Scale Purification of Elastase and Cathepsin G from Human Sputum

A procedure is described which permits the rapid isolation of large amounts of elastase and cathepsin G from purulent sputum. This procedure involves: (1) digestion of sputum with DNase, (2) extraction of the insoluble residue that remains with 1 M NaCl, pH 8, (3) affinity chromatography on Sepharose-bound Trasylol, and (4) separation of the two enzymes by chromatography on CM-Sephadex. Starting with 500 g of sputum it was possible to isolate 175 mg of each of these two enzymes within 7 to 10 days. Active site titration indicated both enzymes to be at least 97% pure. Disc gel electrophoresis in the presence and absence of SDS and amino acid sequence of the N-terminal region support the conclusion that the elastase and cathepsin G isolated from sputum a re identical to the same enzymes isolated directly from the leukocytes of human blood.     

药用级微生物谷氨酰胺转胺酶的纯化工艺研究

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高 选择性和高载量地捕获,再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。     

鸭血超氧化物歧化酶(SOD)的纯化及性质研究

超氧化物歧化酶(E、C、1.15.1.1)是催化超氧阴离子起歧化反应的金属酶类,血红细胞中的SOD属Cu Zn—SOD。本文详细报道了从鸭血分离纯化SOD的改进方法(同时用猪血作对比研究)。设计了热变性、(NH_4)_2SO_4分级监析、低浓乙醇—氯仿短时去除残留血红蛋白、凝胶层析四步纯化方案,获得高产率电泳纯SOD。用紫外扫描,PAGE电泳后考马斯亮蓝和SOD活性杂色,SDS—电泳,HPLC分离和各电泳带组分的N—端测定等技术检测纯度,并进行性质研究。证明所得SOD是均一的;N—端为Ala;分子量和亚分子量各为32,359和16,500dt;并与猪血SOD对比研究其某些性质;对HPLC出现的多个活力峰及PAGE电泳显现的多条活性带进行了讨论。     

人类红细胞葡萄糖-6-磷酸脱氢酶的分离纯化与鉴定

 <正> 葡萄糖-6-磷酸脱氢酶(G6PD,EC1·1·1.49)催化磷酸已糖途径的第一步反应。人红细胞内的G6PD及其催化产生的NADPH对于维持血红蛋白及红细胞膜的稳定性具有非常重要的作用。G6PD的结构变异可影响其活性。纯化人红细胞G6PD对于进一步阐明G6PD的结构与功能的关系有重要意义。本文报道纯化人红细胞的的方法,并对该酶进行鉴定。     

大规模区带离心纯化Vero细胞乙脑疫苗

本文报道一种适合疫苗生产的大规模纯化Ｖｅｒｏ细胞乙脑疫苗的方法。原疫苗经适当浓缩和去除ＤＮＡ处理后,用不连续蔗糖梯度（３６％和６０％）。３２６００ｇ,速率区带离心４ｈ。纯化后疫苗的效力比中国参考疫苗高出６倍以上,补体结合抗原比中国参考疫苗高４～８倍。总蛋白含量低于３０μｇ／ｍＬ,牛血清含量降至０．５μｇ／ｍＬ以下,细胞残余ＤＮＡ低于１００ｐｇ／０．５ｍＬ。用此法连续制备三批纯化疫苗,其纯度和效力均高于日本鼠脑纯化疫苗。此法对于制备其它纯化Ｖｅｒｏ细胞疫苗也具有一定的参考意义。     

Large Scale Isolation and Purification of Eyespot Granules from Euglena gracilis var. bacillaris

Large volumes of eyespot granules were isolated from homogenates of Euglena gracilis Klebs var. bacillaris Pringsheim by flotation centrifugation in a Beckman Ti-15 zonal rotor, and were further purified by centrifugation in a swinging bucket rotor. Examination with the electron microscope showed the eyespot granules to be free from other cellular material. Freezing had no apparent effect on the structure or on the absorption properties of the eyespots. Absorption spectra of pure fractions of eyespot granules free of chloroplast contamination showed the previously reported curves in the range of 360 to 520 nanometers, as well as a peak at 660 to 675 nanometers. The procedure for the large scale isolation of eyespot granules from Euglena gracilis is compared with other methods which have employed conventional centrifugation, and the significance of the use of zonal rotors for isolating large quantities of pure eyespot granules is discussed.     

A Large Scale Procedure for Purification of Desmosine and Isodesmosine

A rapid large scale procedure was devised for the purification of desmosine and isodesmosine from ligamentum nuchae elastin. The method makes use of the hydrophilic nature of the desmosines which preferentially absorbs to cellulose fibers in mixtures of organic solvents. Resolution of the isomers was achieved on a polystyrene resin column.     

Rapid Purification of Glucose-6-phosphate Dehydrogenase from Mammal's Erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6pD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362–3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2′5′ ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2′5′ ADP-Sepharose with KC1 and NADP.

By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2′5′ ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5–4 ml of rabbit blood, which can be performed in about 8 hours and a macro-scale purification starting from 180–200 ml of human blood, which takes a day and a half.     

