Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins. 相似文献
A microfluidic network (μFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations
of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (μCP) step, the protein
was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5
were fabricated spanning a total distance of 320 μm. We tested the response of chick retinal ganglion cell (RGC) axons, which
are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone
in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population,
all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with
axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5
concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during
the detection of a concentration gradient of ephrinA5.
Susanne Lang and Anne C. von Philipsborn contributed equally to this work: S.L. performed the experiments; A.P. evaluated
the data and wrote the paper. 相似文献
The need to develop label‐free biosensing devices that enable rapid analyses of interactions between small molecules/peptides and proteins for post‐genomic studies has increased significantly. We report a simple metal–insulator–metal (MIM) geometry for fabricating a highly sensitive detection platform for biosensing. MIM substrates consisting of an Au–PMMA–Ag nanolayer were extensively studied using both theoretical and experimental approaches. By monitoring reflectivity changes at the normal incidence angle, we observed molecular interactions as the thickness of the biolayer increased on the substrate surface. These interactions included the adsorption of various proteins (Mw=6–150 kD) and interactions between small molecules (Mw≤2 kD) and the immobilized proteins. The interaction of designed monosaccharide‐modified designed peptides with various lectins was also clearly detected. These interactions could not be detected by the conventional Au‐only substrate. Thus, the MIM approach affords a powerful label‐free biosensing device that will aid our understanding of protein interactions and recognition. 相似文献
We report the demonstration of miniaturized capillary isoelectric focusing (CIEF) in plastic microfluidic devices. Conventional CIEF technique was adapted to the microfluidic devices to separate proteins and to detect protein-protein interactions. Both acidic and basic proteins with isoelectric points (pI) ranging from 5.4 to 11.0 were rapidly focused, mobilized, and detected in a 1.2 cm long channel (50 microm deep x 120 microm wide) with a total analysis time of 150 s. In a device with a focusing distance of 4.7 cm, the separation efficiency for a basic protein, lysozyme, was achieved as high as 1.5 x 10(5) plates, corresponding to 3.2 million plates per meter. We also experimentally confirmed that IEF resolution is essentially independent of focusing length when the applied voltage is kept the same and within a range that it does not cause Joule heating. Further, we demonstrated the use of miniaturized CIEF to study the interactions between two pairs of proteins, immunoglobulin G (IgG) with protein G and anti-six histidine (anti-6xHis) with 6xHis-tagged green fluorescent protein (GFP). Using this approach, protein-protein interactions can be detected for as little as 50 fmol of protein. We believe miniaturized CIEF is useful for studying protein-protein interactions when there is a difference in pI between a protein-protein complex and its constitutent proteins. 相似文献
Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins. 相似文献
Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions. 相似文献
The interactions of the negatively charged achiral molecular micelle, poly (sodium N-undecanoyl sulfate) (poly-SUS), with four different proteins using intrinsic and extrinsic fluorescence spectroscopic probes, are studied. A comparison of poly-SUS with the conventional surfactant, sodium dodecyl sulfate (SDS), and the monomeric species, SUS, is also reported. In this work, we observed that poly-SUS preferentially binds to acidic proteins, exhibiting positive cooperativity at concentrations less than 1 mM for all proteins studied. Moreover, it appears that the hydrophobic microdomain formed through polymerization of the terminal vinyl group of the monomer, SUS, is largely responsible for the superior binding capacity of poly-SUS. From these results, we conclude that the interactions of poly-SUS with the acidic proteins are predominantly hydrophobic and postulate that poly-SUS would produce superior interactions relative to SDS at low concentrations in polyacrylamide gel electrophoresis (PAGE). As predicted, use of poly-SUS allowed separation of the His-tagged tumor suppressor protein, p53, at sample buffer concentrations as low as 0.08% w/v (2.9 mM), which is 24 times lower than required for SDS in the standard reducing PAGE protocol. This work highlights the use of poly-SUS as an effective surfactant in 1D biochemical analysis. 相似文献
We report the first example of synthetic chondroitin sulfate (CS) microarrays to rapidly identify glycosaminoglycan-protein interactions and probe the specificity of proteins for distinct sulfation sequences. Using the microarrays, we identify a novel interaction between CS and TNF-alpha, a proinflammatory cytokine involved in rheumatoid arthritis, Crohn's disease, and psoriasis. Moreover, we demonstrate that CS-E tetrasaccharides and polysaccharides enriched in the CS-E sulfation motif can inhibit the activity of this therapeutically important cytokine. We anticipate that carbohydrate microarrays will accelerate our understanding of glycosaminoglycan-protein interactions and the role of sulfation in modulating physiological and disease states. 相似文献
We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay
with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of
hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very
sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic
acid under pH conditions that increase neutral species concentration in the assay reagent in order to enhance the binding
of more CBB dye molecules per protein molecule than in previous CBB-based assays. 相似文献
Despite tremendous advances in our understanding of the molecular basis of diabetes mellitus, substantial gaps still remain
in our understanding of disease pathogenesis and in the development of effective strategies for early diagnosis and treatment.
The proteomic approach has offered many opportunities and challenges in identifying new marker proteins and therapeutic targets,
i.e., using 2D-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry.
