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1.
Chromatography has become an essential tool for the purification of proteins, since most purification schemes involve some forms of this methodology. Recently, using chromatographic matrices prepared from symmetrical aminosquarylium cyanine dyes immobilized on Sepharose via a central alkylamino residue, we were able to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. Following this, we envisioned that the immobilization of an asymmetric squarylium dye bearing an N-carboxyethyl group in one of its ending nuclei, on ethylenediamine-activated Sepharose, through EDC/NHS amidation coupling, could enhance the ligand’s mobility and improve the interactions with the target proteins. The prepared support was found to separate an artificial mixture of BSA, lysozyme, and RNase A. Unexpectedly, the support prepared in the absence of the dye exhibited a separation performance similar to that of the dyed support, contrary to that observed in all previous studies using cyanine dyes as ligands for affinity chromatography, which prompted us to try to determine the structural molecular constitution of the matrix surface. A synthetic route to the final chromatographic support could be devised, which is believed to consist in the cyclization of two nearby ethylenediamine units, involving the inclusion of a succinimide-derived residue between them and the EDC-mediated Lossen rearrangement of an intermediary hydroxamic acid.  相似文献   

2.
An affinity chromatography procedure for the rapid purification of tryptophan-5-monooxygenase from rabbit hindbrains was developed using e-aminocaproyl-D-tryptophan methyl ester-Sepharose-4B gels. The precise requirements for the optimal biospecific interaction between the affinity ligand and the ligate (enzyme) was established from a study of the effects of the variation in the length of the "spacer’’ on the affinity properties of the gel. The enzyme preparation isolated by this procedure was found to be essentially homogeneous and was characterized by a molecular weight of 200,000 ±20,000. SDS-polyacrylamide gel electrophoresis of the enzyme revealed it to be a dimer, the molecular weight of each subunit being approximately 90,000. The specific activity of the enzyme preparation is approxi-mately 7-10 times that of the crude homogenate, but a further fivefold enhancement in the specific activity could be obtained by limited proteolysis with trypsin. The extreme lability of the enzyme could be circumvented by its immobilization on activated Sepharose or by cross-linking with dimethyl suberimidate. The kinetic properties as well as the advantages of such stabilized enzyme preparations are presented.  相似文献   

3.
4.
An affinity or biospecific method of isolating an iron-binding glycoprotein — lactoferrin — from breast milk is described which permits in a single chromatographic operation the isolation of lactoferrin homogeneous according to the results of electrophoresis in polyacrylamide gel. The affinity method of isolating lactoferrin is based on the use of a specific immunosorbent consisting of antibodies to lactoferrin covalently conjugated with cyanogen-bromide-activated Sepharose 4B.  相似文献   

5.
Cyanogen bromide was used to convert pyridine rings in polymers to polyaldehyde. By reaction with NH2-containing substances, the rings are rebuilt, resulting in a pyridinium compound. Thus proteins and other NH2-containing substances can be covalently bound. This method provides a new means for a immobilization technique. Pyridine-gels based on polysaccharide and polyacrylamide matrices, as well as pyridine glass beads, were synthesized and used to study the conditions necessary for coupling. Trypsin and — chymotrypsin were used as test substances for immobilization of proteins. Some properties of the bound protein were studied and compared to native enzyme. Some general results on the applicability of these gels for affinity chromatography are also presented.  相似文献   

6.
A new AMP derivative substituted with spacer arms both at position N6 and C8 of the adenine moiety was synthesized and immobilized to Sepharose. To the immobilized ligand was subsequently coupled C8-substituted ATP in a solid-phase synthesis fashion yielding the bifunctional general ligand AMP-ATP. This affinity material was used in the separation of two major groups of enzymes, dehydrogenases and kinases. It was found that on passage of crude homogenates obtained from mouse kidney through the affinity column, several dehydrogenases and kinases were bound, which could be eluted separately using pulses of NADH and ATP, respectively. In the fractions obtained on NADH elution, lactate dehydrogenase, malate dehydrogenase, and α-glycerol phosphate dehydrogenase were found, whereas ATP eluted 3-phosphoglyceric acid kinase, pyruvate kinase, and aldolase.  相似文献   

7.
Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein–ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein–ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method’s false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein–ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments. Graphical Abstract
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8.
Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

Affinity-based benzotriazole (BTA) probes selectively and covalently label native proteins or endogenous proteins in cells with a fast reaction rate. It is enormously useful for characterizing protein function in biological systems and for drug development.  相似文献   

