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The effect of solute ionization on the retention of weak acids, bases, and ampholytes on octadecylsllica was investlgated both theoretically and experimentally. The retention was attributed l o a reversible association of the solutes with the hydrocarbonaceous ligand of the stationary phase. A phenomenological treatment of the Corresponding equilibria was developed for various types of lonogenic substances. The energetics of the assoclatlon process was analyzed In a rlgorous fashion in the light of the solvophobic theory and a semi-empirical extension of the Debye-Huckel theory to hlgh ionic strength. The predlcted effect of solute loniratlon on the capacity factors was substantiated by experimental data. The observed dependence of the capacity factors on the lonlc strength of the eluent and the hydrophoblc surface of the solute molecules showed good agreement with the theory. The advantages of the technique in the separatlon of biological substances are illustrated.The great majority of biological substances contain ionogenic functions such as carboxylic or amino groups. In most instances, ion exchange chromatography has been the method of choice for the separation of such compounds having similar chemical structure (1,2). Recent developments in high performance liquid chromatography, however, have demonstrated that columns packed with a nonpolar stationary phase, such as octadecylsilica, are also eminently suitable for the separation of weak acids and bases ( 3 ) .Most effort has been focused on the use of "reversed phase" chromatography with eluents containing anionic or cationic surfactants in hydroorganic solvent mixtures; the technique is frequently called "ion-pair" (4, 5 ) or "soap" (6) chromatography. Recently it has been demonstrated (7) that octadecylsilica columns with neat aqueous eluents, which do not contain organic solvents, can also be successfully employed for the separation of polar organic compounds.We have shown ( 3 ) that the physicochemical phenomena underlying the chromatographic process with nonpolar stationary phases can be readily interpreted in the light of the "solvophobic theory" (8-15) and the factors determining solute retention are amenable to an exact theoretical treatment. In this study, the theory is extended to account for the effect of solute ionization on chromatographic retention, both phenomenologically and by a rigorous treatment of the interaction between ionic solutes and the eluent. THEORYThe chromatographic process is viewed as a reversible association of the solute, s, with the hydrocarbonaceous ligand, L, such as an octadecyl function covalently bound to the surface of the stationary phase:The equilibrium constant for the association, KaSSOC, is given by It is assumed that the equilibrium constant of the process with both neutral and ionized solutes is solely determined by solvophobic interactions ( 3 ) , that is, no ionic or hydrogen bonding occurs between the solute and the stationary phase.In this section, we first will present a phenomenological treatment of the...

In order to characterize the thermodynamic constraints on the process of integral membrane protein folding and assembly, we have conducted a biophysical dissection of the structure of bacteriorhodopsin (BR), a prototypical alpha-helical integral membrane protein. Seven polypeptides were synthesized, corresponding to each of the seven transmembrane alpha-helices in BR, and the structure of each individual polypeptide was characterized in reconstituted phospholipid vesicles. Five of the seven polypeptides form stable transmembrane alpha-helices in isolation from the remainder of the tertiary structure of BR. However, using our reconstitution protocols, the polypeptide corresponding to the F helix in BR does not form any stable secondary structure in reconstituted vesicles, and the polypeptide corresponding to the G helix forms a hyperstable beta-sheet structure with its strands oriented perpendicular to the plane of the membrane. [The polypeptide corresponding to the C helix spontaneously equilibrates in a pH-dependent manner between a transmembrane alpha-helical conformation, a peripherally bound nonhelical conformation, and a fully water soluble conformation; the conformational properties of this polypeptide are the subject of the accompanying paper: Hunt et al. (1997) Biochemistry 36, 15177-15192.] Our observations suggest that the folding of alpha-helical integral membrane proteins may proceed spontaneously. However, the preference for a non-native conformation exhibited by two of the polypeptides suggests that the formation of some transmembrane substructures could require external constraints such as the links between the helices, interactions with the rest of the protein, or the involvement of cellular chaperones or translocases. Our results also suggest a strategy for improving the thermodynamic stability of alpha-helical integral membrane proteins, a goal that could facilitate attempts to overexpress and/or refold them.

While all of the peaks are not completely resolved, an increase in column length would most likely improve resolution greatly. Initial chromatograms of the same mixture on a column 750 mm long showed celloheptaose and cellohexaose barely distinguishable as separate peaks. Using the same resin in a 1000-mm column gave the chromatogram in Figure 9. All of the cellodextrins except celloheptaose were easily soluble in 0.10M boric acid adjusted to pH 7.0 with sodium hydroxide. Celloheptaose entered solution completely after several hours stirring at about 40°C. Higher homologs

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