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1.
Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.  相似文献   

2.
发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。  相似文献   

3.
目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。  相似文献   

4.
目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。  相似文献   

5.
经DEAE-Sephacel、UltrogelAcA 34、Heptylam ine-Sepharose 和Bio Scale Q2等四步层析从牛脑皮层膜中同时纯化出抑制型G 蛋白(inhibitory G protein, Gi)和O型G蛋白(other G pro-tein, Go).对Go 的选择性激活使Gi 和Go 的分离大为简化,并使二者的大量制备成为可能.纯化的G 蛋白的SDS-PAGE银染显示单一蛋白带,其结合[35S]GTPγS的活性分别提高68倍和124倍.用圆二色光谱法(CD)测量了纯化的G蛋白的二级结构.  相似文献   

6.
Here, we describe a simple and efficient method for the purification of Escherichia coli outer membrane proteins. We have tested this protocol for the purification of Wza and Osmoporin C (OmpC) proteins. Both proteins were purified to homogeneity, in two steps, by anion exchange and size exclusion chromatography with a final yield of 92.5 mg for the Wza protein and 291.5 mg for the OmpC protein. The purity of the samples was judged by electrophoretic analysis, mass spectrometry, single particle analysis, three-dimensional (3D) crystallisation and X-ray diffraction.  相似文献   

7.
Bovine hemoglobin (bHb) was purified from bovine red blood cells (bRBCs) via anion exchange chromatography preceded by dialysis. This is a fast and effective way to obtain bHb from bRBCs using Q Sepharose XL, a strong anion exchange resin. This resin had double the binding capacity for bHb compared to three other anion exchange resins that were studied in this work. Methemoglobin levels remained below 2% with bHb concentrations between 0.7 and 1.7 mM. The high purity of bHb was confirmed via SDS-PAGE and size exclusion chromatography (SEC).  相似文献   

8.
N-Glycolylneuraminic acid (Neu5Gc), precious sialic acid which could not be synthesized by a chemical method, occurrs in the body of holothuroidea, Gumi Cucumaria echinata. Gumi contains 85% of total sialic acid, as Neu5Gc, in the body. Neu5Gc was purified from dry powder of the body using Dowex 1-x8 (HCOO* form) anion exchange chromatography after mild acid hydrolysis with 0.1 N trifluoroacetic acid. Using GC-MS and 1H-NMR spectroscopy, the purified Neu5Gc was correctly identified to be Neu5Gc. The purity of Neu5Gc was more than 99%. This is the first report of purification and identification of Neu5Gc from holothuroidea by using anion exchange chromatography, GC-MS, and 1H-NMR.  相似文献   

9.
The enzyme Hbp (hemoglobin protease) of the pathogenic Escherichia coli strain EB1 has been purified to homogeneity by gel filtration chromatography. The purified protein is capable of binding heme and shows hemoglobin protease activity. Our method of purification is applicable not only to Hbp but also to other autotransporter proteins and will contribute to a better understanding of the function-structure relationship of this family of proteins.  相似文献   

10.
Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.  相似文献   

11.
抗HIV融合多肽CP32M的制备工艺研究   总被引:1,自引:0,他引:1  
目的:研究抗HIV融合多肽CP32M的制备工艺,为其临床前试验奠定基础。方法:采用固-液相混合策略规模合成目标肽,用离子交换色谱、反相高效液相色谱对粗肽进行纯化。结果:获得了利于提高合成及片段缩合效率的3条优选片段,CP32M粗品经DEAE离子交换色谱及C18反相纯化后纯度高于98%。结论:CP32M按3条片段(Ac-1-9-OH、Fmoc-10-21-OH、H-22-32-NH2)划分,可显著提高片段合成及缩合效率,DEAE阴离子交换色谱能除去大部分杂质,提高了第二步C18反相色谱纯化效率。  相似文献   

