H. S. OLCOTTAND T. D. FONTAINE
1334
paralleled by the effects of pressure on the absorption of light b y similar solutions.
Summary We have found that the absorption of visible light by solutions of aromatic amines in nitro or nitroso compounds is pushed very significantly toward the longer wave lengths when the hydrostatic pressure over the solutions is raised a t constant temperature, and also when the temperature is raised a t constant volume. The absorption of light by these solutions may increase, decrease or remain constant as the temperature is raised a t constant pressure. These results have been correlated into a consistent theory which avoids any assumption of the formation of colored compounds by the hypothesis that the colors of the solutions arise from the mutual polarizations of the molecules when appropriate groups are in
[CONTRIBUTION FROM THE
Vol. 62
close proximity, account being taken of the influence of pressure and temperature on the collision frequency, the distance of closest approach and the effect of short-range attractions between groups. We have also shown from orienting experiments that substituents in the nitro compound and in the amine influence the absorption of light by the solutions in a way that parallels closely their effects on the reducitility of the NOn or NO group on the one hand, and the electron mobility in the amino compound on the other. It is suggested that the polarizations which give rise to the colors of the solutions may be regarded as primary steps in possible reactions, such as oxidation and reduction, involving transfer of electrons from the aromatic 'amine or hydrocarbon to the oxygen of the nitro or nitroso group. WASHINGTON, D. C.
MULTIPLE FELLOWSHIP OF THE
COTTON
RECEIVED FEBRUARY 21, 1940
RESEARCH FOUNDATION,MELLON INSTITUTE ]
Composition of Cottonseeds. 111. Solubility of Proteins in Alkaline Solutions of Neutral Salts' BY H. S. OLCOTTAND T. D. FONTAINE Oil-free cottonseed meats contain approxi- tion by the addition of acid to the isoelectric point mately 8.5% nitrogen (5045% protein). Water (pH 6.5-7.0) was found to be a satisfactory prowill extract 2530% of the nitrogen compounds. cedure but suffered from the disadvantage that Dilute solutions of neutral salts extract 75-85y0 cottonseed pigments are easily oxidized in alkaline of the total. The materials not dissolved by solution, yielding brown products which affect the water but extracted by salt are the cottonseed color of the protein. A consideration of the theories of protein soluglobulins. Osborne and Vorhees2 and Jones and Csonka3 have recorded some of the properties of bility suggested that the globulin might be exthese proteins. Their extraction by salt and al- tracted a t lower PH levels in the presence of low kaline solutions has been described more r e ~ e n t l y . ~concentrations of salt; that is, that the two methMethods were sought for isolating the globulin ods, salt peptization and alkali solution, would be fraction on a large scale. Extraction with salt mutually additive. Experimentally, however, it solutions was efficient but means of recovery was found that the opposite was true; in the therefrom did not appear to be practical. Dilu- presence of low concentrations of salt, the solution required relatively large amounts of water, bility of the protein a t a $H below the maximum and precipitation with acid, though easily effected, for extraction was actually depressed. The pheyielded an insoluble product. The cottonseed nomena observed were of suficient interest to globulins, like several other vegetable globulins, invite further investigation. are very readily denatured in acid solution. Experimental Extraction with alkali a t PH 10.0 and precipitaData were obtained in the following manner. Ether(1) Presented in part before the Division of Biological Chemistry of the American Chemical Society at Boston. Mass., Sept. 11-15, 1939. (2) Osborne and Vorhees, THISJOURNAL, 16, 778 (1894). (3) Jones and Csonka, J. B i d . Chem., 64,673 (1925). (4) Olcott and Fontaine, THISJOURNAL, 61, 2037 (1939).
extracted cottonseed meats4s6were used. A sample was (5) The amount of nitrogen extracted under a given set of conditions vaned for different preparations of cottonseed meal. With the two meals used in the present investigation, alkali at pH 9.0 extracted 83% of the nitrogen from one, 77% from the other.
