Studies on interactions of programmed cell death 5 (PDCD5) and its related peptides with heparin by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used to investigate interactions between heparin and programmed cell death 5 (PDCD5), and between heparin and PDCD5-related peptides. Samples containing PDCD5, PDCD5-related peptides, and heparin at various ratios were incubated at room temperature and then separated by CZE with tris-acetate buffer at pH 7.2. Both qualitative and quantitative characterizations of the binding of PDCD5 and PDCD5-related peptides to heparin were determined. The changes in the signals of PDCD5 and PDCD5-related peptides were monitored by comparing the electropherograms of the mixtures containing PDCD5 and heparin and PDCD5-related peptides and heparin with that of PDCD5 or PDCD5-related peptides only. The binding constant of the interaction between PDCD5 and heparin was calculated as 4.17 × 10⁴ M⁻¹ by Scatchard analysis. Our investigations show that it is possible to characterize the interaction between PDCD5 and heparin quantitatively and the interaction between PDCD5-related peptides and heparin qualitatively using CZE.     

Capillary zone electrophoresis investigation of the interaction between heparin and granulocyte-colony stimulating factor

The interaction between standard heparin, low-molecular-weight heparin (LMWH), and granulocyte-colony stimulating factor (G-CSF) was studied by capillary zone electrophoresis. Both qualitative and quantitative characterizations of the heparin-protein binding were determined. The binding constants of the two different groups of heparins with G-CSF, calculated from the Scatchard plot by regression, were 4.805 x 10(5) M(-1) and 4.579 x 10(5) M(-1), respectively. The two binding constants measured are of the same order of magnitude at 10(5) M(-1), indicating that LMWH contains most of the functional groups bound to G-CSF by standard heparin.     

Quantitative investigation of the interaction between granulocyte-macrophage colony-stimulating factor and heparin by capillary zone electrophoresis

The interactions between granulocyte-macrophage colony-stimulating factor (GM-CSF) and heparin or low-molecular weight heparin (LMWH) were studied by CZE. It was found that GM-CSF could bind to both heparin and LMWH. The binding constants were calculated from Scatchard regression to be (6.5 +/- 0.8) x 10(5)/M and (11.2 +/- 0.7) x 10(5)/M, respectively. The specificity of the interaction between GM-CSF and heparin was also studied by employing another sulfated K carrageenan oligosaccharide as a control. Results showed that K carrageenan oligosaccharide could not interact with GM-CSF, indicating that GM-CSF could specifically interact with heparin.     

Effect of buffer on heparin binding and sensing in competitive aqueous media

Abstract

Although buffer-specific effects on molecular recognition are known in biological science, they remain rare in supramolecular chemistry. The binding between a cationic dye, mallard blue (MalB) and polyanionic heparin in aqueous NaCl (150 mM) is studied in three commonly used buffers (Tris-HCl, HEPES, Phosphate, each 10 mM). Although MalB has a very similar UV–visible spectrum in each buffer, the sensory response towards heparin was different in each case. This can be ascribed to differences in the complex formed. In Tris-HCl which has the least competitive chloride counter-anions, MalB exhibits a hypsochromic shift of 25 nm, assigned to strong binding and aggregation of the dye on heparin. In more competitive HEPES, containing a sulfonate anion, there is weaker binding and less aggregation of MalB along the heparin; the hypsochromic shift is only 15 nm. In phosphate buffer, MalB can interact quite strongly with buffer phosphate anions; although heparin binding is still observed, the hypsochromic shift associated with dye aggregation is only 5 nm. As such, specific buffer interactions with the MalB–heparin complex mediate host–guest binding and sensing. Buffer choice must be made carefully in studies of molecular recognition – we would caution against using phosphate and sulfonate containing buffers when studying electrostatic binding.     

The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.     

