scholarly journals General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1972 ◽  
Vol 127 (4) ◽  
pp. 625-631 ◽  
Author(s):  
K. Mosbach ◽  
H. Guilford ◽  
R. Ohlsson ◽  
M. Scott
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Cyanogen Bromide ◽  
Competitive Inhibitor ◽  
Single Step ◽  
Lactate Dehydrogenase Activity ◽  
Step Procedure ◽  
The Matrix ◽  
Subsequent Elution ◽  
Sepharose 4B

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD+ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.

scholarly journals Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine

1977 ◽  
Vol 165 (1) ◽  
pp. 121-126 ◽  
Author(s):  
G D Smith ◽  
D V Roberts ◽  
A Daday
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibitor ◽  
Chorismate Mutase ◽  
Prephenate Dehydrogenase ◽  
Step Procedure ◽  
One Step ◽  
High Degree ◽  
Sepharose 4B ◽  
Derivatives Of

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

HEPARIN INDEPENDENT PURIFICATION OF ANTITHROMBIN III (AT III) BY IMMUNO-AFFINITY CHROMATOGRAPHY RESULTING IN A FUNCTIONALLY INTACT MOLECULE

1987 ◽  
Author(s):  
M Jørgensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
Heparin Binding ◽  
Purification Method ◽  
Step Procedure ◽  
At Iii ◽  
Sepharose 4B

Previous methods for purification of AT III are based on its heparin-binding capacity. However, in congenital AT III deficiency abnormal inhibitor molecules with impaired binding of heparin and/or thrombin has been reported. The aim of the present study was to develop a purification method based on immuno-affinity chromatography, and thus independent of the heparin binding capacity.Rabbits were immunized with human AT III purified by a three-step procedure involving dextran sulphate precipitation, affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-Sephadex A-50. Rabbit immunoglobulins against human AT III were isolated by affinity chromatography using purified human AT III coupled to CNBr-activated Sepharose 4B. Trace amounts of immunoglobulin against human albumin, IgG and IgM were removed by solid phase immunoadsorption. The highly purified immunoglobulins against human AT III were coupled to CNBr-activated Sepharose 4B. This anti-AT III-Sepharose was used for single-step purification of AT III from plasma. Elution was performed by Na-citrate buffer at pH 3.0 and the eluted fractions immediately neutralized. The recovery was more than 60%.The purified AT III appeared as a single protein band in SDS-poly-acrylamide gel electrophoresis with or without reduction. Affinity purified AT III and AT III purified by the three-step procedure were indistinguishable when analyzed by crossed immunoelectrophoresis in the absence and the presence of heparin isoelectrical focusing in polyacrylamid gel at a pH 4-6.5 gradient, and SDS-polyacrylamide gel electrophoresis. AT III antigen concentration was determined by electroimmunoassay and the reactive site concentration determined by titration with purified human thrombin using Phe-Pip-Arg-Nan (S-2238) as substrate. The ratio (active site conc.)/(antigen conc.) was identical in the two AT III preparations. It is concluded that this single-step immuno-affinity chromatography gives a high recovery from plasma of a highly purified functionally intact AT III molecule. The purification method is independent of the heparin binding capacity of AT III. This is of particular importance for the purification and characterization of abnormal AT III molecules with impaired heparin-binding site.

scholarly journals Purification of cathepsin B by a new form of affinity chromatography

1986 ◽  
Vol 235 (3) ◽  
pp. 731-734 ◽  
Author(s):  
D H Rich ◽  
M A Brown ◽  
A J Barrett
Keyword(s):  
Affinity Chromatography ◽  
Cathepsin B ◽  
Single Step ◽  
Starting Material ◽  
High Yield ◽  
Cysteine Proteinases ◽  
New Form ◽  
Human Cathepsin ◽  
Sepharose 4B

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2′-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

A lactate dehydrogenase-immunoglobulin G1 complex, not blocked by anti-idiotype antibody, in a patient with IgG1-lambda type M-proteinemia.

1987 ◽  
Vol 33 (8) ◽  
pp. 1478-1483 ◽  
Author(s):  
K Fujita ◽  
I Sakurabayashi ◽  
M Kusanagi ◽  
T Kawai
Keyword(s):  
Lactate Dehydrogenase ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
Binding Site ◽  
Binding Capacity ◽  
Isoenzyme Pattern ◽  
M Protein ◽  
Light Chains ◽  
Ldh Isoenzymes ◽  
Sepharose 4B

Abstract The serum of a patient with IgG1-lambda type M-proteinemia showed an abnormal isoenzyme pattern for lactate dehydrogenase (LDH, EC 1.1.1.27). By affinity chromatography, we showed that four isoenzymes (LDH2, LDH3, LDH4, and LDH5) were bound to the M-protein. This complex formation was not blocked by anti-idiotype antibody, even though the binding capacity of IgG was exclusively located in the Fab region of the molecule. Moreover, heavy and light chains of the patient's IgG, obtained by reduction, separately had affinities for each of the LDH isoenzymes. LDH-IgG complex was easily dissociated by affinity chromatography on 5'-AMP-Sepharose 4B or by added NADH. We propose the following hypothesis for the LDH-IgG complex formation: LDH can recognize the gamma-Fab region of IgG at the NAD+ binding site of the molecule, but the affinity of the LDH molecule for immunoglobulin is much weaker than that for NADH or 5'-AMP.