12.
In this study, final anion exchange chromatography in the recombinant human growth hormone (r-hGH) manufacturing process was validated using a validation protocol that was consistent with both policy and standard operation procedure (SOP). Two buffer solutions used in chromatography were first validated and were found to satisfy pre-established acceptance criteria as follows: pH: 8.2, endotoxin: < 0.6 EU/mL, bioburden test: negative. Final anion exchange chromatography was conducted using a DEAE Sepharose FF Resin and eluted with a linear gradient of 30 to 110 mM NaCl in 50 mM Tris-HCl buffer at a flow rate of 15 L/h. Three consecutive batches of hGH solutions were generated via anion exchange chromatography, which was performed within pre-established operating parameters determined through in-process control. When all three batches were assessed by the pre-established sampling plan and tested for quality control, this purification process was shown to satisfy pre-established acceptance criteria; endotoxin: ≤ 0.5 EU/mg, ECP: ≤ 1.4 ppm, IEF: same removal distance, hGH content by Native-PAGE: 100%, purity by HPLC: ≥ 99%, yield by UV scanning: 87 to 89%, hGH monomer protein content by HPLC: 99%. Therefore, the final anion chromatography process was successfully validated in this study, and this method consistently yielded hGH solutions that satisfied pre-established criteria for subsequent processing.  相似文献   

13.
A simple and rapid preparation method for a novel antitumor agent, 2-amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazine-3-one (Phx) was described. The procedure included (1) the reaction of bovine hemolysates with 2-amino-5-methylphenol, (2) one-shot denaturation of hemoglobin and proteins by methanol, and removal of the denatured hemoglobin and proteins, (3) concentration of the reaction products, and (4) purification by a Sephadex column. These procedures yielded Phx in 34% yield.  相似文献   

14.
Methods for the purification of both murine mammary tumor (type B) and murine leukemia (type C) oncornaviral phosphoproteins are described, in which chromatography on alkyl-agarose derivatives is used as the primary fractionation step. Gel filtration or ion exchange chromatography on phosphocellulose was the only subsequent step required for the purification of the type B and type C viral proteins, respectively. The two-step protocols also resulted in the co-purification of a low molecular weight core protein from each virus. Recoveries of the viral proteins purified by this method, based on per cent contribution of individual polypeptides to total virion proteins, were 70% or greater. Radioimmunocompetition analysis of the purified murine mammary tumor virus major core protein as well as analysis of the RNA binding properties of purified low molecular weight type C virus proteins suggests that neither antigenic reactivity nor specific RNA binding characteristics are altered by the purification protocols. The availability of these procedures should aid studies on the possible function and immunochemical properties of the native murine oncornaviral phosphoproteins and may also be extended to the purification of other oncornaviral proteins.  相似文献   

15.
Antibacterial activity of a pepsin-derived bovine hemoglobin fragment   总被引:4,自引:0,他引:4  
Peptic digestion of bovine hemoglobin yields a fragment with antibacterial activity. This peptide was purified to homogeneity by a two-step procedure including anion exchange chromatography and preparative reversed-phase HPLC. Mass determination and fragmentation indicated that this peptide corresponded to the 1-23 fragment of the alpha chain of hemoglobin. The minimum inhibitory concentration and mode of action of this peptide towards Micrococcus luteus strain A270 were determined. Hemolytic assay, interaction with liposomes, and study of its structure in solution were also performed.  相似文献   

16.
An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.  相似文献   

17.
We intend to purify beta-glucuronidase from human liver in a large quantity in order to facilitate the study of its biochemical structure and pathophysiologic roles in cholelithiasis and carcinogenesis. The initial purification procedure involved: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation of protein at 56 degrees C, (4) gel filtration with Bio-Gel P-300 gel, (5) anion exchange chromatography with DEAE agarose, (6) cation exchange chromatography with CM agarose, and (7) hydroxyapatite chromatography (overall yield, 1%; overall purification, 169X). The final product was used to immunize rabbits and BALB/c mice for production of polyclonal and monoclonal antibodies, respectively. The antibodies, mainly IgG, were purified by using gamma-Protein A agarose column chromatography. The purified IgG, after periodate oxidation, was coupled to hydrazide gel by formation of a stable covalent hydrazone bond linkage. The new purification procedure involved the initial first three steps, followed by (4) polyclonal IgG immunoaffinity chromatography and (5) monoclonal IgG immunoaffinity chromatography (overall yield, 6.1%; overall purification, 3720X). Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by electroelution. It is concluded that beta-glucuronidase constitutes 0.016 mg per gram of wet liver tissue and can be obtained on a large scale in a highly purified form within a 2-day cycle.  相似文献   

18.
Abstract

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomuciod, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.  相似文献   

19.
In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.  相似文献   

20.
《Insect Biochemistry》1989,19(5):481-488
Cytochrome P-450, cytochrome b5 and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b5 and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b5 and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm.Further purification of the anion exchange cytochrome b5 fraction, by C8 reverse phase HPLC, resulted in a cytochrome b5 fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa.  相似文献   

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