9.
The binding of competitive inhibitor proflavin by α-chymotrypsin in water-dioxane mixtures over the entire range of thermodynamic activities of water a w was studied. The data on the degree of binding of proflavin were compared to the results on the catalytic activity of the enzyme preliminary incubated in water-dioxane mixtures. An analysis of the behavior of the concentration dependences of these characteristics demonstrated that, at low a w values, the behavior of the interprotein contacts in the enzyme formed during its drying largely governs its functional properties, while at high a w values, they are determined by the interaction of the enzyme with the organic solvent. Interplay of these two factors is responsible for the observed complex shape of the isotherm of binding of proflavin, with the maximum degree of binding being attained at moderate a w values.  相似文献   

10.
The affinity of aspartate aminotransferase to its inhibitors coupled to Sepharose 4 B was tested. The affinity was measured as retardation of the enzyme compared to “inert” bovine serum albumin. Carboxylic ligands of citrate and 2-oxoglutarate bound to aminoethyl-Sepharose were the best of those tested in separation of the proteins. Because the ligands were not essentially hydrophobic and because it was shown that ion-exchange is not significant in the elution conditions used, it was suggested that the separation is based on the recognition of substrate or effector by the enzyme.  相似文献   

11.
A simple method is described for the preparation of dextran-linked coenzyme derivatives. Several different 8-(6-aminohexyl)amino-adenine nucleotide coenzymes and their derivatives were covalently attached to dextran by incubation with bromohydroxypropyl derivatives of dextran at room temperature in an alkaline medium. The polymer-linked adenine nucleotide derivatives were separated from the free coenzyme derivatives by a Sephadex G-50 column. The prepared dextran derivatives have ligand densities ranging from 20 to 100 μmol/g of dextran derivatives depending on the conditions of coupling and derivatives. NMR studies revealed that proton resonances of the polymer-linked coenzymes exhibit short transverse relaxation times (T2) but long longitudinal relaxation times (T1) This phenomenon was interpreted in terms of the anisotropic motions of the dextran-bound coenzyme derivatives in which the fast axial motions and slow restricted transverse motions of the bound coenzyme derivatives are postulated. These observations could properly explain why the polymer-linked coenzymes exhibit lower biological activity, but similar binding affinity to most enzymes.  相似文献   

12.
Human parotid amylase was immobilized by covalent binding to CNBr-activated Sepharose, to Corning GAO-3940 silica glass biomaterial support by the diazonium reaction or reaction with glutaraldehyde, or as a result of the antigen-antibody reaction between rabbit antihuman parotid amylase IgG that was covalently bonded to GAO glass and soluble amylase. The amylase directly bonded to the supports showed constant activity at flow rates of 3-15 ml/min through a 1.76-cm3 (8-mm diameter) support bed, did not lose enzyme into a circulating starch solution, retained its activity in the presence of soluble antiamylase IgG, was optimally active at 35°-40°C, and lost activity at 40°-45°C. When the enzyme was bound by interaction with immobilized antibody, full activity was expressed, but some enzyme was solubilized by a circulating starch solution. Immobilization of either amylase or antiamylase IgG makes dissolution of the antigen-antibody bond difficult.  相似文献   

13.
A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤103 M–1) ligands from moderate and high affinity (>105 M–1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3. Graphical Abstract
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14.
The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution.
  1. An analogous pH dependence of initial activity is found for free and immobilized enzyme.
  2. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent Km values are three to eight times higher for the immobilized (2 mg/g gel) enzyme.
  3. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ?g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration.
  4. The activation energies for free and immobilized fumarase (50 ?g/g gel) were found to be identical between 22 and 32?C and with L-malate as substrate (Ea = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases.
Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ?g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.  相似文献   

15.
Benzoic acid is a competitive inhibitor of the enzyme dopa-oxidase. p-Aminobenzoic acid was coupled by a single-step reaction of the amino group to: (1) CNBr-activated Sepharose 4B: (2) Enzacryl AA, by reaction with thiophosgene (pH 10.0); and (3) CM-Sephadex G-50, modified to azide by the Curtis procedure. These three solid supports were used as affinity adsorbents in the dopa-oxidase purification. The enzyme was obtained from frog epidermis, and was retained at pH 4.7 with 0.1 M acetate buffer. The enzyme elution was carried out using a linear pH gradient with 0.1 M phosphate buffer pH 8.0. We have examined the interaction of the enzyme with the immobilized PABA in relation to the nature of the support, the lengthening of the “arm” of the ligand, bathwise adsorption, and the enzyme activation by immobilized trypsin.  相似文献   

