부산대학교 도서관

Purpose:Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions.Methods:The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.7–5.0), salt concentration (0–400 mM), and ion identity using a series of monovalent, Hofmeister anions (F−, Cl−, Br−, I−, ClO4−, SCN−) and cations (Li+, Na+, K+, Rb+, Cs+). The effects of salt on conformational stability and reduced valence were determined using Fourier-transform infrared spectroscopy, circular dichroism, and capillary electrophoresis/analytical ultracentrifugation.Results:PbA precipitation occurred upon salt addition and could be modulated with solution pH, salt identity & concentration. The precipitation was sensitive to anions, but not cations, and increased with anion size. A reverse Hofmeister effect (SCN−∼ClO4−>I−>Cl−>Br−>F−) was observed with “salting-in” anions being the more effective precipitants. An increase in the precipitation rate below pH 4.3 indicated that protonation of aspartyl and glutamyl side-chains was also important for precipitation. The reversibility of precipitation was excellent (100%) at 4°C but decreased upon storage at 25°C and 37°C; the loss in reversibility correlated with an increase in intermolecular β-sheet content of the precipitate.Conclusion:Salts, employed as buffering, tonicifying, and viscosity modifying agents, may adversely affect the solubility of basic proteins formulated under acidic conditions.