The use of Spheron as a matrix for affinity chromatography of NAD-dependent dehydrogenases

The paper compares several methods of coupling common ligands of dehydrogenases, viz. N6-[(6-aminohexyl)carbamoylmethyl]-AMP and N6-[(6-aminohexyl)carbamoylmethyl]-NAD, to a hydrophilic macroporous glycolmethacrylate gel, Spheron. The affinants coupled best to a CNBr-activated gel and to a gel with hydrazine groups (after activation with nitrous acid). The affinity properties of gels based on Spheron and on Sepharose 4B were similar ( the stability and separation efficiency were almost identical, the binding capacity and the recovery of dehydrogenase activity were somewhat better with the Sepharose). The materials based on Spheron were used in several separation experiments, viz. separation of lactate dehydrogenase form albumin, separation of lactate dehydrogenase from alcohol dehydrogenase under different conditions and separation of isoenzymes of lactate dehydrogenase. Spheron 300 with a coupled affinant was also employed in an attempt to purify a crude alcohol dehydrogenase.

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

Clinical Chemistry ◽

10.1093/clinchem/33.8.1478 ◽

1987 ◽

Vol 33 (8) ◽

pp. 1478-1483 ◽

Cited By ~ 2

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.

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

Clinical Chemistry ◽

10.1093/clinchem/36.1.59 ◽

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.

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Production and certification of secondary enzyme reference materials (ERMs). Part 1: Preparation of the sera and some of their properties.

Clinical Chemistry ◽

10.1093/clinchem/32.10.1901 ◽

1986 ◽

Vol 32 (10) ◽

pp. 1901-1905 ◽

Cited By ~ 6

Author(s):

J C Koedam ◽

G M Steentjes ◽

S Buitenhuis ◽

E Schmidt ◽

R Klauke

Keyword(s):

Creatine Kinase ◽

Lactate Dehydrogenase ◽

Reference Material ◽

Human Serum ◽

Dehydrogenase Activity ◽

Aspartate Aminotransferase ◽

Alanine Aminotransferase ◽

Glutamate Dehydrogenase Activity ◽

The Stability ◽

Clinical Enzymology

Abstract We produced three batches of a human-serum-based enzyme reference material (ERM) enriched with human aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), creatine kinase (EC 2.7.3.2), and lactate dehydrogenase (EC 1.1.1.27). The added enzymes were not exhaustively purified; thus the final ERMs contained some enzymes as contaminants, of which only glutamate dehydrogenase activity might interfere. The stability during storage and after reconstitution was good. The commutability of the four enzymes in the three ERM batches was also good, except when German or Scandinavian methods for aminotransferases were involved. The temperature-conversion factors for the ERMs were equivalent to those for patients' sera. Reactivation after reconstitution was complete within 5 min and was independent of the temperature of the reconstitution fluid. We believe that these secondary ERMs will aid in the transfer of accuracy between well-defined reference methods and daily working methods so that clinical enzymology results will become more comparable from laboratory to laboratory.

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Effect of selected alcohol dehydrogenase inhibitors on the human heart lactate dehydrogenase activity - anin vitrostudy

Acta Physiologica Hungarica ◽

10.1556/aphysiol.91.2004.3-4.7 ◽

2004 ◽

Vol 91 (3-4) ◽

pp. 235-241 ◽

Cited By ~ 3

Author(s):

J Dudka ◽

F Burda ◽

Barbara Madej ◽

Justyna Szumilo ◽

Edyta Tokarska ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Alcohol Dehydrogenase ◽

Dehydrogenase Activity ◽

Human Heart ◽

Lactate Dehydrogenase Activity

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Effect of chorionic gonadotropin on lactate dehydrogenase and alcohol dehydrogenase activity in the pathologically altered liver

Bulletin of Experimental Biology and Medicine ◽

10.1007/bf02444327 ◽

1994 ◽

Vol 117 (6) ◽

pp. 591-592

Author(s):