Effects of Melatonin on Carbonic Anhydrase from Human Erythrocytes In Vitro and from Rat Erythrocytes In Vivo

The in vitro effects of melatonin (N-acetyl-5-methoxytryptamine) on human carbonic anhydrase isozymes (HCA-I and HCA-II) from human erythrocytes and in vivo effects on rat erythrocytes carbonic anhydrase (CA) were determined. Human erythrocyte carbonic anhydrase isozymes were purified by haemolysate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The HCA-I enzyme, having a specific activity of 7337.5?EU/mg protein, was purified 843-fold with a yield of 60% and the HCA-II enzyme, having a specific activity of 17067?EU/mg protein, was purified 1962-fold with a yield of 22.7%. For in vitro experiments, the enzyme activity was minimal at 2×10-4?M melatonin concentration and increased above this concentration. Ten mg?kg-1 melatonin was administered intraperitoneally and showed a stimulatory effect on the enzyme. Time-dependent in vivo studies were conducted for melatonin in Sprague–Dawley type rats. It was found that CA activity in the rat erythrocytes was decreased by the melatonin after 1 and 3 hours to 2500±500.0 and 1875±239.4 respectively which were statistically significant (p<0.05) differences to the control (2660±235.8). However, CA activity was restored to its normal level after 6?h (2666±235.7) (p>0.05) probably due to metabolism of the melatonin. The findings indicate that melatonin may be pharmacologically useful in some diseases.     

Large Scale Purification of Isoinhibitors of Trypsin from Swine Colostrum Using Zinc Chelate Chromatography and Chromatofocusing

Low molecular weight trypsin inhibitors were purified from swine colostrum on a large scale under mild conditions. Ammonium sulfate fractionation and metal chelate chromatography on zinc chelate Sepharose and phenyl Sepharose were used for removal of the bulk of proteins. The inhibitors showed only a weak hydrophobic interaction with phenyl Sepharose even in the presence of 1 M (Nll₄)₂SO4, and advantage was taken of this property to remove the inhibitors from contaminating colostrum proteins which remained tightly adsorbed to phenyl Sepharose under these conditions. The low and high molecular weight inhibitors were then separated by gel filtration on Bio-Gel P-300. The low molecular weight material was eluted in three major inhibitor fractions on DEAE-Sepharose.

Chromatofocusing of these fractions provided greater resolution of the inhibitors, and several previously unreported inhibitor peaks were detected. The six major inhibitors purified by chromatofocusing were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. These inhibitors were composed of a single polypeptide chain with a molecular weight of 18,000 as determined by Sephacryl S-200 gel filtration and polyacrylamide qel electrophoresis in the presence of sodium dodecyl sulfate and e-mercaptoethanol. The specific activities of the pure inhibitors were approximately 30% higher than those previously reported.     

人铜锌超氧化物歧化酶的大规模生产工艺

本文以干扰素生产中废弃的人红细胞为原料,采用乙醇－氯份沉淀→磷酸氢二钾盐析→丙酮沉淀手续和ＤＥＡＥ—５２层析步骤,进行了人铜锌超氧化物歧化酶（ＣｕＺｎ－ＳＯＤ）大规模生产工艺的实验研究。结果表明,生产的酶制剂中含量为９０．６％,比活性在２０００ＭｃＣｏｒｄ—Ｆｒｉｄｏｖｉｃｈ单位／ｍｇ蛋白以上,无菌、无热原质、安全性等项指标符合国家卫生部对血液制品的要求。     

扬子鳄红细胞超氧物歧化酶的纯化及其性质研究

扬子鳄红细胞超氧物歧化酶的纯化及其性质研究周衍茂（安徽教育学院生物系,合肥２３００６１）尹路明（中国科学技术大学生物系,合肥２３００２６）关键词超氧物歧化酶;纯化;扬子鳄;红细胞超氧物歧化酶（的田广泛存在于各类生物组织中,是生物体内超氧自由基有效的清...     

人脑及红细胞膜乙酰胆碱酯酶的芳基酰胺酶活性

采用联合亲和层析法从人小脑及红细胞膜中纯化了ＡＣｈＥ,纯化的人脑及红细胞ＡＣｈＥ在ＳＤＳ－ＰＡＧＥ上呈一主带,分子量约为６６０００。人脑ＡＣｈＥ制备酯酶与酰胺酶比活性分别为１２９９与１４３Ｕ／ｍｇ,人红细胞ＡＣｈＥ制备分别为４５８４与７４７Ｕ／ｍｇ。人脑及红细胞ＡＣｈＥ制备的酯酶与酰胺酶活性最适ｐＨ较接近,在ｐＨ７．５－８．０之间,酯酶活性底物ＡＴＣｈ对其芳基酰胺酶活性有抑制作用。ＩＣ_（５０）分别为１０．２×１０~（－３）及３×１０~（－３）ｍｏｌ／Ｌ。梭曼对其酯酶及酰胺酶活性均有明显抑制作用,说明二者均需活性中心丝氨酸参与。     

Abscisic Acid Transport in Human Erythrocytes

Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [³H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [³H]ABA and [³⁵S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone.     

Heterogeneous Negative Chemography With Human Erythrocytes

A negative chemographic effect, or fading of the latent image, has been found in autoradiographic studies of human red blood cells. The effect is heterogeneous, being restricted to a halo-shaped region over each cell.