Yoshiko Kita

PROTEIN-SOLVENT INTERACTIONS IN PHARMACEUTICAL FORMULATIONS

The stability of proteins is affected by a variety of solvent additives. Sugars, certain amino acids and salts, and polyhydric alcohols stabilize proteins in solution and during freeze-thawing. Urea and guanidine hydrochloride destabilize proteins under either condition. These effects can be explained from the preferential interactions of the cosolvents with the proteins; i.e., the protein stabilizers are preferentially excluded from the proteins, while the destabilizers bind to them. There is a class of compounds, such as polyethylene glycol and 2-methyl-2,4-pentanediol, that destabilize proteins at high temperature but stabilize them during freeze-thawing. Such effects can be accounted for by their preferential exclusion from the native proteins determined at room temperature and from their hydrophobic character, which depends on temperature. During freeze-drying, only a few sugars appear to be effective in protecting proteins from inactivation, as most other stabilizers cannot exert their action on proteins without water. The stabilization is due to hydrogen bonding between the sugars and the dried proteins, the sugars acting as water substitute. Understanding the mechanism of the effects of solvent additives on the protein stability should aid in the development of a suitable formulation for protein.

The basis for toxicity of certain cryoprotectants : a hypothesis

Abstract In this review we offer an explanation for the means by which cryoprotectants stabilize proteins during freezing and destabilize them at physiological temperatures. Dimethylsulfoxide, polyethylene glycol, 2-methyl-2,4-pentanediol, ethanol, and ethylene glycol are preferentially excluded from the hydration shell of the proteins at low temperatures. Thermodynamic analyses reviewed here suggest that this exclusion leads to stabilization. In contrast, we suggest that at higher temperatures these cosolvents; interact hydrophobically with proteins and thus act as protein destabilizers or denaturants. The apparent paradox that compounds such as dimethylsulfoxide are effective cryoprotectants but can be toxic to cells at higher temperatures may be ascribed to the dual temperature-dependent modes of interaction of these molecules with proteins.

NDF/heregulin stimulates the phosphorylation of Her3/erbB3

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193–9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905–4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473–475; (1993) J. Biol. Chem. 268, 18407–18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3‐transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125I‐labeled NDF and then chemically crosslinked. Immunoprecipitation with anti‐Her3 revealed a dense high M W band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero‐oligomerization.

Hydrophobic interaction chromatography in alkaline pH

Hydrophobic interaction chromatography is a very powerful protein purification technique which is dependent on strong salting-out salts to increase the hydrophobic interactions between the protein and the ligand. Ammonium sulfate is the salt most commonly used for this purpose, but it cannot be used at very alkaline pH. Monosodium glutamate was therefore tested as a salt for hydrophobic interaction chromatography at pH 9.5. When ribonuclease A, ovalbumin, and beta-lactoglobulin were individually applied to a phenyl superose column in 2 M monosodium glutamate, all three proteins bound to the column and could be subsequently eluted by decreasing the salt concentration. Using this salt, it was possible to separate commercially obtained beta-lactoglobulin into authentic protein and contaminants and to purify the individual proteins from a mixture of ovalbumin and beta-lactoglobulin. These results demonstrate that monosodium glutamate is a useful salt for hydrophobic interaction chromatography. Guanidine and sodium sulfate and sodium aspartate were also examined at the same pH, demonstrating that they also resulted in the binding and elution of the proteins examined.

Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.

A light scattering/size exclusion chromatography method for studying the stoichiometry of a protein-protein complex

Publisher Summary This chapter discusses the light scattering/size exclusion chromatography method to study the stoichiometry of a protein–protein complex. Interactions of a protein with itself or other macromolecules play important roles in diverse biological processes. These interactions are characterized by their affinity and stoichiometry. The size exclusion chromatography (SEC) is used to estimate the molecular weight of a complex and therefore determine the stoichiometry. During the formation of a complex, the binding of an additional protein may not affect the overall hydrodynamic size, or conversely, it may change the carbohydrates conformation that influences the elution position. The molecular weight from a light scattering measurement is independent of the elution position of a protein or complex, and reflects only the polypeptide if the extinction coefficient of the polypeptide alone is used in the analysis. These characteristics make the combination of light scattering with SEC an easy, accurate, and reliable technique. The chapter focuses on the determination of the stoichiometry of complexes containing at least one glycosylated protein.