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Results for “Chromatography, Affinity”

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At least 19 recordsLinked to original sources
PubMed1983

Effects of whole blood storage on results for glycosylated hemoglobin as measured by ion-exchange chromatography, affinity chromatography, and colorimetry.

After storage of whole blood at either 4 or 20 degrees C, results for glycosylated hemoglobin by ion-exchange chromatography ("high-performance" liquid and mini-column chromatography), thiobarbituric acid colorimetry, and affinity chromatography were compared. At 4 degrees C, all methods gave acceptable results for samples stored for as long as a week. At 20 degrees C, the colorimetric and affinity methods also showed sample stability for a week or more. The ion-exchange methods were associated with a marked increase in values for glycosylated hemoglobin after a few days of storage. Evidently, care in details of sample collection and handling is especially important for ion-exchange methods, and the colorimetric and affinity methods have advantages over ion exchange in situations where long delays between sample collection and assay are unavoidable.

Blood Preservation↗
PubMed1975

Determination of sperm-antibodies in sera of sterile women by affinity-chromatography (affinity-chromatography of sperm-antibodies).

The total amount of humoral sperm antibody protein is determined following its isolation by affinity-chromatography on spermatozoa. Serum proteins are eluted by Sorensen phosphate buffer. Antibodies attached to spermatozoa suspended in Sephadex G 75 columns resist elution by Sorensen buffer. They can be quantitatively washed out by 8 ml Tris HCl-buffer pH 8,9 and be determined as proteins by the Folin-Ciocalteu method.

Adsorption↗
PubMedDate not supplied

Colorimetric versus radioimmunological measurement of glycated and non-glycated serum albumin after affinity chromatography.

Affinity chromatography by m-aminophenylboronic acid has been proposed for routine measurement of glycated albumin. We assayed glycated and non-glycated fractions of serum albumin (HSA) eluted by affinity chromatography columns by both a specific RIA method for the human serum albumin (HSA) and by a colorimetric method. Sixteen diabetic patients presented a significantly higher percentage of glycated-HSA than 7 control subjects with both methods, and a strong correlation was found between the values obtained with the two methods. RIA was able to detect a significant concentration of glycated-HSA in all normal subjects, while the colorimetric method was not. The accuracy of separation between the glycated and non-glycated fractions of albumin was tested using [14C]glucose as tracer. When [14C]glycated-HSA purified by Sephadex G25 filtration was chromatographed using the m-aminophenylboronic acid, only 5.3% of the total 14C-radioactivity present in the solution was recovered in the bound fraction, while 44.0% was eluted in non-protein-bound fraction and 54.7% was retained in the column. Our findings confirm that affinity chromatography by m-aminophenylboronic acid can be a useful tool in the monitoring of short glycemic control of diabetic patients. Our data also indicate that the affinity chromatography with m-aminophenylboronic acid does not accurately discriminate between glycated and non-glycated fraction of HSA.

Adult↗
PubMedDate not supplied

Thermostable carbohydrate-binding modules in affinity chromatography.

Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydrate-binding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based oligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65 degrees C. With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM-microM range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates.

Amino Acid Sequence↗
PubMed2004

Fractionation of the naringinase complex from Aspergillus terreus by dye affinity chromatography.

Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).

Adsorption↗
PubMed1992

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.

Binding Sites↗
PubMed1992

Trypsin and affinity chromatography.

Affinity adsorbents for trypsin which were prepared by immobilizing product-type ligands, that is, peptides having C-terminal arginine, proved to be effective not only for preparative purposes but also for basic research on molecular recognition. The properties of the binding site of trypsin were revealed by chromatographic experiments. Quantitative analysis based on the theory of frontal affinity chromatography proved to be extremely effective. As an extension of the product-type ligands, peptide argininals were also used and information on the mechanism of action of these inhibitors was obtained. Anhydrotrypsin, which lost the hydroxyl group of Ser183, was found to gain increased binding ability for product-type compounds. This inactivated enzyme was also used as an immobilized ligand and the unique affinity adsorbent thus prepared proved to be extremely effective for the separation of peptides and recombinant proteins based on their C-terminal structures. High-performance affinity chromatography of trypsin and related enzymes using a polymer-based support was also developed.

