Stability of Helix-Rich Proteins at High Concentrations

Abstract

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.

Temperature scanning FTIR analysis of secondary structures of proteins embedded in amorphous sugar matrix

“…Therefore, the impact of a wide range of temperature change to the conformational change of protein has been extensively investigated. Circular dichroism (CD) spectroscopy [3][4][5] and differential scanning calorimetry (DSC) [5][6][7][8] of protein solutions enable in situ analysis of conformational changes and denaturation induced by alterations in temperature. Secondary structures present at different temperatures can be measured using a temperature-controlled CD spectrometer.…”

“…[5][6][7][8] However, protein-based pharmaceuticals often exist in solid, dried matrices formed by stabilizers and excipients and are subjected to high temperature in some cases, which includes spray drying. Proteins in aqueous solution are dried to prolong shelf life and improve handling properties.…”

Self-crowding induced phase separation in protein dispersions

“…(5), and f Conf by Eq. (6). Note that for κ = 1, we retrieve the dimensionless free energy density for a monodisperse Carnahan-Starling model.…”

“…This includes changes in physico-chemical solution conditions, 1 binding to other proteins, [2][3][4][5] and competition for water at elevated protein concentrations. 6 The response of the structure of protein molecules to such changes in local environment can affect the way they interact with other proteins in the solution, e.g., because their effective volume, which includes tightly bound hydration water, changes. This implies that in crowded environments, where the free volume is limited due to the presence of a high concentration of macromolecules, such as is the case in the cell, 7,8 interactions between proteins may be different from that in a dilute solution.…”

“…9 Crowding stabilises the compact (native) protein conformation for the same reason. 6,[14][15][16] How precisely crowding influences protein conformation and how this in turn influences the phase and aggregation behaviour remain rather poorly understood, despite a considerable research effort in both fields. 9,17 This is remarkable because applications in the food 18 and pharmaceutics industry 19 often involve dense protein formulations that are difficult to process and that exhibit undesirable processes such as syneresis, i.e., the expulsion of water.…”