Adsorption of charged macromolecules at a gold electrode

Using an optical reflectometer with impinging-jet system, the adsorption from aqueous solution onto gold of three charged macromolecules has been studied: the strong linear-chain polyelectrolyte polyvinyl pyridine (PVP(+)), the fifth-generation poly(propylene imine) dendrimer DAB-64, which has a pH-dependent charge and a relatively fixed shape, and the protein lysozyme, of which both the charge and the structure-stability are dependent on solution composition. Experimental conditions that have been varied include the adsorbate concentration, electrolyte concentration, pH, and externally applied potential across the gold/solution interface. Making use of the earlier established dependency of the double layer potential of the gold substrate on solution conditions and externally applied potential, the results of measurements as a function of pH and as a function of external potential control are compared. The total set of results enables us to draw conclusions with respect to the relative importance of electrostatic interactions for the adsorption process. PVP(+) adsorption follows the electric potential of the gold/solution interface and is further determined by a rather strong nonelectrostatic affinity between segments and surface. The adsorption behavior of DAB-64 is not quite understood, but electrostatic interactions with the gold surface seem to play a minor role. For lysozyme, surface-induced conformational changes dominate the adsorption process. The extent of spreading of the molecules decreases with increasing polarity of the surface, resulting in a minimum in adsorbed amount around the point of zero potential of the gold.     

Determination of the interchangeable heavy-metal fraction in soils by isotope dilution mass spectrometry

An isotope dilution technique using enriched stable isotopes is applied to determine the interchangeable heavy-metal fraction in soils. Metals in two soil samples are extracted at constant pH, with water, NH₄NO₃, and EDTA. A spike of enriched stable isotopes is added to the suspension of sample and eluant at the beginning of the extraction. The heavy-metal fraction which exchanges with the added spike during the extraction is called the interchangeable fraction. The extractable heavy-metal fractions are obtained from the heavy-metal concentrations in the eluates. Isotope ratios and concentrations are determined by HR-ICP-MS. The isotope dilution technique described enables both the extractable and the interchangeable heavy-metal fractions to be determined in the same experiment. The combination of both results gives additional information on elemental availability under different conditions that cannot be obtained by analyzing the extractable heavy-metal fractions alone. It is demonstrated that in some cases different eluants just shift the distribution of the interchangeable fraction of an element between the solid and liquid phases (e.g., Pb and Cd in a topsoil sample) while the amount of the interchangeable fraction itself remains constant. For other elements, as Ni, Zn, and Cr, the use of different eluants (different pH, complexing agents) sometimes enlarges the interchangeable fraction. Received: 8 December 1998 / Revised: 30 June 1999 / Accepted: 2 July 1999     

Kaon interferometry in heavy-ion collisions at the CERN SPS

K⁺K⁺ and K^–K^– correlations from S+Pb collisions at 200 GeV/c per nucleon and K⁺K⁺ correlations from p+Pb collisions at 450 GeV/c per nucleon, are presented as measured by the focusing spectrometer of the NA44 experiment at CERN. Multidimensional fits are performed in order to characterize the kaon-emission volume, which is found to be smaller than the pion-emission volume.     

Synthesis of functionalised nucleosides for incorporation into nucleic acid-based serine protease mimics

The synthesis of nucleosides modified with an extra imidazole, carboxyl and hydroxyl group is described. These nucleosides can be incorporated into an oligonucleotide duplex, thus generating a novel type of serine protease mimic.     

Presence of thiamine pyrophosphate in mammalian peroxisomes

Background

Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the α-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.

Results

Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 ± 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.

Conclusion

Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.     