Animals↗
PubMed1977

The purification of antimorphine antibodies by affinity chromatography.

Affinity chromatography was employed successfully to purify extensively antimorphine antibodies produced in rabbits. Morphine succinylated at position 6 was attached to bovine serum albumin to make it immunogenic and to Sepharose beads for affinity chromatography. A crude gamma-globulin fraction of antimorphine serum, prepared by salt fractionation, was used for purification. After unbound gamma-globulin was washed off the column with phosphate-buffered saline, two populations of antimorphine antibodies were eluted in succession by two low pH buffers. Each of these two purified antibody fractions had an index of heterogeneity of approximately 1. Immunoelectrophoresis indicated that the antibodies were immunoglobulin G and that they formed a single precipitation band with antirabbit serum. The average association constants for morphine of the two purified antibody fractions were similar and ranged from 4.5 to 4.8 X 10(7) LITERS/MOL-1. A 67-fold purification of the antimorphine antibodies could be achieved by a single pass through an affinity column.

Antibodies↗
PubMed2003

Design and selection of ligands for affinity chromatography.

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.

Chromatography, Affinity↗
PubMed1988

Thermodynamic characterization of the interactions of methotrexate with dihydrofolate reductase by quantitative affinity chromatography.

Affinity chromatography on methotrexate-Sepharose has been used to evaluate dissociation constants for interactions of methotrexate with dihydrofolate reductase from Lactobacillus casei. Equilibrium constants of 0.25 microM and 0.6 nM were obtained for dissociation of the inhibitor from the enzyme-methotrexate and enzyme-NADPH-methotrexate complexes, respectively, these estimates being in good agreement with the corresponding published values for dihydrofolate reductase from Streptococcus faecium. By employing a different method for evaluating the thermodynamic dissociation constant for the enzyme-NADPH-methotrexate interaction, this investigation provides independent support for the inference drawn from published fluorescence quenching studies that the interaction of methotrexate with dihydrofolate reductase-NADPH complex is governed by a dissociation constant in the vicinity of 600 pM, a value slightly higher than, but approaching, the inhibition constant of 50-60 pM obtained by enzyme kinetic techniques.

Chromatography, Affinity↗
PubMedDate not supplied

Further results on lipase-colipase interactions studied by affinity chromatography.

Affinity chromatography of lipase on a colipase-coupled gel was studied in the present paper. The elution volume of the associable lipase increased when the loaded amount decreased. A KD value of 1.9 X 10(-6) M at pH 6.2 was thus deduced. A minimum value of 1.5 X 10(-6) M was obtained at pH 5.1-5.3. Mixed micelles associated with coupled colipase, but no modifications of lipase-colipase interactions took place when mixed micelles were added to the elution buffer. DMMA-modified coupled colipase failed to interact with lipase, owing to the specific orientation of the modified cofactor in the gel.

Animals↗
PubMed1975

A general method of purification of adenosine deaminase by affinity chromatography.

Affinity chromatography has been used to purify adenosine deaminase from various sources: calf spleen, calf intestinal mucosa, chicken duodena and human erythrocytes. For this purpose a specific inhibitor, 9-(p-aminobenzyl) adenine, was synthesized and covalently joined to agarose. Adenosine deaminase is selectively retained by such an inhibitor-resin when highly impure solutions are chromatographed through it. After elution from the resin with guanylurea, a competitive inhibitor, the enzyme is homogeneous and can be recovered in yields of 80 percent or more and the same number of multiple forms of the enzyme is present in the purified preparation and in the crude extract.