The differential protein expressions were analyzed in alloxan-induced diabetic rats treated with Cynodon dactylon leaf extract. The plant extract was administered for 15 days that resulted in a significant increase in plasma insulin and
C-peptide levels. We have also identified four differentially expressed proteins from rat plasma. These four diabetes-associated
proteins were broadly classified into three groups as per their function: (1) lipid metabolism-associated protein (Apo A-IV),
(2) antioxidant activity-related proteins [preprohaptoglobin and heat shock proteins B8 (HspB8)], and (3) muscle function-related
protein (TPM3). Apo A-IV, HspB8, and preprohaptoglobin may play a key role in the recovery of diabetes mellitus and also prevent
the diabetes-associated complications such as prevention of oxidative stress due to free radical and free hemoglobin. These
results show the value of proteomic approach in identifying the potential markers that may eventually serve as diagnostic
markers or therapeutic targets. 相似文献
The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction solutions, that are compatible with the first-dimensional separation step, such as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), do not solubilize all proteins in a sample. We studied the effect of various common, strong detergents and chaotropes, widely used as solubilizing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and guanidine hydrochloride, on the solubilization of the total and membrane proteins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Use of sodium dodecyl sulfate, lithium dodecyl sulfate or guanidine hydrochloride for the solubilization of total proteins of the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detected in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/CHAPS system. When the dry immobilized pH gradient strips were rehydrated in a solution containing the proteins of the membrane fraction solubilized with sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of protein spots were detected in comparison with strips that were rehydrated in the urea/CHAPS solution. However, no improvement was observed in comparison with protein application in sample cups. The additional proteins detected with the use of strong detergents and chaotropes are in the majority difficult to solubilize and less hydrophobic proteins. 相似文献
It is known that proliferation and survival of neural stem/progenitor cells in vitro not only depend on exogenous factors, but also on autocrine factors secreted into the conditioned medium. It is also well known that the identification of bioactive proteins secreted into the conditioned medium poses a substantial challenge. Recently, neural stem/progenitor cells were shown to secrete a survival factor, cystatin C, into the conditioned medium. Here, we demonstrate an approach to identify other low molecular weight proteins in conditioned medium from cultured adult rat hippocampal progenitor cells. A combination of preparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry was utilized in the analysis. We were able to identify a number of proteins, which include Rho-guanine nucleotide dissociation inhibitor 1, phosphatidylethanolamine binding protein (PEBP), also termed Raf-1 kinase interacting protein, polyubiquitin, immunophilin FK506 binding protein 12 (FKBP12) and cystatin C. The presence of PEBP and FKBP12 in conditioned medium was confirmed immunologically. All nestin-positive progenitor cells showed immunoreactivity for antibodies against PEBP and FKBP12. To our knowledge we are the first to use this preparative proteomic approach to search for stem cell factors in conditioned medium. The method could be used to identify novel bioactive proteins secreted by stem/progenitor cells in vitro. Identification of bioactive proteins in vitro is of potential importance for the understanding of the regulatory mechanisms of the cells in vivo. 相似文献
EphrinA1 is a tyrosine kinase receptor localized in the cellular membrane of healthy cardiomyocytes, the expression of which is lost upon myocardial infarction (MI). Intra-cardiac injection of the recombinant form of ephrinA1 (ephrinA1-Fc) at the time of ligation in mice has shown beneficial effects by reducing infarct size and myocardial necrosis post-MI. To date, immunohistochemistry and Western blotting comprise the only experimental approaches utilized to localize and quantify relative changes of ephrinA1 in sections and homogenates of whole left ventricle, respectively. Herein, we used matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) coupled with a time-of-flight mass spectrometer (MALDI/TOF MS) to identify intact as well as tryptic fragments of ephrinA1 in healthy controls and acutely infarcted murine hearts. The purpose of the present study was 3-fold: (1) to spatially resolve the molecular distribution of endogenous ephrinA1, (2) to determine the anatomical expression profile of endogenous ephrinA1 after acute MI, and (3) to identify molecular targets of ephrinA1-Fc action post-MI. The tryptic fragments detected were identified as the ephrinA1-isoform with 38% and 34% sequence coverage and Mascot scores of 25 for the control and MI hearts, respectively. By using MALDI-MSI, we have been able to simultaneously measure the distribution and spatial localization of ephrinA1, as well as additional cardiac proteins, thus offering valuable information for the elucidation of molecular partners, mediators, and targets of ephrinA1 action in cardiac muscle.
Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions. 相似文献
Nanomaterials hold promise as multifunctional diagnostic and therapeutic agents. However, the effective application of nanomaterials is hampered by limited understanding and control over their interactions with complex biological systems. When a nanomaterial enters a physiological environment, it rapidly adsorbs proteins forming what is known as the protein 'corona'. The protein corona alters the size and interfacial composition of a nanomaterial, giving it a biological identity that is distinct from its synthetic identity. The biological identity determines the physiological response including signalling, kinetics, transport, accumulation, and toxicity. The structure and composition of the protein corona depends on the synthetic identity of the nanomaterial (size, shape, and composition), the nature of the physiological environment (blood, interstitial fluid, cell cytoplasm, etc.), and the duration of exposure. In this critical review, we discuss the formation of the protein corona, its structure and composition, and its influence on the physiological response. We also present an 'adsorbome' of 125 plasma proteins that are known to associate with nanomaterials. We further describe how the protein corona is related to the synthetic identity of a nanomaterial, and highlight efforts to control protein-nanomaterial interactions. We conclude by discussing gaps in the understanding of protein-nanomaterial interactions along with strategies to fill them (167 references). 相似文献