Protein in nonaqueous solvents

V. Physical and Chemical Properties of Proteins in Nonaqueous Solvents. 59... 

B. Biochemical Reactions of Proteins in Nonaqueous Solvents and Solvent... 

In preliminary surveys of the solubilities of proteins in nonaqueous solvents, the solvent has generally been added directly to the solid protein. After suitable agitation, those mixtures that appeared homogeneous were classed as solutions. In this manner a number of interesting binary systems have been discovered. Some proteins which are not very soluble in aqueous media are quite soluble in a wide range of suitable nonaqueous... 

The presence of counterions bearing hydrocarbon-miscible tails, such as the trichloroacetate anion just mentioned, or tetraalkylammonium cations, instead of the usual inorganic ions, may be an aid in solubilizing proteins in nonaqueous solvents. 

At this juncture, it is useful to discuss the experimental methods that are of value in studying and separating the various kinds of interactions in macromolecular systems. A variety of experimental methods have been applied to the determination of protein structure and conformation in solution, and these have been summarized by Kauzmann (1959). In the discussion which follows, emphasis is placed on those methods which have so far been of most use in studies of proteins in nonaqueous solvents, and these remarks should be considered as supplementary to the Kauzmann summary. 

It has recently been found (Simmons et al., 1961) that a pronounced Cotton effect exists in solutions of helical polypeptides and proteins in the wavelength region from about 260 mg to 220 mg. A large trough in rotation occurs at 233 mg which is removed when the helix is disrupted. The magnitude of [a]233 may therefore be found to serve as an independent measure of the helical contents of proteins. This effect has not yet been extensively applied to proteins in nonaqueous solvents, but it should prove to be of great interest for proteins dissolved in those solvents which are sufficiently transparent in this region of the spectrum. 

In view of these results, two observations may be made (a) as a corollary to studies of protein ultraviolet spectra in any particular nonaqueous solvent, the spectral properties of relevant simple compounds in that solvent must be investigated and (5) any changes in protein spectra produced as a result of modification of the native protein conformation in a particular nonaqueous solvent must be superimposed on changes resulting simply from the replacement of the aqueous environment by the nonaqueous one of generally different polarizability and refractive index. In the extreme case, for example, it may make little or no difference spectrally whether the aromatic chromophores remain internally bound within the protein molecule, or whether they become exposed to the solvent, and hence no useful information about protein conformations can be expected. More studies have to be made to clarify to what extent spectral changes can be useful in the investigation of proteins in nonaqueous solvents. 

With these remarks we now proceed to discuss the experimental studies that have been made to date on the conformational properties of polypeptides and proteins in nonaqueous solvents. 

E. Proteins in Nonaqueous Solvents 1. Weakly Frolic Solvents... 

V. Physical anb Chemical Properties op Proteins IN Nonaqueous Solvents... 

Although most recent studies of proteins in nonaqueous solvents, and the major part of this article, have been devoted to problems of protein molecular conformations, there are many other aspects of these systems which might profitably be investigated. A few of these aspects are discussed briefly in this section. 

The solubility of proteins in nonaqueous solvents makes it possible to study them in homogeneous solution at temperatures below the freezing point of aqueous solutions, since many pure nonaqueous solvents have freezing points well below 0°C (Table I). Freezing temperatures can be lowered even further by the use of solvent mixtures. This opens a new dimension in protein chemistry the significance of which can only dimly be appreciated at the present time. Several possible problems of interest may be mentioned, however. 

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