Adenine↗
PubMed1973

Isolation of Aleutian mink disease virus by affinity chromatography.

Affinity chromatography was used to isolate the Aleutian disease virus of mink. Dissociation of the immunoadsorbent-virus complex with 0.75 molar sodium chloride and then with a glycine-hydrochloride gradient released infective particles resembling picornaviruses. The elution profile suggests that two different types of virus-antibody complexes are formed, one dissociated by sodium chloride and another that requires glycine-hydrochloride in addition to sodium chloride for release of virus.

Adsorption↗
PubMed1977

Purification of equine infectious anemia virus antigen by affinity chromatography.

Affinity chromatography was performed to obtain highly purified antigen from equine infectious anemia (EIA) virus. After crude antigen was concentrated by polyethylene glycol precipitation of culture fluids from equine dermal cells persistently infected with EIA virus, and after the virus was disrupted with ether, it was added to a column of cyanogen bromide-activated Sepharose 4B to which EIA-specific antibody had been conjugated. The antigen was effectively released from the column with 5M MgCl2 and proved to be highly purified. Passive hemagglutination tests on sera from EAI infections were carried out, using the purified antigen. Results indicated that the passive hemagglutination test with the antigen was a specific laboratory test with high sensitivity for EIA infection.

Animals↗
PubMedDate not supplied

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.

Chromatography, Affinity↗
PubMed1989

Interaction of lactate dehydrogenase with structurally related triazine dyes using affinity partitioning and affinity chromatography.

Affinity partitioning in aqueous two-phase systems consisting of dextran and dye-liganded polyethylene glycol was employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle (E.C. 1.1.1.27) with Procion Red HE-3B and four structurally related derivatives of this dye in order to follow the significance of the terminal rings of Procion Red HE-3B for the strength of interaction. The study revealed that the arrangement of the two 1-amino-8-naphthol-3,6-disulphonic acid rings seems to be a prerequisite for the interaction of azonaphthol dyes with LDH. The negatively charged sulfonic acid group at the terminal rings of Procion Red HE-3B enhances the affinity of the ligand for LDH significantly. The removal of this sulphonic acid group or splitting off the complete terminal rings decreases the affinity to LDH and improves the competitive effect of NAD+. The results of affinity partitioning are compared with those of affinity chromatography and kinetic data. The usefulness and the choice of parameters of affinity partitioning as an analytical tool to predict the chromatographic behaviour of dye ligands are discussed.

Animals↗
PubMed2005

Design, synthesis, and screening of biomimetic ligands for affinity chromatography.

Affinity chromatography is ideally suited to the purification of pharmaceutical proteins due to its unique bio-specificity characteristics. Tailor-made affinity ligands that represent a promising class of synthetic affinity ligands have been developed to target specific proteins and designed to mimic peptidal templates, natural biological recognition motifs, or complementary surface-exposed residues. These biomimetic ligands have been generated by a combination of rational design, combinatorial library synthesis, and subsequent screening of potential leads against target proteins. Small ligands based on a triazine scaffold also present exceptional selectivity and stability, which allows their use in harsh manufacturing environments.

Biomimetics↗
PubMed2001

Quantitative structure/retention relationships in affinity chromatography.

Affinity chromatography (AC) followed by quantitative structure/retention relationships (QSRR) analysis provides information on both the analytes and the macromolecules forming the stationary phases. QSRR equations derived for test series of analytes (often drugs) are interpreted in terms of structural requirements of the specific binding sites on macromolecules. Chromatographically demonstrated differences in analyte/macromolecule interactions may be relevant to molecular pharmacology and rational drug design. Multiple regression analysis of appropriately designed sets of affinity-chromatographic data may help increase the speed and efficiency of search as for new drugs and reduce the need for in vivo screening. Specific high-performance affinity-chromatographic separations can be optimized by rational selection of chiral columns, the characteristics of which are provided by QSRR.

Binding Sites↗