Rapid Single-Step Kinetic Colorimetric Assay for Lactate Dehydrogenase in Serum

1973 ◽  
Vol 19 (2) ◽  
pp. 223-227 ◽  
Author(s):  
Charles C Allain ◽  
Carl P Henson ◽  
M Keith Nadel ◽  
Adam J Knoblesdorff
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Dynamic Range ◽  
Colorimetric Assay ◽  
Single Step ◽  
Tetrazolium Salt ◽  
Lactate Dehydrogenase Activity ◽  
Tetrazolium Chloride ◽  
System A ◽  
Reducing Substances

Abstract We report an improved kinetic colorimetric system for measuring lactate dehydrogenase activity in serum. In the system a tetrazolium salt, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride, is used as the chromogenic indicator of dehydrogenase activity, with diaphorase serving as the electron transfer agent. All ingredients required for an assay are combined in a single dry reagent that is stable at room temperature. The method is 2.5 times as sensitive as the ultraviolet method of Wacker and has a dynamic range three times that of the ultraviolet method. Reducing substances in serum do not affect the results. Precision, range of linearity, and stability of reagent after reconstitution are excellent. Results for fresh sera correlated well with those obtained by the "A-Gent" ultraviolet method (Wacker method at 37°C) and with the SMA 12/60.

Purification of Escherichia coli Chloramphenicol Acetyltransferase by Affinity Chromatography

1974 ◽  
Vol 52 (12) ◽  
pp. 1087-1090 ◽  
Author(s):  
Michel Guitard ◽  
Réjean Daigneault
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Nitro Group ◽  
Chloramphenicol Acetyltransferase ◽  
Single Step ◽  
Isolation Procedure ◽  
Amino Group ◽  
R Factor ◽  
Sepharose 4B

Chloramphenicol acetyltransferase (CATase) was purified by affinity chromatography from Escherichia coli W677/HJR66, an R-factor-bearing mutant. The chloramphenicol aryl nitro group had to be reduced to an amino group prior to its coupling to the Sepharose 4B matrix. The single-step isolation procedure resulted in a 237-fold purification of CATase with over 65% recovery of the enzyme.

Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates

1990 ◽  
Vol 36 (1) ◽  
pp. 59-64
Author(s):  
R N Weijers ◽  
R de Bruijn ◽  
J Mulder ◽  
H Kruijswijk
Keyword(s):  
Amino Acid ◽  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
B Lymphocytes ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Fluorescein Isothiocyanate ◽  
Molar Ratio ◽  
The Stability ◽  
Sepharose 4B

Abstract Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

scholarly journals Affinity chromatography of pig heart fumarase

1979 ◽  
Vol 177 (1) ◽  
pp. 115-119 ◽  
Author(s):  
S Chaudhuri ◽  
E W Thomas
Keyword(s):  
Affinity Chromatography ◽  
Aromatic Ring ◽  
Fumaric Acid ◽  
Competitive Inhibitor ◽  
Sepharose 4B

2-(5′-Phenylpentyl)fumaric acid was shown to be a competitive inhibitor (Ki 0.5 mM) of pig heart fumarase. After nitration of the aromatic ring, reduction to the amine and diazotization, the acid was attached via azo linkages to a Sepharose 4B-tyramine matrix. The resulting adsorbent was used for the affinity chromatography of crude fumarase, purifications of approx. 20-fold being obtained by specific elution with 0.01 M-citrate.

Purification by exploitation of activity

2001 ◽  
Author(s):  
S. Angal ◽  
Simon D. Roe
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Careful Consideration ◽  
Single Step ◽  
The Matrix ◽  
Crude Mixture ◽  
Support Matrix ◽  
P Proteins ◽  
The Many ◽  
Major Factors

Proteins carry out their biological functions through one or more binding activities and, consequently, contain binding sites for interaction with other biomolecules, called ligands. Ligands may be small molecules such as substrates for enzymes or larger molecules such as peptide hormones. The interaction of a binding site with a ligand is determined by the overall size and shape of the ligand as well as the number and distribution of complementary surfaces. These complementary surfaces may involve a combination of charged and hydrophobic moieties and exhibit other short-range molecular interactions such as hydrogen bonds. This binding activity of a protein, which is stereoselective and often of a high affinity, can be exploited for the purification of the protein in a technique commonly known as affinity chromatography. The operation of affinity chromatography involves the following steps: (a) Choice of an appropriate ligand. (b) Immobilization of the ligand onto a support matrix. (c) Contacting the protein mixture of interest with the matrix. (d) Removal of non-specifically bound proteins. (e) Elution of the protein of interest in a purified form. At best, affinity chromatography is the most powerful technique for protein purification since its high selectivity can, in principle, allow purification of a single protein of low abundance from a crude mixture of proteins at higher concentrations. Secondly, if the affinity of the ligand for the protein is sufficiently high, the technique offers simultaneous concentration from a large volume. In practice, such single-step purifications are not common and successful affinity chromatography requires careful consideration of a number of parameters involved. The remainder of this chapter attempts to guide the experimenter in the selection and use of affinity adsorbents for protein purification. For more extensive information on this technique the reader is advised to consult the many excellent texts on this subject as well as proceedings of symposia. The construction of an affinity adsorbent for the purification of a particular protein involves three major factors: (a) Choice of a suitable ligand. (b) Selection of a support matrix and spacer. (c) Attachment of the ligand to a support matrix. The criteria for making these decisions are discussed in the following sections.

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