Electrochemical determination of heparin using methylene blue probe and study on competition of Ba with methylene blue for binding heparin

Cyclic voltammetric investigation of the interaction of methylene blue (MB) with heparin (hep) at a gold electrode is presented. The combination of MB with heparin formed a nonelectroactive complex MB-hep, which resulted in the peak current decrease of MB. The anodic peak current difference of MB was found to be proportional to the concentration of heparin in the range of 0.666-64.5 μg mL⁻¹ with a detection limit of 270 ng mL⁻¹ and a satisfactory result was obtained for the determination of heparin in injection samples. The equilibrium constant for MB-hep complex was calculated to be 7.32 × 10⁵. The dynamic process of competition of Ba²⁺ with methylene blue for binding heparin was monitored using quartz crystal microbalance (QCM) technique. The reaction rate constant between Ba²⁺ and MB-hep was estimated to be 0.0022 s⁻¹.     

Further characterization of the binding of heparin to granulocyte colony-stimulating factor: importance of sulfate groups

Heparin mediates fundamental biological mechanisms through interaction with proteins. Previously, we have shown that standard heparin binds to granulocyte colony-stimulating factor (G-CSF) with an affinity of 4.8 x 10(5) M(-1). To further study the structural features in heparin that are responsible for this interaction, we studied the bindings of G-CSF and N-desulfated and 2,3-O-desulfated heparin by CZE. Results showed that the N-desulfated heparin had a similar affinity for G-CSF ((5.4 +/- 0.9) x 10(5) M(-1)), but the 2,3-O-desulfated heparin had a 1000-fold lower affinity ((3.4 +/- 1.2) x 10(2) M(-1)) in comparison to standard heparin. The results showed that 2,3-O-sulfate groups are more important than N-sulfate groups in heparin-G-CSF interaction.     

Contributions of hydroxyethyl groups to the DNA binding affinities of anthracene probes

Contributions of hydroxyethyl functions to the DNA binding affinities of substituted anthracenes are evaluated by calorimetry and spectroscopy. Isothermal titration calorimetry indicated that binding of the ligands to calf thymus DNA (5 mM Tris buffer, 50 mM NaCl, pH 7.2, 25 degrees C) is exothermic. The binding constants increased from 1.5 x 10(4) to 1.7 x 10(6) M(-1) as a function of increase in the number of hydroxyethyl functions (0-4). DNA binding was accompanied by red-shifted absorption (approximately 630 cm(-1)), strong hypochromism (>65%), positive induced-circular dichroism bands, and negative linear dichroism signals. DNA binding, in general, increased the helix stabilities to a significant extent (DeltaT(m) approximately 7 degrees C, DeltaDeltaH approximately 3 kcal/mol, DeltaDeltaS approximately 6-20 cal/K.mol). The binding constants showed a strong correlation with the number of hydroxyethyl groups present on the anthracene ring system. Analysis of the binding data using the hydrophobicity parameter (Log P) showed a poor correlation between the binding affinity and hydrophobicity. This observation was also supported by a comparison of the affinities of probes carrying N-ethyl (Kb = 0.8 x 10(5) M(-1)) versus N-hydroxyethyl side chains (Kb = 5.5 x 10(5) M(-1)). These are the very first examples of a strong quantitative correlation between the DNA binding affinity of a probe and the number of hydroxyethyl groups present on the probe. These quantitative findings are useful in the rational design of new ligands for high-affinity binding to DNA.     

Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.     

In situ growth of nanogold on quartz crystal microbalance and its application in the interaction between heparin and antithrombin III

A novel biosensor for detecting antithrombin III (AT III) was constructed based on in situ growth of nanogold on the gold electrode of quartz crystal microbalance (QCM). The growth process of nanogold was monitored by QCM in real time. Heparin was used as the affinity ligand and immobilized onto the nanogold modified gold electrode. A flow injection analysis-quartz crystal microbalance (FIA-QCM) system was used to investigate the relationship between nanogold growth and the AT III response. Along with the nanogold particle growth within initial 5 min, the amount of heparin immobilized onto the nanogold modified electrode increased quickly. Correspondingly, the frequency response to AT III binding increased rapidly at the same time. After that, both the immobilized amount of heparin and the sensor response to AT III decreased gradually. Compared with the directly immobilized large nanogold particles, the in situ grown particles with the same size occupy more sensor surface, resulting in higher frequency shifts to AT III in the interaction study between heparin and AT III. The obtained constants of AT III binding to immobilized heparin are k(ass)=(1.65+/-0.12)x10(3) L/mols, k diss=(2.63+/-0.18)x10(-2) 1/s and K(A)=(6.27+/-0.42)x10(4) L/mol.     

