In this study, we report the use of ionic liquids as modifiers in the separation of achiral and chiral analytes in micellar electrokinetic chromatography. In this investigation, polymeric surfactants and ionic liquids were added to a low-conducting buffer solution. The polymeric surfactants used in this study were poly(sodium N-undecylelinic sulfate) and poly(sodium oleyl-l-leucylvalinate). The ionic liquids used in this study were chosen because of their high conductivity, hydrophobicity, and good solvating properties. Thus, it was expected that these ionic liquids would have the ability to assist in the separation of hydrophobic mixtures while maintaining adequate background current. Three analyte mixtures were separated using various buffer combinations of polymeric surfactant and ionic liquids. The ionic liquids were shown to improve the resolution and peak efficiency of the analytes while maintaining adequate background current.
Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The K M of sphingosine kinase for its fluorescently labeled substrate was 38 ± 18 μM with a V max of 0.4 ± 0.2 μM/min and a k cat of 3900 s −1 . Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration. [1][2][3][4][5][6] These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die. 7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis. 4,[7][8][9] S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2. 6,[9][10][11] In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine. 6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies. 7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases. 10,[15][16][17][18] Thus, sphingosine signaling is proving to be extremely important in clinical medicine. [17][18][19][20][21] *Corresponding authors. Phone: 919-966-2291 (C.E.S. and N.L.A.). Fax: 919-962-2388 (C.E.S. and N.L.A.). cesims@unc.edu (C.E.S.); nlallbri@unc.edu (N.L.A. Although S1P plays a major role as an extracellular signaling molecule, it is predominately syn...
Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer-ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5'-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (ΔG(unfold)) between these two aptamers yields a ΔG of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (K(d)) of 2.0 ± 0.2 μM, a value consistent with the K(d) obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 μM) and close to that determined by affinity chromatography in the literature (6 ± 3 μM). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing.
Phosphatidyl inositol 4,5-bisphosphate (PIP2) and phosphatidyl inositol 3,4,5-trisphosphate (PIP3) labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) on the acyl chain or a phosphatidyl ethanolamine head group were separated by CE with LIF detection. Several methods and capillary-coating procedures were tested for the separation of these phosphatidyl inositol phosphates (PIPs) at 20 degrees C. Separation of the PIPs in less than 20 min with excellent resolution was achieved using a buffer containing sodium deoxycholate (SDC), 1-propanol, MgCl2 and the polymer coating reagent, EOTrol LR. The efficiency of the optimized method was as high as 1.3x10(5) plates. The dependence of the separation on the concentration of 1-propanol, SDC, and MgCl2 was determined. The separation of PIP2 and PIP3 was primarily due to differential binding of the lipids to Mg2+ rather than to different solubilities in the micellar phase. The role of the SDC was to prevent adsorption of the hydrophobic lipids to the capillary wall and thus enhance the efficiency. The fluorescent PIPs are of value for both in vitro and in vivo assays of phospholipid metabolism. In particular, the use of these lipids with the optimized capillary-based separation will be of utility for drug screening as well as cell-based assays.
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