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Mei Yuan, Xiaolan Yang, Yuwei Li, Hongbo Liu, Jun Pu, Chang-Guo Zhan, Fei Liao
Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20...
June 2016: Applied Biochemistry and Biotechnology
Helga Margrét Pálsdóttir, Ágústa Gudmundsdóttir
This report describes the isolation and partial characterization of the novel group III trypsin Y from the pyloric caeca of Atlantic cod. Other Atlantic cod trypsins have been used as food processing aids with good results. Trypsin Y was purified by p-aminobenzamidine affinity chromatography and characterized by SDS-PAGE and western blot analysis, as well as by activity measurements towards synthetic substrates. Identification of trypsin Y was done with polyclonal antibodies raised towards the recombinant form of the enzyme and by MALDI-TOF mass spectrometry...
November 15, 2008: Food Chemistry
Betsaida Bibo-Verdugo, Liliana Rojo-Arreola, Maria A Navarrete-del-Toro, Fernando García-Carreño
A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3...
August 2015: Marine Biotechnology
Nathan J Alves, Jeffrey A Kline
The potent fibrinolytic enzyme, plasmin has numerous clinical applications for recannulizing vessels obstructed by thrombus. Despite its diminutive size, 91 kDa, success in the recombinant expression of this serine protease has been limited. For this reason, a truncated non-glycosylated plasmin variant was developed capable of being expressed and purified from E. coli. This mutated plasmin, known as δ-plasmin, eliminates four of the five kringle domains present on native plasmin, retaining only kringle 1 fused directly to the unmodified catalytic domain of plasmin...
February 13, 2015: Biochemical and Biophysical Research Communications
Brian L Henry, Umesh R Desai
Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used...
November 2014: Thrombosis Research
Serap Beyaztaş, Oktay Arslan
A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20...
June 2015: Journal of Enzyme Inhibition and Medicinal Chemistry
Elena I Klimova, Marcos Flores-Alamo, Tatiana Klimova, Sandra Cortez Maya, Irina P Beletskaya
Acetamidine hydrochloride and p-aminobenzamidine dihydrochloride interact with 3-ferrocenylmethylidene-2,4-pentanedione at 80-82 °C in the presence of K2CO3 in the water-alcohol medium in two tautomeric forms (the amidoimine and enediamine ones) with formation of mixtures of pyrimidine and piperidone derivatives and polymeric coordination complexes of potassium ferrocenyl(hexahydro)pyrimidoxides. The structure of the resultant compounds is elucidated on the basis of IR, 1H- and 13C-NMR spectroscopy, mass spectrometry and elemental analysis data...
2013: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Ezio Fasoli, Yiaslin Ruiz Reyes, Osiris Martinez Guzman, Alexandra Rosado, Vivian Rodriguez Cruz, Amaris Borges, Edmarie Martinez, Vibha Bansal
Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: (i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; (ii) the affinity ligand (pABA) density on membrane surface (5-25nmol/cm(2))...
July 1, 2013: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
N Pozzi, L Acquasaliente, R Frasson, A Cristiani, S Moro, A Banzato, V Pengo, G L Scaglione, A Arcovito, R De Cristofaro, V De Filippis
BACKGROUND: This work was aimed at characterizing the interaction of β(2)-glycoprotein I (β(2)GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade. METHODS: The β(2)GPI-thrombin interaction was studied by surface plasmon resonance (SPR), fluorescence, and molecular modeling. The effect of β(2)GPI on the procoagulant (fibrin generation and platelet aggregation) and anticoagulant (protein C activation) functions of thrombin were investigated with turbidimetric, immunocytofluorimetric and enzymatic assays...
June 2013: Journal of Thrombosis and Haemostasis: JTH
Alberta N A Aryee, Benjamin K Simpson
Grey mullet (Mugil cephalus) lipase was isolated using para-aminobenzamidine agarose and immobilized on octyl Sepharose CL-4B (o-Sep). Immobilized grey mullet lipase (GMLi) had a 10 °C higher optimum temperature compared to the free enzyme and showed remarkable thermal stability. GMLi was most active within the pH range of 8.0-9.5 with an optimum at 8.5. Immobilization also enhanced the storage stability and reusability of the enzyme with minimal changes in efficiency during repeated batches. GMLi showed variable stabilities in various organic solvents...