Capillary electrophoresis and enzyme solid phase assay for examining the purity of a synthetic heparin proteoglycan-like conjugate and identifying binding to basic fibroblast growth factor

Interaction of basic fibroblast growth factor (bFGF) with heparin/heparan sulfate proteoglycans protects the growth factor against proteolytic degradation and is essential for its cellular activity. Although the structural requirements of heparin and heparan sulfate for the high-affinity binding to bFGF have been extensively examined, studies on intact heparin proteoglycans are limited. In this report, the purity and the binding ability of a heparin proteoglycan-like molecule-the heparin-bovine serum albumin (heparin-BSA) conjugate-was examined using capillary zone electrophoresis (CZE). Furthermore, the affinity of bFGF binding to the heparin-BSA conjugate was studied using an enzyme solid-phase assay. Chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and variously sulfated disaccharides derived from heparin and heparan sulfate were also studied for their ability to compete with the binding of bFGF to heparin. Heparin-BSA conjugate was synthesized by reductive amination and, following precipitation with 1.5 vols of ethanol-sodium acetate, it was obtained free of contaminating heparin. Heparin-BSA-bFGF conjugate was obtained following incubation of heparin-BSA with bFGF for 2 h at 37 degrees C. Intact heparin, heparin-BSA and heparin-BSA-bFGF conjugates were completely resolved by CZE using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Competitive solid phase assay showed that, among the glycosaminoglycans tested, heparin exhibits the highest affinity binding to bFGF (IC(50) = 6.4 nM). Heparan sulfate showed a lower affinity as compared with that of heparin, whereas all other glycosaminoglycans and heparin/heparan sulfate-derived disaccharides tested showed minute effects. The developed CZE method is rapid and accurate and can be easily used to identify bFGF-interacting heparin preparations of biopharmaceutical importance.     

Investigation on the binding site in heparin by spectrophotometry

Heparin has a variety of biological activities, most of them due to heparin’s high sulfate groups. To gain insight into the mechanism of activation of the spectroscopic probe with sulfate groups of heparin in vitro, we have used a cationic dye by a spectrophotometric method. It is considered that the combination of heparin with methylene blue is due to noncovalent binding forces. Dye binding requires an organic chain structure form with sulfate groups. The solution equilibria of the reaction system are discussed. A new linear regression equation has been proposed, in which the maximum binding number N expresses the binding ability of methylene blue (MB) with sulfate groups of heparin. The linear regression equation can estimate this parameter.     

Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion

Von Willebrand factor (VWF) binding and platelet adhesion to subendothelial collagens are initial events in thrombus formation at sites of vascular injury. These events are often studied in vitro using flow assays designed to mimic vascular hemodynamics. Flow assays commonly employ collagen-functionalized substrates, but a lack of standardized methods of surface ligation limits their widespread use as a clinical diagnostic. Here, we report the use of collagen thin films (CTF) in flow assays. Thin films were grown on hydrophobic substrates from type I collagen solutions of increasing concentration (10, 100, and 1000 μg/mL). We found that the corresponding increase in fiber surface area determined the amount of VWF binding and platelet adhesion. The association rate constant (k(a)) of plasma VWF binding at a wall shear stress of 45 dyn/cm(2) was 0.3 × 10(5), 1.8 × 10(5), and 1.6 × 10(5) M(-1) s(-1) for CTF grown from 10, 100, and 1000 μg/mL solutions, respectively. We observed a 5-fold increase in VWF binding capacity with each 10-fold increase in collagen solution concentration. The association rates of Ser1731Thr and His1786Asp VWF mutants with collagen binding deficiencies were 9% and 22%, respectively, of wild-type rates. Using microfluidic devices for blood flow assays, we observed that CTF supported platelet adhesion at a wall shear rate of 1000 s(-1). CTF grown from 10 and 100 μg/mL solutions had variable levels of platelet surface coverage between multiple normal donors. However, CTF substrates grown from 1000 μg/mL solutions had reproducible surface coverage levels (74 ± 17%) between normal donors, and there was significantly diminished surface coverage from two type 1 von Willebrand disease patients (8.0% and 24%). These results demonstrate that collagen thin films are homogeneous and reproducible substrates that can measure dysfunctions in VWF binding and platelet adhesion under flow in a clinical microfluidic assay format.     

