Effect of polyols on polyethylene glycol (PEG)-induced precipitation of proteins: Impact on solubility, stability and conformation.

Effect of polyols on the solubility of bovine serum albumin (BSA) in the presence of polyethylene glycols (PEGs) was investigated in order to strengthen the understanding of the observed effects of polyols and PEGs on protein properties in solution. Effect of polyols and/or PEGs on the thermodynamic (conformational) stability of BSA was measured using DSC and circular dichroism (CD). Glucose, sucrose, raffinose, glycerol and sorbitol, all reduced the extent of protein precipitation. Solubility of BSA in the presence of ethylene glycol increased in the case of PEG 1450 and PEG 8000, but was unaffected in the case of PEG 400. DSC studies indicated that smaller PEGs have destabilizing influence on protein structure. CD studies showed that smaller PEGs (ethylene glycol) induce subtle unfolding while stabilizing polyols induce subtle compaction. Results show that, effect of polyols on the apparent solubility of the protein correlates with their effect on the thermodynamic stability of the protein, smaller PEGs are not appropriate for estimating the activity of proteins in saturated solutions, and subtle changes in protein conformation can significantly affect protein precipitation. Though smaller PEGs have weak attractive interactions with protein molecules, perturbation of protein structure by PEGs can be balanced by utilizing appropriate stabilizing solutes.

[1]  B. Chang,et al.  Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: role in restricted conformational mobility and compaction of native state. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. N. Timasheff,et al.  Mechanism of the stabilization of ribonuclease a by sorbitol: Preferential hydration is greater for the denatured than for the native protein , 1997, Protein science : a publication of the Protein Society.

[3]  N. Choulis,et al.  A controlled release matrix using a mixture of hydrophilic and hydrophobic polymers , 1991 .

[4]  A. McPherson.  Use of polyethylene glycol in the crystallization of macromolecules. , 1985, Methods in Enzymology.

[5]  D. Fisher,et al.  PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimisation of coupling techniques. , 1998, International journal of hematology.

[6]  D. Davies,et al.  Protein crystallization: the growth of large-scale single crystals. , 1984, Methods in enzymology.

[7]  K. Gekko,et al.  Thermodynamic and kinetic examination of protein stabilization by glycerol. , 1981, Biochemistry.

[8]  D. Mcclements.  Modulation of Globular Protein Functionality by Weakly Interacting Cosolvents , 2002, Critical reviews in food science and nutrition.

[9]  D Oakenfull,et al.  Increased thermal stability of proteins in the presence of sugars and polyols. , 1979, Biochemistry.

[10]  K. Gekko,et al.  Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. , 1981, Biochemistry.

[11]  B. Farruggia,et al.  Thermal features of the bovine serum albumin unfolding by polyethylene glycols. , 1999, International journal of biological macromolecules.

[12]  T. Arakawa,et al.  Mechanism of poly(ethylene glycol) interaction with proteins. , 1985, Biochemistry.

[13]  J. Peris,et al.  Pharmacokinetics of the time-dependent elimination of all-trans-retinoic acid in rats , 2004, AAPS PharmSci.

[14]  J. Lee,et al.  The stabilization of proteins by sucrose. , 1981, The Journal of biological chemistry.

[15]  B. Farruggia,et al.  Destabilization of human serum albumin by polyethylene glycols studied by thermodynamical equilibrium and kinetic approaches. , 1997, International journal of biological macromolecules.

[16]  R. Bhat,et al.  Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols , 1992, Protein science : a publication of the Protein Society.

[17]  D. Atha,et al.  Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume. , 1981, The Journal of biological chemistry.

[18]  L. Paleg,et al.  Proline and glycine betaine influence protein solvation. , 1984, Plant physiology.

[19]  B. P. Naidu,et al.  Rationalization of the effects of compatible solutes on protein stability in terms of thermodynamic nonideality. , 1992, Archives of biochemistry and biophysics.

[20]  D. Kalonia,et al.  Polyethylene glycol-induced precipitation of interferon alpha-2a followed by vacuum drying: Development of a novel process for obtaining a dry, stable powder , 2004, AAPS PharmSci.