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Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-␤ (A␤)peptide. Elucidating the underlying Cu(II)-induced A␤ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of A␤ 1-40 using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):A␤ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of A␤, because three different kinetic scenarios were observed depending on the Cu(II):A␤ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):A␤ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):A␤ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with A␤ 1-42 were compatible with the conclusions for A␤ 1-40 even though the low solubility of A␤ 1-42 precluded examination under the same conditions as for the A␤ 1-40 . Experiments with A␤ 1-16 and A␤ 1-28 showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced A␤ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of A␤ 1-40 depending on the Cu(II):A␤ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.Extracellular cerebral plaques composed mainly of amyloid ␤-peptide (A␤) 1-40 and 1-42 fibrils are a histopathological hallmark of Alzheimer disease (AD) 3 (1, 2). These plaques contain elevated levels of metals, in particular zinc and copper (3). Also, it has been shown that these metal ions can promote the aggregation of A␤ in vitro (4, 5). This implies a key role of the metal ions in the A␤-mediated pathology of AD (6), although the subject is still under much debate (7,8), and a role of amyloid-independent pathways in AD neurodegeneration have recently been reviewed (9).Specifically regarding the role of metal ions in the amyloidmediated pathology of AD, it is believed that Zn(II) acts as a neuroprotector (10, 11), whereas Cu(II) is considered to mediate neurotoxicity (12)(13)(14). The latter effect is thought to occur through early stage soluble A␤ and A␤-Cu oligomeric intermediates (15-18) that may be involved in the formation of reactive oxygen species (6). It was discovered that Cu(II) may be released postsynaptically at glutamatergic synapses in hippocampus (19,20), the site for initial A␤ deposition in AD. This provides an explanation for how Cu(II) can interact with A␤. Seemingly in contrast to the support for the neurotoxic effects of Cu(II),...

The understanding of protein adsorption at charged surfaces is important for a wide range of scientific disciplines including surface engineering, separation sciences and pharmaceutical sciences. Compared to chemical entities having a permanent charge, the adsorption of small ampholytes and proteins is more complicated as the pH near a charged surface can be significantly different from the value in bulk solution. In this work, we have developed a phenomenological adsorption model which takes into account the combined role of interfacial ion distribution, interfacial charge regulation of amino acids in the proximity of the surface, electroneutrality, and mass balance. The model is straightforward to apply to a given set of experimental conditions as most model parameters are obtained from bulk properties and therefore easy to estimate or are directly measurable. The model provides a detailed understanding of the importance of surface charge on adsorption and in particular of how changes in surface charge, concentration, and surface area may affect adsorption behavior. The model is successfully used to explain the experimental adsorption behavior of the two model proteins lysozyme and α-lactalbumin. It is demonstrated that it is possible to predict the pH and surface charge dependent adsorption behavior from experimental or theoretical estimates of a preferred orientation of a protein at a solid charged interface.

Capillary electrophoresis frontal analysis: Principles and applications for the study of drug-plasma protein bindingCapillary electrophoresis is a well-established technique for the study of noncovalent interactions. Various approaches exist and capillary electrophoresis-frontal analysis provides an interesting alternative to the migration shift affinity capillary electrophoresis methods and conventional methods. The present work reviews the principles on which the frontal analysis method is founded. Advantages and limitations of capillary electrophoresis frontal analysis in comparison with both conventional and other capillary electrophoresis based methods for quantification of binding interactions are discussed. Investigations utilizing capillary electrophoresis-frontal analysis have focused on the interaction of drugs with plasma proteins. These studies, primarily addressing the binding of drugs to human serum albumin, a 1 -acid glycoprotein, and lipoproteins are reviewed together with some recent developments in capillary electrophoresisfrontal analysis methodology.

The interaction between an intact protein and two lipophilic ions at an oil-water interface has been investigated using cyclic voltammetry, impedance based techniques and a newly developed method in which the biphasic oil-water system is analyzed by biphasic electrospray ionization mass spectrometry (BESI-MS), using a dualchannel electrospray emitter. It is found that the protein forms interfacial complexes with the lipophilic ions and that it specifically requires the presence of the oil-water interface to be formed under the experimental conditions. Furthermore, impedance based techniques and BESI-MS with a common ion to polarize the interface indicated that the Galvani potential difference across the oil-water interface significantly influences the interfacial complexation degree. The ability to investigate protein-ligand complexes formed at polarized liquid-liquid interfaces is thus a new analytical method for assessing potential dependent interfacial complexation using a structure elucidating detection principle.The behavior of biological macromolecules at liquid-liquid interfaces has important implications in the pharmaceutical sciences. It is for example well-known that proteins adsorb at a wide variety of interfaces, 1-4 a process that can have unwanted consequences such as a loss of therapeutic activity due to denaturation and aggregation. 4,5 Two-phase systems are at equilibrium generally associated with an interfacial potential difference, 6 in the following referred to as the Galvani potential difference between a water (w) and oil (o) phase at equilibrium; ∆ o w φ. The value of ∆ o w φ can influence adsorption kinetics and/ or adsorption isotherms. 7 Electrochemistry at Interfaces between two immiscible electrolyte solutions (ITIES) provides an effective methodology for studying potential dependent adsorption and ion transfer processes. [8][9][10][11][12] In relation to protein research, electrochemistry at ITIES have been used to study protein adsorption, 13 protein adsorption kinetics, 7 the effects on transfer of aqueous ions, 14 and the assisted transfer of proteins to the oil phase. [15][16][17][18] Recently, electrochemistry at ITIES has been used for detecting proteins in solution by means of a protein assisted transfer of organic anions into the water phase at positive potentials. [19][20][21][22] A suggested mechanism for the organic anion transfer involves the formation of a protein-anion complex at the interface. 22 Usually electrospray ionization mass spectrometry (ESI-MS) involves an aqueous solution which by application of a high voltage is sprayed into a mass analyzer. Recently, the development of a new biphasic electrospray interface has allowed interfacial complexes in two phase systems to be analyzed using a mass spectrometer; the new technique is termed biphasic electrospray mass spectrometry (BESI-MS). [23][24][25][26] The combined use of BESI-MS and electrochemistry at ITIES is attractive as it allows studying quantitative mechanistic aspects of adsorption and ion transfer pro...