December 2012: Applied Biochemistry and Biotechnology
Lisbeth M Andersen, Peter A Andreasen, Ivan Svendsen, Janneke Keemink, Henrik Østergaard, Egon Persson
In the absence of its cofactor tissue factor (TF), coagulation factor VIIa (FVIIa) predominantly exists in a zymogen-like, catalytically incompetent state. Here we demonstrate that conformation-specific monoclonal antibodies (mAbs) can be used to characterize structural features determining the activity of FVIIa. We isolated two classes of mAbs, which both increased the catalytic efficiency of FVIIa more than 150-fold. The effects of the antibodies were retained with a FVIIa variant, which has been shown to be inert to allosteric activation by the natural activator TF, suggesting that the antibodies and TF employ distinct mechanisms of activation...
March 16, 2012: Journal of Biological Chemistry
Tomas Erban
BACKGROUND: p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca(2+)-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process...
2011: PloS One
Gulay Bayramoglu, M Yakup Arica
In this work, a new methodology is developed for selection of affinity ligands towards the enzyme "trypsin" using quartz crystals microbalance (QCM) technique. To achieve this goal, the surface amination of gold plated QCM crystals was achieved in 13.56 MHz plasma polymerization system by using ethylenediamine. Three different ligands (i.e., 4-aminobenzamidine, 4-aminobenzoic acid, and phenylalanine) were immobilized on the aminated QCM crystals surface via glutaraldehyde coupling. All three ligand immobilized QCM crystals were characterized and compared under different experimental conditions...
March 2012: Bioprocess and Biosystems Engineering
Ya-Chi Su, Tara N Miller, Duraiswamy Navaneetham, Robert T Schoonmaker, Dipali Sinha, Peter N Walsh
To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI...
September 9, 2011: Journal of Biological Chemistry
Weiling Niu, Zhiwei Chen, Prafull S Gandhi, Austin D Vogt, Nicola Pozzi, Leslie A Pelc, Fatima Zapata, Enrico Di Cera
Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E)...
July 26, 2011: Biochemistry
Chung-Yang Kao, Chia-Ni Lin, Yung-Li Yang, Nobuko Hamaguchi, Shu-Jhu Yang, Ming-Ching Shen, Jau-Tsuen Kao, Shu-Wha Lin
A patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material- reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo...
April 2011: Thrombosis and Haemostasis
Olalla Vázquez, Mateo I Sánchez, José L Mascareñas, M Eugenio Vázquez
Orthogonal attachment of a DOTA[Ln(3+)] complex or a coumarin fluorophore to appropriately functionalized bis-4-aminobenzamidines yields compounds that experience A/T-selective, dsDNA-dependent energy transfer processes, and elicit long wavelength emission of light.
August 14, 2010: Chemical Communications: Chem Comm
Marina Marcuschi, Talita S Espósito, Maurício F M Machado, Izaura Y Hirata, Marcelo F M Machado, Márcia V Silva, Luiz B Carvalho, Vitor Oliveira, Ranilson S Bezerra
An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9...
June 4, 2010: Biochemical and Biophysical Research Communications
Burcu Okutucu, Ali Zeytunluoglu, Figen Zihnioglu
New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase...
2010: Preparative Biochemistry & Biotechnology
Olalla Vázquez, Mateo I Sánchez, José Martínez-Costas, M Eugenio Vázquez, José L Mascareñas
N(1),N(3)-Bis(4-amidinophenyl)propane-1,3-diamine (BAPPA, R = H) is a bisaminobenzamidine fluorogenic derivative that displays a large increase in its emission fluorescence when bound to the minor groove of specific A/T DNA sites (K(d) for an AATT site approximately 79 +/- 6 nM). Moreover, the structural characteristics of BAPPA allow the easy introduction of functional groups that protrude out of the DNA surface.
January 15, 2010: Organic Letters
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