June, 1940
SOLUBILITY OF PROTEINS I N
ALKALINE SOLUTIONS
OF SALTS
1333
but only 22% could be peptized a t the same pH in the presence of 0.05 N sodium chloride. A solution of the protein a t pH 9.0 or 9.5 could be partially precipitated by the addition of the correct amount of salt. The effect of several other inorganic salts on the solubility of the cottonseed proteins was investigated 100 under similar conditions (Fig. 3). In the case of the sodium salts, the important part played by the anion was evident. In contrast to the chlocd ride, the sulfate and iodide caused only a slight 80 depression in the solubility curve. Sodium fluoride is a poor peptizing agent; whereas, in neutral F4 solution, most inorganic salts (0.5 N)peptize 75 * to 8570 of the nitrogen of cottonseed meal, so'3 60 dium fluoride peptizes only 57%. The results obtained with potassium chloride and sulfate 4 c were almost identical with those observed with the corresponding sodium salts (Table I). J 40 The effect of alkaline earth salts was more 8 pronounced. Inasmuch as water alone extracts b a 25 to 30y0 of cottonseed nitrogen, the 0.03-0.05 N solutions of calcium chloride and magnesium 20 0.1 0.3 0.5 sulfate apparently suppressed entirely the soluhility of the globulin. This fact was also denionConcentration of NaC1, normality. protein. A quantitaFig. 1.-Effect of sodium chloride concentration on the extraction strated with the of cottonseed proteins from the meal at different PH levels. Sodium tive method for the separation Of the globulin from an alkaline extract of cottonseed meal is hydroxide was used to bring the mixtures to the proper pH. suggested, based upon the addition of calcium, magnesium, or barium salts in the correct concentration. stand, with occasional shaking, for sixteen to twenty-four hours. To determine the amount of protein extracted, the The precipitation is not dependent upon a decrease in PH. The data for experiments typical of those which have suspension was centrifuged, filtered, and aliquots were analyzed for nitrogen by a modified Kjeldahl method. PH been plotted on the graphs are shown in Table I. Preliminary runs were used to determine how much alkali was was determined with the glass electrode.6 The effect of sodium chloride on the extraction of the necessary to bring the mixture to the required PH. It nitrogen compounds of cottonseed a t various pH 8(' levels is indicated in Fig. 1. The solubility of the protein a t pH 10 was depressed only slightly by the presence of salt. At pH 9, the amount of Z protein extracted reached a minimum in 0.05-0.07 N salt, the solubility again increasing with increas- g 6(' ing concentration of salt. At pH 8, the antagonistic effect of the salt was only slightly evident. 4o The lowest curve represents the extractability of the cottonseed proteins in solutions of salt to which ;;j no alkali had been added. The p H of such extracts varied from 6.7 in the absence of salt to 6.4 2 in 0.5 N solution. c: 2(' That the behavior of cottonseed meal under the conditions described could be attributed specifically to the globulins was demonqtrated by dupli- C eating the experimental procedure with the isoi) lated protein (Fig. 2). The material was prepared 0.1 0.3 0.5 hy leaching cottonseed meal with water to remove Concentration of NaCI. normalitv. water-so1ub1e constituents, then extracting the Fig. 2.-Effect of sodium chloride on the solubility of a cottonseed residue with 0.5 N sodium chloride. After centrifglobulin preparation at different PH levels. ugation, the protein was precipitated by dilution with 5 volumes of water. The precipitate was repeatedly will be noted that increasing amounts of alkali were needed washed with distilled water to remove residual salts, with increasing concentrations of salt.' separated each time by centrifugation, and finally dried The effect of sodium and calcium chlorides, in the conin thin layers on a glass plate in a current of air. Sixty- centrations resulting in minimum solubility, on the extracfour % of the product so obtained was soluble a t p H 9.0, __-___ (7) This result was observed in every series of experiments. The
shaken thoroughly with 40 times its weight of water containing sufficient salt and alkali (as indicated by preliminary titrations) to bring the mixture to the required pH and salt concentration. Several drops of chloroform were added to prevent spoilage and the mixtures were allowed to
-z
c)
g
e
z
3 5
,z 5
E
(6) Coleman electrometer, model 3 D .
I complete data have been omitted for the sake of brevity.
H. S. OLCOTTAND T. D. FONTAINE
1336
Vol. 62
TABLE I EXTRACTION OF NITROGENOUS SUBSTANCES FROM ETHER-EXTRACTED COTTONSEED’ Salt concn.,b N
Y N a C 1 NaOH Miliiequv. pH
--
ZtT
NaOH Mil!ieqmv.
KC1-
pH
7 -
%N
extd.
NaOH M$ieqruv.
NaaSO4-
pH
%N
extd.
-KaSOdNaOH Mi!liequv. p H
--
%N
extd.