Macromolecule-macromolecule interaction in drug distribution. II. Effect of alpha-globulin on saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes in primary culture.

The uptake of fractionated [3H]heparin was investigated in rat parenchymal hepatocytes in primary culture. The initial uptake of fractionated [3H]heparin was found to be saturable with the maximum uptake velocity (Vmax) of 10.1 +/- 1.46 pmol/min/mg protein and the Michaelis constant (Km) of 284 +/- 47.9 nM. The effect of alpha-globulin, the major protein binding to fractionated [3H]heparin, on the saturable uptake profile of fractionated [3H]heparin was also investigated. The uptake clearance was reduced, depending on the concentration of fractionated [3H]heparin, by the addition of 1 mg/ml alpha-globulin. We assumed that fractionated 3H-heparin bound to alpha-globulin was not available for uptake and that the reduction in the uptake clearance was solely attributable to the saturable binding of fractionated [3H]heparin to alpha-globulin. The uptake clearance versus concentration profile was analyzed to obtain the dissociation constant (Kd) of 31.8 nM and the capacity (n) of 0.047 for the binding of fractionated [3H]heparin to alpha-globulin. The saturable binding of fractionated [3H]heparin to alpha-globulin was supported by in vitro binding experiments using gel chromatography, in which bound fractionated [3H]heparin decreased with the concentration of fractionated [3H]heparin in the presence of alpha-globulin. In conclusion, the present study demonstrated the saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes and the saturable binding of fractionated [3H]heparin to alpha-globulin. The saturable uptake may suggest the involvement of a specific transport system such as receptor-mediated endocytosis.     

Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: the effect of hapten density

Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.     

A resonance light scattering ratiometry applied for binding study of organic small molecules with biopolymer

Resonance light scattering (RLS) technique is a creative application of light scattering signals detected by using a common spectrofluorometer, but it has drawbacks such as the fluctuation of signals caused by poorly quantified or variable factors. Herein we develop a RLS ratiometry to overcome the drawbacks of the technique and apply to measure the binding nature of organic small molecules (OSM) with biopolymer using the binding of cation porphyrins with heparin (HP) as an example. In near neutral solution, cationic porphyrins meso-tetrakis [(trimethylammoniumyl) phenyl] porphyrin (TAPP) and meso-tetra (4-methylpyridy) porphyrin (TMPyP-4) interact with heparin, resulting in hypochromatic effect, and enhanced RLS signals. Linear relationship could be established between the ratio of enhanced RLS signals at two wavelengths, where the maximum and minimum are available in the ratio curve of UV-vis spectrum of porphyrin to that of heparin-porphyrin complex, and the logarithm of heparin concentration, and thus a wide dynamic range detection method of biopolymers could be developed. In comparison with RLS method, this RLS ratiometric one is less affected by environmental conditions such as pH, ionic strength. The mechanism of these interactions was investigated based on the charge density distribution of the two porphyrin molecules and it could be concluded that the enhanced RLS intensity is proportionally promoted by the charge capacity of components in the complex. Additionally, the binding number and binding constant were measured scientifically by Scatchard plot.     

