Liquid–liquid phase separation (LLPS) of proteins has recently been associated with the onset of numerous diseases. Despite several studies in this area of protein aggregation, buffer-specific effects always seem to be overlooked. In this study we investigated the influence of buffers on the phase stability of hen egg-white lysozyme (HEWL) and its respective protein–protein interactions by measuring the cloud point temperature, second virial coefficient, and interaction diffusion coefficient of several HEWL–buffer solutions (MOPS, phosphate, HEPES, cacodylate) at pH 7.0. The results indicate that the buffer molecules, depending on their hydration, adsorb on the protein surface, and modulate their electrostatic stability. The obtained information was used to extend the recently developed coarse-grained protein model to incorporate buffer-specific effects. Treated by Wertheim’s perturbation theory the model qualitatively correctly predicted the experimentally observed phase separation of all investigated HEWL–buffer solutions, and further allowed us to predict the phase stability of protein formulations even in experimentally unattainable conditions. Since the theory can be straightforwardly extended to include multiple components it presents a useful tool to study protein aggregation in crowded cell-like systems.

Amyloid fibrils, highly ordered protein aggregates, play an important role in the onset of several neurological disorders. Many studies have assessed amyloid fibril formation under specific solution conditions, but they all lack an important phenomena in biological solutions—buffer specific effects. We have focused on the formation of hen egg-white lysozyme (HEWL) fibrils in aqueous solutions of different buffers in both acidic and basic pH range. By means of UV-Vis spectroscopy, fluorescence measurements and CD spectroscopy, we have managed to show that fibrillization of HEWL is affected by buffer identity (glycine, TRIS, phosphate, KCl-HCl, cacodylate, HEPES, acetate), solution pH, sample incubation (agitated vs. static) and added excipients (NaCl and PEG). HEWL only forms amyloid fibrils at pH = 2.0 under agitated conditions in glycine and KCl-HCl buffers of high enough ionic strength. Phosphate buffer on the other hand stabilizes the HEWL molecules. Similar stabilization effect was achieved by addition of PEG12000 molecules to the solution.

Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable...

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules “connect” through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient.

Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.

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We present here a freely available web-based database, called BioMThermDB 1.0, of thermophysical and dynamic properties of various proteins and their aqueous solutions. It contains the hydrodynamic radius, electrophoretic mobility, zeta potential, self-diffusion coefficient, solution viscosity, and cloud-point temperature, as well as the conditions for those determinations and details of the experimental method. It can facilitate the meta-analysis and visualization of data, can enable comparisons, and may be useful for comparing theoretical model predictions with experiments.

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