NaOH Mi$eqmv.
MgSO4-
pH
% N extd.
100
concentration of sodium chloride in solutions alkaline t o the isoelectric point. 5 However, in the series in which the largest + 80 amount of alkali was used, a depression of solubility is recorded a t a concentration 2 of 0.025 N sodium chloride, as shown in + .E 60 Table 11. This discrepancy is not com* mented upon in the original paper nor by 0 + Greenlo in her interpretation of the rej 40 sults. If the data are significant, they A-NOCI 8 - *OIS0, would indicate that, within a limited pH 8-No1 b range, low concentrations of salt may -wosq cu .-COCI, 20 antagonize rather than increase the solu0.1 0.3 0.5 bility of casein in alkali. There is no Concentration of salt, normality. suggestion in Green’slO solubility deterFig. 3.-A comparison of the effect of several inorganic salts on the minations of hemoglobin in alkaline salt extraction of cottonseed proteins from the meal a t PH 9.0. solutions of any parallelism with the phethe pH range between 8 and 10, while that of calcium chlo- nomena described in this paper. The pH, how-
-zf:
I
c
%
0
.
ride does not disappear until a fiH of 11 is attained. The results shown graphically in Fig. 5 were obtained with ether-extracted hempseed meal under experimental conditions similar to those used with cottonseed meal. The absolute amounts of nitrogenous substances peptized differ from those observed with cottonseed meal but the shapes of the curves are somewhat similar. The antagonistic effect of sodium sulfate was more marked than in the case of cottonseed meal but was still less than that of sodium chloride.
Discussion The phenomena of protein solubility have recently been reviewed by Edsall.* According to (8) Schmidt, “The Chemistry of the Amino Acids and Proteins,” Charles C. Thomas, Springfield, Ill., Chapter XVI, 1938.
TABLE I1 EFFECTOF SALTON THE SOLUBILITY OF CASEININ ALKALI“ (Data of Sorensen and Sladek)g % ’ of total casein in Concn. of NaCl, N
pH (cor.)
solution
0.01 5.50 69.3 .025 5.40 63.5 .05 5.31 65.4 .10 5.24 82.2 .25 5.17 97.5 a In each case 91.6 milligram equivalents of casein nitrogen and 0.85 milligram equivalent of surplus NaOH were present in 1000 cc. of the mixture. (9) Swensen and Sladek, Compt. rend. ;raw. lab. Carlsbcrg, 17, No. 14 (1927-1929). (10) Green, J . Bioi. Chew., 99, 517 (1931).
June, 1940
SOLUBILITY OF PROTEINS IN
ALKALINESOLUTIONS O F SALTS
1337
ever, did not vary by more than 1 PH unit from the isoelectric point, nor were salts other than phosphate buffers investigated. I n the case of the cottonseed globulin, the region of maximum
That the results obtained with soybean meal are a reflection of the behavior of the soybean globulin is indicated by the data shown graphically in Fig. 7. I n this case the equilibrium was approached from the solution in dilute alkali. 100 A fresh precipitate of purified soybean globulin13 was washed thoroughly with water, then dissolved by the careful addi80 tion of sodium hydroxide to p H 6.7. Upon the addition of different amounts aJ E of sodium chloride to aliquot portions, g 60 some cloudiness appeared but no precipis tate could be removed by centrifugation. E m Quantities of acid sufficient to lower the c 2 40 pH to approximately 5.7 were then added * to each aliquot, the several samples were J centrifuged, and portions of the super4 20 natant solution were analyzed for nitro$: gen. The sodium chloride concentration for minimum solubility appeared to be 0 0.1 to 0.15 NI4 or about twice that indi2 4 6 8 10 cated by Smith, et a1.,12 for the meal. PH . However, the solution contained approxiFig. 4.-Extraction of proteins from cottonseed meal. Sulfuric acid mately 3% protein, whereas with the and sodium hydroxide were used to adjust PH. meal, a t maximum solubility, the protein antagonism appears to be 2 p H units higher than was present to the extent of only-l.25%: The the isoelectric point of the protein. data for calcium chloride were obtained after the Edsall" observed that a clear gel of myosin in salt had been added to the solution a t p H 6.7. alkaline solution was precipitated by the addition In this case, no further addition of acid was necesof a small amount of salt. Upon the addition of further salt the precipitate dissolved. No quantitative data were recorded. The data of Smith, Brother and Circle12 can be interpreted as indicating a direct parallelism between soybean meal and cottonseed meal proteins (Fig. 6). More protein can be extracted from soybean meal with distilled water than with dilute salt solutions. Minimum solubility occurs a t a A - YoCI concentration of 0.1 N sodium chloride (1 x - usso, part meal:40 parts water). The isoelectric point of the principal soybean protein lies in the PH range 4.2-4.7; therefore the solu0.1 0.3 0.5 bility behavior in neutral salt solutions Concentration of salt, normality. (PH 6.7-6.0) was measured a t approxi- Fig. 5.-Effect of several inorganic salts on the extraction a t PH 9.0 mately 2 PH units above the isoelectric of the proteins from ether-extracted hempseed meal. point. While there was no attempt to maintain a constant PH level, the variations were sary. The concentration of calcium chloride probably not large enough to change (13) Kindly supplied by Dr. N. J. Beaber, Mellon Institute. (14) The salt concentrations are approximations. I t waa not dethe character of the solubility curves.
