Search results for " concentration"
showing 10 items of 1684 documents
Amyloid Fibrils Formation of Concanavalin A at Basic pH
2011
Mechanisms of partial unfolding and aggregation of proteins are of extreme interest in view of the fact that several human pathologies are characterized by the formation and deposition of protein-insoluble material, mainly composed of amyloid fibrils. Here we report on an experimental study on the heat-induced aggregation mechanisms, at basic pH, of concanavalin A (ConA), used as a model system. Thioflavin T (ThT) fluorescence and multiangle light scattering allowed us to detect different intertwined steps in the formation of ConA aggregates. In particular, the ThT fluorescence increase, observed in the first phase of aggregation, reveals the formation of intermolecular β-sheet structure wh…
Kinetics of different processes in human insulin amyloid formation.
2007
Human insulin has long been known to form amyloid fibrils under given conditions. The molecular basis of insulin aggregation is relevant for modeling the amyloidogenesis process, which is involved in many pathologies, as well as for improving delivery systems, used for diabetes treatments. Insulin aggregation displays a wide variety of morphologies, from small oligomeric filaments to huge floccules, and therefore different specific processes are likely to be intertwined in the overall aggregation. In the present work, we studied the aggregation kinetics of human insulin at low pH and different temperatures and concentrations. The structure and the morphogenesis of aggregates on a wide range…
Thermodynamic versus Conformational Metastability in Fibril-Forming Lysozyme Solutions
2012
The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation is a crucial aspect for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions of lysozyme at acidic pH and low ionic strength. The amyloid formation occurs after a long lag time and is preceded by the formation of oligomers, which seems to be off-pathway with respect to fibrillation. By measuring the osmotic isothermal compressibility and the collective diffusion coefficient of lysozyme in solution, we observe that the monomeric solution is kept in a thermodynamically metastable state by strong electrosta…
Thioflavin T Hydroxylation at Basic pH and Its Effect on Amyloid Fibril Detection
2008
The fluorescent dye thioflavin T (ThT) is commonly used for in situ amyloid fibril detection. In this work, we focused on the spectroscopic properties and chemical stability of ThT in aqueous solution as a function of pH, temperature, and dye concentration. A reversible hydroxylation process occurs in alkaline solutions, which was characterized using a combination of UV-vis absorption spectroscopy, proton NMR, and density functional theory (DFT). On the basis of these studies, we propose a chemical structure for the hydroxylated form. Finally, by means of fluorescence spectroscopy, ThT hydroxylation effects on in situ amyloid detection have been investigated, providing new insights on the e…
The kinetic behavior of insulin fibrillation is determined by heterogeneous nucleation pathways
2005
When subjected to acidic conditions and high temperature, insulin is known to produce fibrils that display the common properties of disease amyloids. Thus, clarifying the mechanisms of insulin fibrillation can help the general understanding of amyloidal aggregation. Insulin fibrillation exhibits a very sharp time dependence, with a pronounced lag phase and subsequent explosive growth of amyloidal aggregates. Here we show that the initial stages of this process can be well described by exponential growth of the fibrillated proteins. This indicates that the process is mainly controlled by a secondary nucleation pathway.
Polysorbate 80 controls Morphology, structure and stability of human insulin Amyloid-Like spherulites
2022
AbstractAmyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics s…
High-pressure flow-injection assembly. Indirect determination of glycine by atomic absorption spectrometry.
1991
A procedure for the determination of glycine is described. The method is based on the reaction of the analyte with finely powdered, solid copper(II) carbonate in a continuous-flow assembly. The optimum experimental conditions of pH, temperature, sample volume, flow-rate, column length and internal diameter, and the linear range of calibration, were studied. Interference from foreign substances that accompany this amino acid in pharmaceutical formulations was studied, and the method was applied to the determination of glycine.
Spectrophotometric determination of promazine with an oxidative column in FIA manifolds
1992
Abstract A simple flow-injection spectrophotometric method for the determination of promazine is described. The two proposed procedures are based on the oxidation of analyte with a manganese dioxide column. Concentrations of promazine in the ranges 2–20 and 1–6 are determined with a relative standard deviation of 1.0%. The injection rates are 62 and 80 samples h −1 , respectively. The influence of foreign species and the determination of promazine in a pharmaceutical formulation are also reported.
Capillary electrokinetic fractionation mass spectrometry (CEkF/MS): Technology setup and application to metabolite fractionation from complex samples…
2014
Capillary electrokinetic fractionation (CEkF) is investigated as a new, simple, and robust approach for semipreparative and analytical sample analysis based on pKa -dependant pH-driven electrophoretic mobility. CEkF was optimized with contactless conductivity detection and conducted with 10 kV reverse voltage for 10 min, then coupled on/at-line to ESI/MS. We propose a semi-empirical model with 14 representative compounds based on the correlation between sample/medium pH regulating the partial charge, the electrokinetic loading of the capillary and intensity (I) of analytes. According to the model, an empirical function (I = f (pH)) could be derived to calculate the acid dissociation constan…
Noninvasive sampling of phenylalanine by reverse iontophoresis.
1999
While iontophoresis is typically associated with drug delivery across the skin, the symmetry of the technique permits its application to the essentially noninvasive withdrawal of biologically important analytes from the subcutaneous space to the body's surface. The identification of other substances which can be monitored by this procedure, and to its optimization and development as a more general clinical chemistry tool, is a long-term objective. In this paper, we describe a preliminary in vitro investigation into the feasibility of extracting and analyzing the amino acid, phenylalanine, with the ultimate aim to develop a diagnostic test for phenylketonuria, a potentially fatal metabolic d…