Determination of dissociation constants for a heparin-binding domain of amyloid precursor protein and heparins or heparan sulfate by affinity capillary electrophoresis

Dynamic affinity capillary electrophoresis (ACE) was used for determining the binding constants between heparin-like glycosaminoglycans and the (96-110) heparin-binding domain of amyloid precursor protein (APP). The migration time shift of the (96-110) APP peptide was monitored as the concentration of heparin was increased in the background electrolyte. The compounds investigated included low-molecular-weight heparin, porcine mucosa heparin, and heparan sulfate. Change in mobility as a function of glycosaminoglycan concentration was plotted using both linear regression (Scatchard analysis) and nonlinear regression. Dissociation constants (K(d)) were determined and compared for both sets of analyses with the low-molecular-weight heparin giving the most reproducible results and best fit with a K(d) value of 3.9 microM.     

Effects of sulfate position on heparin octasaccharide binding to CCL2 examined by tandem mass spectrometry

Chemokine-glycosaminoglycan (GAG) interactions have been shown to be essential for in vivo chemokine signaling, which functions in such diverse processes as inflammation, development, and cancer metastasis. Despite the importance of these interactions, the saccharide sequence dependency of chemokine-GAG interactions is poorly understood. In a recent study, FT-ICR mass spectrometry was used to show that the chemokine CCL2 (monocyte chemoattractant protein 1) binds only to the 11- and 12-sulfated components of a heparin octasaccharide library. Although the exact structure of the fully sulfated, 12-sulfated octasaccharide is known, the 11-sulfated species could have a number of sulfated disaccharide sequences. In the current study, the composition of the 11-sulfated heparin octasaccharides, as well as the composition of CCL2 affinity purified 11-sulfated heparin octasaccharides, were examined by tandem MS. Of the three possible singly desulfated disaccharides, one species, III-S, is enriched by CCL2 affinity purification, indicating that the 11-sulfated heparin octasaccharides containing this disaccharide are preferentially bound to CCL2. These data suggest that 2-O and N sulfation of heparin may be of greater importance to CCL2-heparin binding than 6-O sulfation.     

Anion binding studies of fluorinated expanded calixpyrroles

The anion binding properties of fluorinated calix[n]pyrroles (n = 4-6) in aprotic solvents (acetonitrile and DMSO) and modified reaction conditions allowing for the synthesis and isolation of the hitherto missing dodecafluorocalix[6]pyrrole from the condensation of 3,4-difluoro-1H-pyrrole and acetone are described. In acetonitrile solution containing 2% water, the association constants for the 1:1 binding interaction between octafluorocalix[4]pyrrole and chloride anion obtained with isothermal titration calorimetry (ITC) and (1)H NMR titration methods were found to match reasonably well. As compared to its nonfluorinated congener, octafluorocalix[4]pyrrole was found to display enhanced binding affinities for several representative anions in pure acetonitrile as judged from ITC analyses. Similar analyses of the fluorinated calix[n]pyrroles revealed an increase in the relative affinity for bromide over chloride with increasing macrocycle size, as manifest in a decrease in the binding ratio K(a(Cl))/K(a(Br)). Anion binding studies in the solid state, involving single-crystal X-ray diffraction analyses of the chloride and acetate anion complexes of octafluorocalix[4]pyrrole and decafluorocalix[5]pyrrole, respectively, confirmed the expected hydrogen bond interactions between the pyrrolic NH protons and the bound anions.     

Proteomics-Based Discovery of First-in-Class Chemical Probes for Programmed Cell Death Protein 2 (PDCD2)

Chemical probes are essential tools for understanding biological systems and for credentialing potential biomedical targets. Programmed cell death 2 (PDCD2) is a member of the B-cell lymphoma 2 (Bcl-2) family of proteins, which are critical regulators of apoptosis. Here we report the discovery and characterization of 10 e , a first-in-class small molecule degrader of PDCD2. We discovered this PDCD2 degrader by serendipity using a chemical proteomics approach, in contrast to the conventional approach for making bivalent degraders starting from a known binding ligand targeting the protein of interest. Using 10 e as a pharmacological probe, we demonstrate that PDCD2 functions as a critical regulator of cell growth by modulating the progression of the cell cycle in T lymphoblasts. Our work provides a useful pharmacological probe for investigating PDCD2 function and highlights the use of chemical proteomics to discover selective small molecule degraders of unanticipated targets.