scholarly journals Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

1973 ◽  
Vol 135 (4) ◽  
pp. 595-607 ◽  
Author(s):  
Standish Barry ◽  
Pádraig O'Carra
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Xanthine Dehydrogenase ◽  
Mechanistic Studies ◽  
Amino Groups ◽  
Enzyme Binding ◽  
Specific Affinity ◽  
Spacer Arm ◽  
Primary Amino ◽  
Derivatives Of

1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+(probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.

Purification of Penicillin Amidohydrolase, an Enzyme for Semisynthetic Procedures

1992 ◽  
Vol 57 (10) ◽  
pp. 2187-2191 ◽  
Author(s):  
Jiří Jiráček ◽  
Tomislav Barth ◽  
Jiří Velek ◽  
Ivo Bláha ◽  
Jan Pospíšek ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Amino Groups ◽  
Primary Amino

Penicillin amidohydrolase (EC 3.5.1.11.) is one of the few enzymes used successfully for deprotection of primary amino groups of semisynthetic peptides. The available material is usually contamined by endo- and exopeptidases. We managed to prepare the enzyme devoid of trypsin- and chymotrypsin-like activities using affinity chromatography with specific ligands: Gly-D-Phe-Phe-Tyr-Thr-Pro-Lys-Thr (the fF peptide) and Leu-Gly-Val-D-Arg-Arg-Gly-Phe (the rR peptide). For further purification of the enzyme affinity chromatography with N-phenylacetyl-D-tert-Leu as a ligand was used.

Trägergebundene Serotoninderivate und Affinitätschromatographie synaptischer Membranproteine / Resin Linked Derivatives of Serotonin and Affinity Chromatography of Proteins Isolated from Synaptic Membranes

1974 ◽  
Vol 29 (5-6) ◽  
pp. 248-256 ◽  
Author(s):  
Frank Seela ◽  
Wolfgang Wesemann
Keyword(s):  
Affinity Chromatography ◽  
Nerve Ending ◽  
Serotonin Receptor ◽  
Oxalyl Chloride ◽  
Synaptic Membranes ◽  
Amino Group ◽  
Indole Derivatives ◽  
Primary Amino ◽  
Affinity Resin ◽  
Derivatives Of

An affinity resin for the isolation of the serotonin receptor from nerve ending membranes was prepared by coupling tryptamine, 5-methoxy-tryptamine or serotonin to CNBr-activated Sepharose 4 B. The tryptamine derivatives, N-6-(aminohexyl)-tryptamines, were obtained by subsequent con­ densation of the corresponding indole derivatives with oxalyl chloride and t-aminocaproamide and reduction with LiAlH4. The 6-aminohexyl-group was used as spacer to link covalently the primary amino-group of tryptamine to agarose. - An extract of membrane proteins isolated from rat brain was resolved on this affinity resin into two fractions as compared with only one fraction using unsubstituted Sepharose 4 B.

scholarly journals Preparation of adenosine nucleotide derivatives suitable for affinity chromatography

1974 ◽  
Vol 139 (3) ◽  
pp. 609-623 ◽  
Author(s):  
Ian P. Trayer ◽  
Hylary R. Trayer ◽  
David A. P. Small ◽  
Robin C. Bottomley
Keyword(s):  
Affinity Chromatography ◽  
Synthetic Methods ◽  
Partial Purification ◽  
General Applicability ◽  
Reduced Pressure ◽  
Adenine Ring ◽  
Adenosine Phosphate ◽  
Spacer Arm ◽  
Primary Amino ◽  
Adenosine Nucleotide