Stabilization of protein-loaded starch microgel by polyelectrolytes

The interaction of biocompatible polyelectrolytes (chargeable poly(amino acids)) with oxidized starch microgel particles has been studied. The aim was to form a polyelectrolyte complex layer around the outer shell of microgel particles filled with functional ingredients to slow down the release of the ingredients from the gel and make this process less sensitive to salt. First, the distribution of positively charged poly(l-lysine) (PLL) of two different molecular weights ("small", 15-30 kDa, and "large", 30-70 kDa) in the negatively charged gel particles was measured. The small PLL distributes homogeneously throughout the gel particles, but the large PLL forms a shell; i.e., its concentration at the outer layer of the particles was found to be much higher than in their core. This shell formation does not occur at a relatively high salt concentration (0.07 M). The large PLL was selected for further study. It was found that upon addition of PLL to lysozyme-loaded gel particles the protein is exchanged by PLL. The exchange rate increases with increasing pH, in line with the increasing electrostatic attraction between the gel and the polyelectrolyte. Therefore, it was decided to use also a negatively charged poly(amino acid), poly(L-glutamic acid) (PGA), to form together with PLL a stable polyelectrolyte complex shell around the gel particles. This approach turned out to be successful, and the PLL/PGA complex layer effectively slows down the release of lysozyme from the microgel particles at 0.05 M salt. In addition, it was found that the PLL/PGA layer protects the gel particle from degradation by α-amylase.     

The electrical double layer on gold probed by electrokinetic and surface force measurements

Gold surfaces, obtained by vacuum deposition of 15-nm gold films on glass and silica wafers, were studied in aqueous solutions by streaming potential measurements and colloidal-probe AFM force measurements. In the force measurements both a bare and a gold-coated silica particle (6 microm in diameter) have been used as colloidal probes. From the streaming potential measurements we determined the zeta-potential of the gold surface, while from the force measurements the diffuse double-layer potential psi(d) was obtained by fitting the data to the DLVO theory or to the nonlinear Poisson-Boltzmann equation. Measured interactions were found to be entirely due to overlap of electric double layers with no indication of attractive Van der Waals forces. Results of both types of measurements are in good agreement. The double layer potential strongly depends on the pH, probably as a result of the presence of oxide species on the gold surface. Insight in the double layer potential of polarizable interfaces such as the gold/electrolyte solution interface is the first step for understanding the effect of externally applied potentials on the adsorption behavior of charged species.     

Effectiveness of Bioactive Compound as Antibacterial and Anti-Quorum Sensing Agent from Myrmecodia pendans: An In Silico Study

Background: antibiotic resistance encourages the development of new therapies, or the discovery of novel antibacterial agents. Previous research revealed that Myrmecodia pendans (Sarang Semut) contain potential antibacterial agents. However, specific proteins inhibited by them have not yet been identified as either proteins targeted by antibiotics or proteins that have a role in the quorum-sensing system. This study aims to investigate and predict the action mode of antibacterial compounds with specific proteins by following the molecular docking approach. Methods: butein (1), biflavonoid (2), 3″-methoxyepicatechin-3-O-epicatechin (3), 2-dodecyl-4-hydroxylbenzaldehyde (4), 2-dodecyl-4-hydroxylbenzaldehyde (5), pomolic acid (6), betulin (7), and sitosterol-(6′-O-tridecanoil)-3-O-β-D-glucopyranoside (8) from M. pendans act as the ligand. Antibiotics or substrates in each protein were used as a positive control. To screen the bioactivity of compounds, ligands were analyzed by Prediction of Activity Spectra for Substances (PASS) program. They were docked with 12 proteins by AutoDock Vina in the PyRx 0.8 software application. Those proteins are penicillin-binding protein (PBP), MurB, Sortase A (SrtA), deoxyribonucleic acid (DNA) gyrase, ribonucleic acid (RNA) polymerase, ribosomal protein, Cytolysin M (ClyM), FsrB, gelatinase binding-activating pheromone (GBAP), and PgrX retrieved from UniProt. The docking results were analyzed by the ProteinsPlus and Discovery Studio software applications. Results: most compounds have Pa value over 0.5 against proteins in the cell wall. In nearly all proteins, biflavonoid (2) has the strongest binding affinity. However, compound 2 binds only three residues, so that 2 is the non-competitive inhibitor. Conclusion: compound 2 can be a lead compound for an antibacterial agent in each pathway.