ELECTROKINETIC PROPERTIES O F PROTEINS*
I. Isoelectric Point and Solubility of Wheat Proteins in Aqueous Solutions of Ethanol BY TY. M. MARTIN**
Introduction This investigation was undertaken with the object of obtaining some information concerning the proteins extracted from wheat gluten by ethanol solutions of various concentrations. Solubility has long served as a differentiating property in the isolation and purification of the proteins. They are prepared, according to the prevailing methods, by extracting plant and animal substances with reagents in which they are soluble. Specific reagents are designated as solvents for the various proteins. The composition and concentration of the latter are fixed arbitrarily, and it is usually assumed that the preparations obtained by reprecipitation from such reagents are chemically pure compounds. Chemical entities. The fallacy of our methodology in this regard has been clearly indicated by the experimental studies of Gortner, Hoffman and Sinclair‘ in which they have shown that the percentages of protein extracted from wheat flour with normal solutions of KF, KCl KBr and K I were 13, 2 3 , 3 7 and 63, respectively. GortneP and his coworkers’ were the first to emphasize t’he unsatisfactory state of our present methods involving the solubility properties of the proteins when they ask : “Whal salts and what concentrations?” Obviously the quantity and the nature of the prot,ein extracted are dependent upon the composition, concentration and the temperature of the solvent used as an extractive. This view is also supported by the data of Bailey and B l i ~ h Blish , ~ and SandstedtP Hoffman and Gortner,5 Haugaard and Johnson6 and by a number of other workers who have been interested in this phase of the problem. Apparently no attempt has been made to define or name the proteinaceous material obtained when the concentration of the reagent designated as the solvent for a given protein is altered slightly from that arbitrarily stated. Would z different protein be obtained for each different concentration of * Contrihution from the Chemistry Department, University of Montana, Agricultural Experiment Station, Bozeman. Paper No. 3, Journal Series. * * llssistant Professor, Chemistry Department, University of Montana, Agricultural Experiment Station, Bozeman. R. .4. Gortner, K. F. Hoffman, and W.B. Sinclair: Colloid Symposium BIonograph, 5 , PP. 179-198, (1928).
R. A. Gortner: .‘Outlines of Biochemistry” (1929). C. H . Bailey and &I. J. Blish: J. Biol. Chern., 23, 345-357 (191j). a hI. J. Biish and R. hI. Sandstedt: Cereal Chem., 6,494-503 (1929). 5 W.F. Hoffman and R. A. Gortner: Cereal Chem., 4, 221-229 (1927). G. Haugaard and A. H. Johnson: Comptes-rendus des travaux du Laboratoire Carlsberg, 18 ( z ) , 1-138 (1930). ?
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reagent used in its preparation? What are the effects of composition and concentration of solvent on the properties of the protein extracted? In other words, can the proteinaceous materials isolated on the basis of their differences in solubility in arbitrary reagents be logically regarded as chemical compounds? Perhaps they are fragnients of the compounds as they existed in nature; or they may be polymers, or condensation products of the undenatured proteins. The effect of different solvent's on the physical properties of molecules is well known. 11any of the simple and relatively stable compounds are mononiolecular i n one solvent and di- or polymolecular in others. hssociated molecules are cornmonly known to exist under certain conditions with respect to temperature and chemical character of solvent. The complex nature of the proteins and their extreme lability might be expected to render them even more sensitive to the influence of external forces. Much of the confusion in protein research rnay undoubtedly be attributed to the tendency, on the part of many workers in this field, to look upon proteins as static bodies, and not as dynamic structures whose propert'ies are inseparably related to the environmental forces acting upon them. In their ultracentrifugal studies Svedberg? and his coworkers have shown that the state of aggregation of protein particles is determined by the chemical properties of the liquids in which they are dispersed. Although a constant value was obtained for the apparent molecular weight of each protein in a given reagent, it was found to vary with changes in the chemical composition of the latter. Recent studies have indicated that protein sols which were hitherto regarded as homogeneous systems, are probably mixtures of two or more components in equilibrium. That is, the dispersed particles are not all in the same stat,e of aggregation. 8orensen8 has expressed the view that these Components do not exist as a simple rnixt'ure, but as a system whose equilibrium is influenced by environmental forces due to the temperature, composition and concentration of the suspensions medium. Recent work at the Carlsberg Laboratories, more especially t>hatof Haugaard and ,Johnson6 on the fractionation of gliadin, further confirms this view. Since it is not possible to apply the usual analytical method of study to the proteins on account of their instabilit,v toward chemical reagents, it is proposed to introduce a new angle of attack on the problem through studies on their electrokinetic properties by streaming-potential methods. By the latter, the intermolecular force relations of protein systems may be studied in the absence of the influence of reagents. The advantages are obvious. 'The Svedberg and Bertil Sjogren: J . Am. Chem. Sor., 52, 279-287 (1930); 52, 2855-63 (1930); 51, 3594-604 (1929); 50, 3318-32 (1928); Bertil, Sjogren, and The Svedherg: 52, 3279-X~(1930); 52, 36jo-54 (1930); F. Xrish1iam)irti and The Svedherg: 52, 2897906 (1930); Bertil, Sjogren, and Romauld Spyholskl: 52, 4100-04 (1930); The Svedberg, I,. bl. Carpenter, and D. C. Carpenter: 52, 211-52 (1930); 52, 701-10 (1930); The Svedberg and A . J. Ytamm: 51, 2170-85, (1929); The Svedberg, and S . B. Lewis: 50, j2j-36 (1928); The Svedherg and Eugen Chirnoaga: Bul. chim. soc. ramona stiinte, 31, 90.1-3, 23-52 (1929); J. Am. Chern. So?., 51, 1399-411 (1928); The Svedberg and J. B. Nichols: 49, 292034 (1929); 48, 3081-92 (1926); 45, 2910-17 (192.3); The Svedberg and Robin Fahreus: 48, 430-38 (1926); The Svedberg and Herman Rind: 46, 2677-93 ( 1 9 2 4 ) . * S. P. 1,. Sorensen: Comptes-rendus du Lahoratoire Carlsberg, 15, No. I I (1923); 16, SI,. 8 (1926).
ELECTROKINETIC PROPERTIES O F PROTEINS
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The Problem As a basis for subsequent studies on the physical and chemical aspects of protein solubility, the initial work has been devoted very largely to the development of apparatus and methods for determining the