Methods of synthesizing a series of chemically-defined AMP, ADP, ATP, adenylyl imidodiphosphate and pyrophosphate derivatives suitable for affinity chromatography are extensively described. Each derivative has a single primary amino group at the end of a hexamethylene ‘spacer’ chain for attachment to CNBr-activated agarose. The synthesis of the derivative where the ‘spacer’ arm is attached directly to the 8 position of the adenine ring to produce 8-(6-aminohexyl)amino-AMP involves the direct bromination of AMP in the 8 position followed by displacement of the halogen by 1,6-diaminohexane. This monophosphate derivative can then be converted into the corresponding di- or triphosphate forms by direct phosphate condensation with carbonyl di-imidazole. A second series of adenosine phosphate derivatives with the phosphate moieties unsubstituted has been similarly prepared from N6-(6-aminohexyl)-AMP (Guilford et al., 1972). A third type of ligand has been synthesized by condensing the phosphoryl imidazolide of AMP with 6-aminohex-1-yl phosphate. This compound, P1-(6-aminohex-1-yl) P2-(5′-adenosyl) pyrophosphate, has an unsubstituted adenine ring. The synthesis of a fourth type of ligand, 6-aminohex-1-yl pyrophosphate, was done by heating 6-aminohexan-1-ol with crystalline pyrophosphoric acid under reduced pressure. The structures of the synthesized compounds were confirmed by chemical, electrophoretic and chromatographic methods and by u.v. spectrometry. The general applicability of the synthetic methods used is discussed in relation to the preparation of other affinity adsorbents. Examples are given where these derivatives have been successful in reversibly binding dehydrogenases, kinases and myosin and its proteolytic subfragments. The partial purification of rat liver glucokinase on an ADP derivative is shown.

scholarly journals CRYSTALLINE ACETYL DERIVATIVES OF PEPSIN

1934 ◽  
Vol 18 (1) ◽  
pp. 35-67 ◽  
Author(s):  
Roger M. Herriott ◽  
John H. Northrop
Keyword(s):  
Specific Activity ◽  
Amino Nitrogen ◽  
Strong Acid ◽  
Acetyl Derivative ◽  
Crystal Form ◽  
Amino Groups ◽  
Acetyl Content ◽  
Titration Curves ◽  
Primary Amino ◽  
Derivatives Of

Crystalline pepsin has been acetylated by the action of ketene in aqueous solution at pH 4.07–5.5. As acetylation proceeds the activity decreases, the decrease being more rapid at pH 5.0–5.5 than at 4.0–4.5. Three acetyl derivatives have been isolated from the reaction mixture and obtained in crystalline form. The crystal form of these derivatives is similar to that of pepsin. Fractionation and solubility determinations show that these preparations are not mixtures or solid solutions of the original pepsin with an inactive derivative. A compound which contains three or four acetyl groups and which has lost all of its original primary amino groups can be isolated after short acetylation. It has the same activity as the original pepsin. A second derivative containing six to eleven acetyl groups has also been isolated. It has about 60 per cent of the activity of the original pepsin. A third derivative having twenty to thirty acetyl groups and about 10 per cent of the activity of original pepsin can be isolated after prolonged acetylation. The 60 per cent active derivative on standing in strong acid solution loses some of its acetyl groups and at the same time regains the activity of the original pepsin. The compound obtained in this way is probably the same as the completely active three acetyl derivative obtained by mild acetylation. These results show that acetylation of three or four of the primary amino groups of pepsin causes no change in the specific activity of the enzyme but that the introduction of acetyl groups in other parts of the molecule results in a marked loss in activity. The solubilities, amino nitrogen content, acetyl content, isoelectric point, and the specific activity have been determined by a variety of methods and found to be different from the corresponding properties of crystalline pepsin. The pH-activity curves, acid and alkali inactivation, and titration curves were not significantly different from the same respective properties of pepsin.

Aldehyde gold clusters for molecular labeling

1995 ◽  
Vol 53 ◽  
pp. 858-859
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya
Keyword(s):  
Covalent Bond ◽  
Aldehyde Group ◽  
Gold Clusters ◽  
Side Reactions ◽  
Amino Groups ◽  
Sodium Cyanoborohydride ◽  
Schiff’S Base ◽  
Protein Molecules ◽  
Hydroxysuccinimide Esters ◽  
Primary Amino

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

scholarly journals Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography

1975 ◽  
Vol 151 (3) ◽  
pp. 631-636 ◽  
Author(s):  
R I Brinkworth ◽  
C J Masters ◽  
D J Winzor
Keyword(s):  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
Equilibrium Constants ◽  
Mouse Tissue ◽  
Rabbit Muscle ◽  
Muscle Lactate ◽  
Sodium Phosphate Buffer ◽  
A Value ◽  
Series Of Experiments ◽  
Reduced Nicotinamide Adenine Dinucleotide

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 × 104M−1 for the interaction of enzyme with NADH at 5°C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 × 105M−1, 3 × 105M−1, 4 × 105M−1, 7 × 105M−1 and 2 × 106M−1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.

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