Purification of a human plasma membrane glycoprotein from human red blood cells by affinity chromatography using a monoclonal antibody

Using the monoclonal antibody LICR-LON-Fib75.1 coupled to Sepharose as an affinity chromatography column, a membrane glycoprotein with an apparent molecular weight of 18,000 on sodium dodecyl sulfate-polyacrylamide gels has been purified from human red blood cells. The purified protein contained 25% carbohydrate by weight, the predominant sugars being galactose, mannose, and glucosamine. Amino acid analysis indicated that the protein was relatively rich in aspartate, glutamate, valine, and leucine and had a low proline and methionine content. The molecule could be removed from intact red blood cells by trypsin and could be labeled with iodine by lactoperoxidase-catalyzed cell surface iodination of red blood cells. The protein could also be labeled using the lipidsoluble photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine) and partitioned into the lower phase of the phase-separable detergent Triton X-114. During size-exclusion chromatography in different detergents alterations were observed in the apparent molecular weight of the protein. These results suggest that this Fib75.1-binding protein is an external red blood cell membrane glycoprotein which is capable of binding detergent. Proteins with a similar molecular weight have also been isolated from two human tumor cell lines by immunoprecipitation with this monoclonal antibody.     

Metabolic profiling of human urine by CE-MS using a positively charged capillary coating and comparison with UPLC-MS

The potential of capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) using capillaries coated with a triple layer of polybrene-dextran sulfate-polybrene (PB-DS-PB) was evaluated for metabolic profiling of human urine. The method covers various metabolite classes and stable metabolic profiles of urine samples were obtained with favourable migration time repeatability (RSDs <1%). The PB-DS-PB CE-TOF-MS method was used for the analysis of human urine samples from 30 males and 30 females, which had been previously analyzed by reversed-phase UPLC-TOF-MS. Multivariate data analysis of the obtained data provided clear distinction between urine samples from males and females, emphasizing gender differences in metabolic signatures. Nearly all compounds responsible for male-female classification in CE-TOF-MS were different from the classifying compounds in UPLC-TOF-MS. Almost all compounds causing classification in the CE-TOF-MS study were highly polar and did not exhibit retention in the reversed-phase UPLC system. In addition, the CE-TOF-MS classifiers had an m/z value in the range of 50-150, whereas 95% of the classifying features found with UPLC-TOF-MS had an m/z value above 150. The CE-TOF-MS method therefore appears to be highly complementary to the UPLC-TOF-MS method providing classification based on different classes of metabolites.     

Purification of phosphotransacetylase by affinity chromatography.

Phosphotransacetylase from Clostridium kluyveri was purified using a C⁸-(6-aminohexyl)-amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-fold and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5′-dithiobis(2-nitrobenzoic acid) is described.     

Purification of normal human urinary N-acetyl-beta-hexosaminidase A by affinity chromatography.

N-Acetyl-beta-hexosaminidase A was purified 1000-fold from human urine by chromatography on DEAE-Sephadex followed by concanavalin A--Sepharose affinity chromatography. The optimal pH range was 4.4--4.5 for both the N-acetylglucosamine and N-acetylgalactosamine derivatives. The Km values were 0.51 mM and 0.28 mM respectively for the N-acetylglucosamine and N-acetylgalactosamine derivatives. The glycoprotein nature of the urinary enzyme was established by its affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the molecule.     

Purification of a beta-D-galactosidase from bovine liver by affinity chromatography

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.     

Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography

Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by $\text{M}$ hydroxylamine in 0.1 $\text{M}$ potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 $\text{M}$ potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography.

An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10(-4) M, and the Vmax was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10(-5) M. Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed.     

Purification of lysozyme by multistage affinity filtration

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins.     

Urinary trypsin-inhibitor: extraction from pregnancy urine by affinity chromatography.

Affinity chromatography has led to the extraction of a trypsin inhibitor from urines of pregnant women, with a recovery of about 85%. The inhibitor obtained by this method is fairly pure, but heterogeneous, according to several criteria. The heterogeneity is not the result of the extraction procedure, but it seems to pre-exist in the urine itself.     

Cathepsin D from human leukocytes. Purification by affinity chromatography and properties of the enzyme

Cathepsin D of human leukocytes was isolated and characterized. Purified leukocytes were lysed under nitrogen pressure and the proteinase activity precipitated by centrifugation at 48,000 x g. The precipitate was extracted by various buffers. The yield of cathepsin D was almost pH-independent but could be increased by Triton X-100. Employing gel chromatography the activity was found at a molecular mass close to 42,000 Da. Purification of the enzyme was performed by a two-step procedure using pepstatin-Sepharose chromatography and ion exchange chromatography. Three multiple forms of the enzyme were separated by ion exchange chromatography. The isoelectric points of the three forms of the enzyme were close to pH 5.0. The enzyme showed the typical characteristics of the acid proteinase cathepsin D. Enzyme activity was influenced by heavy metals such as Hg2 and Fe3 as well as by typical inhibitors for carboxyl-proteinases such as diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(4-nitrophenoxy)propane and 4-bromo-phenacylbromide. An immunological comparison with cathepsin D from human liver by immunodiffusion and immunoelectrophoresis indicates identity of the two enzymes.     

Purification of prephenate dehydratase from Corynebacterium glutamicum by affinity chromatography.

Prephenate dehydratase has been purified from the wild type strain Corynebacterium glutamicum by affinity chromatography. Three ligands, L-Trp, L-Tyr, and L-Phe have been tested as well as conditions for elution. L-Phe is the most specific ligand: it leads to a purification factor of 11 in one step using step gradients of NaCl in Tris-HCl buffer at pH 7.5.     

Purification of alcohol dehydrogenase from Drosophila by general-ligand affinity chromatography.

A method for the purification of alcohol dehydrogenase from Drosophila melanogaster is described. The method makes use of 8-(6-aminohexyl)amino-5'-AMP, immobilized on Sepharose 4B, as an affinity ligand. Since alcohol dehydrogenase from Drosophila shows weak affinity for this column, a novel technique was developed to separate alcohol dehydrogenase from both unbound proteins and more strongly bound enzymes. The purification procedure is simple to operate and give a homogeneous preparation in good yield after only three steps.