16.
UDP-galactose ovomucoid galactosyltransferase, a membrane-bound enzyme involved in the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences on glycoproteins, has been solubilized and purified from rat ventral prostate Golgi membranes. Solubilization was effected by treatment of the particulate fraction with Triton X-100 (0.5% v/v) and MnCl2 (25 mM). The solubilized enzyme was purified by affinity chromatography on hen ovomucoid-sepharose column. The purified galactosyltransferase showed three protein bands of approx. 74,000, 60,000, and 54,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity eluted at approx. 70,000 daltons with a broad shoulder between 60,000 and 50,000 daltons. Isoelectric focusing of the purified enzyme resolved at least five active bands with pHi of 9, 7.4, 6.75, 6.1, and 4.8.  相似文献   

17.
We describe the conformational behavior of histone deacetylase 8 (HDAC8) using molecular dynamics (MD) simulations. HDAC8 conformers were used for the docking studies using some known HDAC inhibitors (HDACi) suberoylanilide hydroxamic acid (SAHA), valproic acid (VPA), aroyl-pyrrole-hydroxy-amide (APHA-8) and tubacin to explore their interactions, binding modes, free energy values. The MD simulation show that HDAC8 make important surface changes at the catalytic site (CS) entrance as well as at two entrances locations in the 14-Å tunnel. In addition, we identify an alternate entrance to the 14-Å tunnel named adjacent to the catalytic site pocket (ACSP). By using docking studies, it was possible to elucidate the importance of hydrophobic and π–π interactions that are the most important for the ligand–HDAC8 complex structural stabilization. In conclusion, the ligand flexibility, molecular weight and chemical moieties (hydroxamic acid, aryl and aliphatic moieties) are the principal properties required to increase the binding affinity on HDAC8.  相似文献   

18.
Biswajit Kalita 《Tetrahedron》2004,60(47):10771-10778
Derivatives of iminodiacetic acid (IDA) are important as ligands for metal ions, having numerous applications in separations, sensing, catalysis and medicine. This report describes the preparation of two types of IDA derivatives (1, 2) that could be covalently attached to a polymer or protein surface via a variable length spacer chain. The parent compounds 1 (R′=H) were easily prepared via N-alkylation of dimethyl iminodiacetate with esters of 6-bromo-hexanoic acid and subsequent selective ester hydrolysis. Metal complexes of IDA derivatives having an α-dienyl side chain are required for the selection of histidine-rich proteins with potential lipoxygenase activity. The α-hexadienyl side chain of IDA derivative 2 was selectively introduced in the reaction of (2,4-hexadienyl acetate)Fe(CO)3 with a glycine-derived TMS-enol ester. Subsequent demetallation, followed by N-carboxymethylation, N-deacylation, N-alkylation with a trichloroethyl 6-halohexanoate, and TCE-ester cleavage provided the desired α-hexadienyl IDA derivative 2. Amide formation with IDA acid 1b demonstrates the feasibility of conjugating the IDA ligands to polymers and proteins while Ni(II)-complexation with the derived IDA triacid 1e shows the complexing ability of the tethered IDA ligand.  相似文献   

19.
The reaction of the amino group of α-chymotrypsin with poly(N,N-diethylacrylamides) bearing terminal carboxyl groups which have the degree of polymerization ranging from 30 to 180 and which possess an LCST of 34–29°C affords polymer derivatives of the enzyme. It is found that, upon an increase in the temperature of the aqueous solution of the resulting derivatives to 40°C, the derivative with a degree of polymerization of 180 precipitates at 34°C, while the derivatives with a degree of polymerization of 30–80 remain in solution. The activity of α-chymotrypsin as a part of the derivatives with a degree of polymerization of 30 does not change with increasing temperature, whereas the activity of the enzyme as a part of the derivatives with degrees of polymerization of 60 and 80 decays almost to zero near the LCST of the initial polymers. Such a change in the enzyme activity is reversible (the activity fully recovers with a decrease in temperature).  相似文献   

20.
Micrococcal nuclease has been covalently attached to CNBr-activated Sepharose 4B by coupling through three different enzyme functions: (a) amino groups; (b) carboxyl groups; and (c) tyrosyl or histidyl residues. On the basis of coupling yield and catalytic efficiency, Sepharose-(NH2) nuclease derivatives were chosen for further activity andstability studies. The activity of the insoluble enzyme has been evaluated with macromolecular (DNA) and small (synthetic nucleotide) substrates; with the latter the enzyme retains 70% of native enzyme activity. Good enhancement of enzyme stability in the 4–40°C range has been observed.  相似文献   

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