Yu. V. Zimin ◽

I. M. Solopaeva

Keyword(s):

Lactate Dehydrogenase ◽

Alcohol Dehydrogenase ◽

Dehydrogenase Activity ◽

Chorionic Gonadotropin ◽

Alcohol Dehydrogenase Activity ◽

Altered Liver

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The Effect of Fibrinopeptide B Release on Temperature Dependent Fibrin Association

10.1055/s-0039-1682613 ◽

1977 ◽

Author(s):

W. Edgar ◽

C.R.M. Prentice

Keyword(s):

Affinity Chromatography ◽

Clot Formation ◽

Effect Of Temperature ◽

Fibrinopeptide A ◽

Temperature Dependent ◽

Fibrin Polymerization ◽

Soluble Fibrin ◽

Agkistrodon Contortrix ◽

The Stability ◽

Sepharose 4B

The effect of temperature on soluble fibrin complexes was studied using Biogel filtration and chromatography on fibrinogen-sepharose 4B at 20°C and 37°C. Complexes were formed by plasmin digestion of non-crosslinked fibrin produced by thrombin, ancrod, thrombin in 2M urea, or ancrod plus Agkistrodon contortrix venom. Thrombin removes fibrinopeptides A and B, ancrod removes fibrinopeptide A, while A. contortrix enzyme removes first fibrinopeptide B, followed by A. Complexes containing neither fibrinopeptide A or B, formed by digestion of fibrin produced by thrombin, or by ancrod plus Agkistrodon contortrix, were stable at 37°C. In contrast, complexes which retained fibrinopeptide B, formed from fibrin produced by ancrod or by thrombin in 2M urea, were unstable at 37°C. Fibrin polymerization was necessary for the stability of fibrin complexes. Complexes from plasmin digests of fibrin produced by ancrod plus A. contortrix enzyme in 2M urea, where no clot formation occurred, were unstable at 37°C. Using affinity chromatography, plasmin digests of thrombin-fibrin bound to fibrinogen-sepharose at 37°C, whereas those from ancrod-fibrin did not. A second set of polymerization sites in fibrinogen are proposed, distinct from the N-DSK and carboxyterminal sites. These are activated by removal of fibrinopeptide B and require clot formation.

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Human Skeletal Muscle Lactate Dehydrogenase Activity in the Presence of Some Alcohol Dehydrogenase Inhibitors

Basic & Clinical Pharmacology & Toxicology ◽

10.1111/j.1742-7843.2004.pto950108.x ◽

2004 ◽

Vol 95 (1) ◽

pp. 38-42 ◽

Cited By ~ 9

Author(s):

Jaroslaw Dudka ◽

Franciszek Burdan ◽

Agnieszka Korobowicz ◽

Robert Klepacz ◽

Elzbieta Korobowicz

Keyword(s):

Skeletal Muscle ◽

Lactate Dehydrogenase ◽

Alcohol Dehydrogenase ◽

Dehydrogenase Activity ◽

Human Skeletal Muscle ◽

Muscle Lactate ◽

Lactate Dehydrogenase Activity

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

Biochemical Journal ◽

10.1042/bj1270625 ◽

1972 ◽

Vol 127 (4) ◽

pp. 625-631 ◽

Cited By ~ 169

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.

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

Biochemical Journal ◽

10.1042/bj1790479 ◽

1979 ◽

Vol 179 (3) ◽

pp. 479-482 ◽

Cited By ~ 7

Author(s):

A J Brown ◽

C Y Lee

Keyword(s):

Drosophila Melanogaster ◽

Alcohol Dehydrogenase ◽

Affinity Chromatography ◽

Good Yield ◽

Purification Procedure ◽

Ligand Affinity ◽

Novel Technique ◽

Affinity Ligand ◽

Sepharose 4B ◽

Homogeneous Preparation

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.

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