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Affinity Binding (affinity + binding)
Terms modified by Affinity Binding Selected AbstractsControlling Affinity Binding with Peptide-Functionalized Poly(ethylene glycol) HydrogelsADVANCED FUNCTIONAL MATERIALS, Issue 14 2009Chien-Chi Lin Abstract Poly(ethylene glycol) (PEG) hydrogels functionalized with peptide moieties have been widely used in regenerative medicine applications. While many studies have suggested the importance of affinity binding within PEG hydrogels, the relationships between the structures of the peptide motifs and their binding to protein therapeutics remain largely unexplored, especially in the recently developed thiol-acrylate photopolymerization systems. Herein, Förster resonance energy transfer (FRET) and thiol-acrylate photopolymerizations are employed to investigate how the architectures of affinity peptides in crosslinked hydrogels affect their binding to diffusible proteins. The binding between diffusible streptavidin and biotinylated peptide immobilized to PEG hydrogel network was used as a model system to reveal the interplay between affinity binding and peptide sequences/architectures. In addition, peptides with different structures are designed to enhance affinity binding within PEG hydrogels and to provide tunable affinity-based controlled delivery of basic fibroblast growth factor (bFGF). This study demonstrates the importance of affinity binding in controlling the availability of hydrogel-encapsulated proteins and provides strategies for enhancing affinity binding of protein therapeutics to bound peptide moieties in thiol-acrylate photopolymerized PEG hydrogels. The results presented herein should be useful to the design and fabrication of hydrogels that retain and exhibit sustained release of growth factors for promoting tissue regeneration. [source] Organic Phase PPO Biosensors Prepared by Multilayer Deposition of Enzyme and Alginate Through Avidin-Biotin InteractionsELECTROANALYSIS, Issue 24 2004S. Cosnier Abstract Films of electrogenerated polypyrrole and hydrophilic alginate, both functionalized with biotin moieties, were used to allow for the transfer of polyphenol oxidase activity in organic media. Enzyme electrodes, based on multilayered structures, were protected at the molecular level by the affinity binding of alginate as a hydrophilic additive, and were then transferred into chlorobenzene, dichloromethane, chloroform, ethyl acetate or acetonitrile. The biosensor performance for the detection of catechol at ,0.2,V was investigated, highlighting the main influence of the hydrophobicity of the solvent and, to a lesser extent, the dielectric constant. The effect of the substrate hydrophobicity on the biosensor response was examined in chlorobenzene. [source] Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesisFEBS JOURNAL, Issue 18 2007Petra Ml, ochová Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1, site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N -acetyl- l -aspartyl- l -glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1, pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the ,fine-tuning' of GCPII substrate specificity. [source] Study of the subunit interactions in myosin phosphatase by surface plasmon resonanceFEBS JOURNAL, Issue 6 2000Attila Tóth The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1,511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11,296 > MYPT11,38 > MYPT123,38. No binding was detected with MYPT11,34, suggesting a critical role for residues 35,38, i.e. the PP1c binding motif. Binding of residues 1,22 was inferred from: a higher affinity binding to PP1c for MYPT11,38 compared to MYPT123,38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11,38, but not by MYPT123,38. Residues 40,296 (ankyrin repeats) in MYPT11,296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nm), whereas MYPT11,38, MYPT123,38 or MYPT11,34 were without effect. MYPT140,511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c,MYPT11,38 and PP1c,MYPT123,38. The inhibitory effect of MYPT140,511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11,38. The binding of MYPT1304,511 to complexes of PP1c and MYPT11,38, or MYPT11,296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner. [source] Controlling Affinity Binding with Peptide-Functionalized Poly(ethylene glycol) HydrogelsADVANCED FUNCTIONAL MATERIALS, Issue 14 2009Chien-Chi Lin Abstract Poly(ethylene glycol) (PEG) hydrogels functionalized with peptide moieties have been widely used in regenerative medicine applications. While many studies have suggested the importance of affinity binding within PEG hydrogels, the relationships between the structures of the peptide motifs and their binding to protein therapeutics remain largely unexplored, especially in the recently developed thiol-acrylate photopolymerization systems. Herein, Förster resonance energy transfer (FRET) and thiol-acrylate photopolymerizations are employed to investigate how the architectures of affinity peptides in crosslinked hydrogels affect their binding to diffusible proteins. The binding between diffusible streptavidin and biotinylated peptide immobilized to PEG hydrogel network was used as a model system to reveal the interplay between affinity binding and peptide sequences/architectures. In addition, peptides with different structures are designed to enhance affinity binding within PEG hydrogels and to provide tunable affinity-based controlled