The systematic characterization by aqueous column chromatography of solutes which affect protein stability.
We have systematically characterized, by aqueous column chromatography on a size exclusion cross-linked dextran gel (Sephadex G-10), 12 solutes, 11 of which are known to affect protein stability. Six are chaotropes (water structure breakers) and destabilize proteins, while five are polar kosmotropes (polar water structure makers) and stabilize proteins. Analysis of the chromatographic behavior of these neutral (ethylene glycol, urea), positively charged (Tris, guanidine, as the hydrochloride salts) and negatively charged (SO2-4, HPO2-4, F-, Cl-, Br-, Cl3CCO-2, I-, SCN-, as the sodium salts, in order of elution) solutes at pH 7 as a function of sample concentration (up to 0.6 M), supporting electrolyte, and temperature yields four conclusions, based largely on the behavior of the anions. Chaotropes adsorb to the gel according to their position in the Hofmeister series, with the most chaotropic species adsorbing most strongly. ++Chaotropes adsorb to the gel less strongly in the presence of chaotropes (a salting in effect) and more strongly in the presence of polar kosmotropes (a salting out effect). Polar kosmotropes do not adsorb to the gel, and are sieved through the gel according to their position in the Hofmeister series, with the most kosmotropic species having the largest relative hydrodynamic radii. The hydrodynamic radii of polar kosmotropes is increased by chaotropes and decreased by polar kosmotropes. These results suggest that a chaotrope interacts with the first layer of immediately adjacent water molecules somewhat less strongly than would bulk water in its place; a polar kosmotrope, more strongly.
D000581:Amidohydrolases / Q000302:isolation & purification*; D000838:Anions; D002412:Cations; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel / Q000379:methods; D004080:Dihydroorotase / Q000302:isolation & purification* / Q000378:metabolism; D004573:Electrolytes; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D012996:Solutions; D013696:Temperature; D014867:Water
D000838:Anions; D002412:Cations; D004573:Electrolytes; D012996:Solutions; D014867:Water; D000581:Amidohydrolases; D004080:Dihydroorotase