Interaction of ribonuclease A with aqueous 2-methyl-2,4-pentanediol at pH 5.8

The solubilities of lysozyme, α‐chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25°C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting‐out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for α‐chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient κe to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus κe is a constant. For α‐chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient κe is independent of the initial protein concentration. However, for both lysozyme and α‐chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior. © 1992 John Wiley & Sons, Inc.

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“…Abundant experimental results for ~3 for various salts and proteins have been reported ( 1,2,3,4,6,7,8,18,30,31,32,37 …”

Section: The Theoretical Basis Of This Equation Is Not Clearmentioning

confidence: 99%

Some characteristics of protein precipitation by salts

Shih

Prausnitz

Blanch

1992

Biotech & Bioengineering

150

114

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“…P olyhydric alcohols and polyethylene glycols are commonly used as reagents to crystallize proteins. Cosolvents and precipitants of this type are known to promote the preferential hydration of proteins, creating a thermodynamic disequilibrium that can result in precipitation (1,2) or, in favorable circumstances, crystallization. It is also well established, although not widely appreciated, that the same cosolvents can destabilize the tertiary structures of proteins (3).…”

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confidence: 99%

Cosolvent-induced transformation of a death domain tertiary structure

Xiao¹,

Gardner

Sprang

2002

Proc. Natl. Acad. Sci. U.S.A.

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The death domain (DD) of the protein kinase Pelle adopts a six-helix bundle fold in the crystal structure of the complex with its dimerization partner, Tube-DD. However, in crystals obtained from a solution of 45% 2-methyl-2,4-pentanediol (MPD), the C-terminal half of Pelle-DD folds into a single helix, and the N-terminal half of the molecule is disordered. The helical segment forms an antiparallel dimer with the corresponding helix of a symmetry-related molecule, and together they form extensive lattice interactions similar in number, composition, and buried surface to those in the six-helix bundle of the native fold. Secondary structure analysis by heteronuclear nuclear magnetic resonance spectroscopy (NMR) demonstrates that Pelle-DD adopts a six-helix bundle fold in aqueous solution. The fold is perturbed by MPD, with the largest chemical shift changes in one helix and two loop regions that encompass the Tube-DD binding site. Pelle-DD is stable to urea denaturation with a folding free energy of 7.9 kcal͞mol at 25°C but is destabilized, with loss of urea binding sites, in the presence of MPD. The data are consistent with a cosolvent denaturation model in which MPD denatures the N terminus of Pelle-DD but induces the C terminus to form a more compact structure and aggregate. A similar perturbation in vivo might occur at the plasma membrane and could have consequences for Pelle-mediated regulation. Generally, crystallographers should be aware that high concentrations of MPD or related cosolvents can alter the tertiary structure of susceptible proteins.

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“…) and of having a good affinity for nonpolar residues (Pittz & Bello, 1971;Arakawa et al, 1990b). The preferential exclusion of MPD is believed to be due to repulsion from charges on the surface of globular proteins (Pittz & Timasheff, 1978).…”

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confidence: 99%

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols

1992

Self Cite

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The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and 6-lactoglobulin with polyethylene glycols (PEGS) of molecular weight 200-6,OOO have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein.The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by R, = a M 0 , 5 s . The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24,6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.

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Interaction of ribonuclease A with aqueous 2-methyl-2,4-pentanediol at pH 5.8

Cited by 137 publications

References 72 publications

Some characteristics of protein precipitation by salts

Some characteristics of protein precipitation by salts

Cosolvent-induced transformation of a death domain tertiary structure

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols

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