delivery of basic fibroblast growth factor (bFGF). This study demonstrates the importance of affinity binding in controlling the availability of hydrogel-encapsulated proteins and provides strategies for enhancing affinity binding of protein therapeutics to bound peptide moieties in thiol-acrylate photopolymerized PEG hydrogels. The results presented herein should be useful to the design and fabrication of hydrogels that retain and exhibit sustained release of growth factors for promoting tissue regeneration. [source] A genome-inspired DNA ligand for the affinity capture of insulin and insulin-like growth factor-2JOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2009Junfeng Xiao Abstract The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here, we report on interactions of insulin and the highly homologous insulin-like growth factor-2 (IGF-2) with ILPR variants a, h, and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI MS and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with KD , 10,13 M and KD , 10,7 M, which was not observed for insulin with variant h (KD , 10,8 M) or IGF-2 with either variant (KDs , 10,9 M). The results provide a basis for the design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery. [source] Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2009Anne K. Callesen Abstract Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting. [source] Apolipoproteins and , Amyloid Transport PathwayPSYCHOGERIATRICS, Issue 3 2002Kouzin Kamino Abstract: Cholesterol metabolism has been viewed as an important step in the development of Alzheimer's disease, since it was shown that the ,4 allele of apolipoprotein E (APOE) gene is a genetic risk and modifies age-at-onset of Alzheimer's disease. Although the knowledge of the effect of cholesterol in the neuronal cell has been recently accumulated, the link between systemic and brain cholesterol metabolism remains to be elucidated. Lipoproteins in cerebrospinal fluid (CSF) are fractionated only to high-density lipoprotein (HDL), and contain apolipoprotein (apo) A-I, E, A-II, and J. Whereas apoE is produced in the brain, apoA-I and apoA-II in cerebrospinal fluid, the major components of plasma HDL cholesterol, originate from plasma. Plasma HDL is thought to act in reverse cholesterol transport, and in vitro experiments indicated that these apolipoproteins and albumin show a high affinity binding to , amyloid. In patients with Alzheimer's disease, plasma apoA-I and apoA-II levels are significantly decreased, which is possibly related to the deposition of , amyloid in the brain, and to the , amyloid transport pathway. [source] Ex Vivo Biocompatibility of Avidin-Agarose: A New Device for Direct Adsorption of Biotinylated Antibodies from Human Whole BloodARTIFICIAL ORGANS, Issue 9 2000T. Bosch Abstract: Radioimmunotherapy using radiolabeled antitumor antibodies (RAA) is limited by the toxicity of unbound antibodies in the circulation. Removal of excessive antibodies by affinity-adsorption could therefore allow the administration of increased dosages of RAA while decreasing their adverse effects. Recently, avidin-agarose (AA) minicolumns were used in animal experiments for the removal of biotinylated antibodies from whole blood exploiting the high affinity binding of biotin to avidin (pK 1015 M,1). This study was performed to evaluate the ex vivo biocompatibility of AA minicolumns with human blood. Ten ml AA minicolumns were perfused online ex vivo in the single pass mode with fresh blood from 8 healthy donors at a flow rate of 6.25 ml/min. The anticoagulation consisted of 0.5 IU heparin plus 0.0,2.1 mg citrate per ml of blood. In Part 1 of the study (40 min perfusion, n = 4), the optimal anticoagulation was found to be 0.5 IU heparin plus about 1 mg citrate per ml of blood. In Part 2 of the study, four 80 min test-runs were performed. No signs of hemolysis were found, and the thrombogenicity of the AA gel was negligible. Cell counts and column inlet pressures remained constant; toward the end of the 80 min test-runs, some activation of blood cells (elastase, ,-thromboglobulin), the complement system (C3a, C5a) and the plasmatic coagulation (thrombin-antithrombin complex) was detectable. A moderate initial bradykinin release rapidly subsided to very low levels. In summary, AA minicolumns showed good biocompatibility upon contact with human whole blood and merit further investigation in a closed-loop system for a potential application of direct tumor antibody removal by hemoperfusion. [source] Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved-core alginate microspheresBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Ayesha Chaudhary Abstract The feasibility of dissolved-core alginate-templated fluorescent microspheres as "smart tattoo" glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye-labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution-phase counterparts. The glucose sensitivity of the encapsulated TRITC-Con A/FITC-dextran (500,kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC-apo-GOx/FITC-dextran (500,kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0,50,mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120,s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0,50,mM glucose, using near-infrared dyes (Alexa Fluor-647-labeled dextran as donor and QSY-21-conjugated apo-GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075,1085. © 2009 Wiley Periodicals, Inc. [source] | |||||||||||||