Additivity of the Specific Effects of Additives on Protein Phase Behavior

Abstract

Protein phase behavior and protein-protein interactions can be tuned by additives. We experimentally determined the phase behavior of lysozyme solutions, namely, the cloud-point temperature (CPT), in the presence of two additives, sodium chloride (NaCl) and guanidine hydrochloride (GuHCl). Their concentrations are chosen to maintain the secondary structure, as verified by CD spectroscopy. Our data indicate that the salts affect the CPT through electrostatic screening and salt-specific contributions. At high salt concentrations, the CPT is a linear function of the additive concentration for the salts NaCl and GuHCl as well as for a nonionic additive, glycerol, and a solvent, dimethyl sulfoxide (DMSO). Their molar temperature increments, which rank their specific effects on the CPT (NaCl > 0 > DMSO > glycerol > GuHCl), are found to be essentially independent of the protein concentration. In particular, the specific effects of NaCl and GuHCl in mixtures are found to be additive, indicating the absence of synergies or suppressions between both salts. Thus, molar temperature increments represent a characteristic measure for the specific effects of additives on protein interactions, which is easily accessible in lab experiments and which will help to characterize the effects of additives on protein interactions and phase behavior.

The crystallization enthalpy and entropy of protein solutions: microcalorimetry, van't Hoff determination and linearized Poisson–Boltzmann model of tetragonal lysozyme crystals

“…The final protein concentration (typically 65 mg mL À1 ) was determined by UV-vis absorption spectroscopy and/or refractometry. 65 For some experiments, higher protein concentrations (up to 150 mg mL À1 ) were needed, which were achieved by ultrafiltration, as described previously. 65 Samples were prepared by mixing appropriate amounts of lysozyme, buffer and salt stock solutions.…”

“…65 For some experiments, higher protein concentrations (up to 150 mg mL À1 ) were needed, which were achieved by ultrafiltration, as described previously. 65 Samples were prepared by mixing appropriate amounts of lysozyme, buffer and salt stock solutions. Sample preparation was conducted at room temperature (21 AE 2) 1C, above the solution cloud-points.…”

“…The state diagram contains data on solubility (black filled circles 36 ) and metastable liquid-liquid phase separation (LLPS, gray filled squares), where the latter have been extrapolated from cloud-point measurements at fixed protein or salt concentration. 65,67 corresponding to supersaturations c p /c eq \ 10. Experiments with smaller c s or c p than indicated were not successful.…”

“…Fig.1State diagram of lysozyme solutions at pH 4.5 and 20.0 1C in the salt concentration c s vs. molar protein concentration c p (or, equivalently, mass protein concentration r p ) plane: solubility (black filled circles36 ) and cloud-point data (gray filled squares65,67 ). Lines are guides to the eye.…”

Adsorption isotherms of charged nanoparticles

“…It is well known that salt can modify significantly the interaction between biomolecules in aqueous suspensions, affecting their stability [1][2][3][4][5][6][7][8][9][10][11]. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory [12] attributes the stability of suspensions to the competition between electrostatic and dispersive, van der Waals (vdW), forces.…”