z
Y
9.4
z
(11) Edsall, J. Bioi. Chem., 89, 289 (1930). (12) Smith. Circle and Brother, THISJOURNAL, 60, 1316 (1938).
termined how much of the sodium hydroxide used to bring the protein into solution had been required to neutralize adsorbed sulfuric acid.
1338
H. S. OLCOTTAND T. D. FONTAINE
0.1 0.3 0.5 Concentration of salt, normality. Fig. 6.-Data of Smith, Brother and Circle12 on the extraction of soybean proteins by aqueous salt solutions. The pH was not held constant but probably varied from 6.7 to 6.3 with sodium chloride and from 6.7 to 5.2 with calcium chloride.
Vol. 62
0.1 0.3 0.5 Concentration of salt, normality. Fig. 7.-Effects of salts on the solubility of a soybean globulin preparation. The pH varied from 5.8 to 5.63 in the sodium chloride series and from 6.48 to 6.25 in the calcium chloride series.
necessary to ensure minimum solubility was also approximately twice that required for the meal. As the proteins discussed in this communication, The solubility of cystine in water is increased in the presence of salt. Cystine is also soluble in except myosin, are known to be mixtures of sevdilute alkali. This resemblance to the behavior of eral individual entities, the possibility that the globulins, which has been discussed by C ~ h n , ' ~solubility phenomena observed with sodium chlosuggested that the antagonism might be observ- ride may be ascribed to a fractionation requires able with the amino acid. However, in a short further investigation. A comparison of the physiseries of determinations, in which the sodium cal and chemical properties of the precipitate and chloride concentration was varied from 0.1 to 2.0 the soluble portion would be of interest. The N and the PH maintained a t 5.0, the solubility was data obtained with hempseed meal suggest that found to vary directly with the salt concentration. edestin would be a suitable purified protein for a A complete interpretation of the physical and more critical investigation of the phenomenon. Inasmuch as it is not possible a t present to conchemical reactions involved in the antagonism of small amounts of salts to alkaline extraction of the tinue this work, the attention of other investigaseveral globulins does not seem possible a t pres- tors is invited to the possibilities for study of proent. In the case of the alkaline earth salts, the tein behavior suggested by these observations. inference that metallic proteinates are precipiSummary tated appears justified. The insoluble proteinate either may again be peptized in the presence of At PH 9.0 small amounts of sodium chloride higher concentrations of salt or may react to form markedly depress the solubility of cottonseed more soluble compounds containing larger protein, as determined by extraction studies with amounts of calcium, barium, or magnesium or cottonseed meal. Greater or smaller concentraconsisting of double salts of the metallic protein- tions of salt permit the peptization of larger ate and the salt. amounts of the total nitrogen. The alkaline In the case of the sodium and potassium salts, earth salts are particularly effective precipitants ; the anion plays an important r01e.l~ Thus the under the conditions used, 0.03-0.05 N calcium sodium proteinate which is insoluble in sodium or magnesium salt solutions suppress the soluchloride solution (0.05-0.1 N a t PH 9.0), is soluble bility of the globulin a t pH 9.0 completely. The in sodium sulfate or sodium iodide when these effects of several other salts on the solubility of salts are present a t the same concentration. the nitrogenous constituents of cottonseed meal (15) Cohn, Chem. Rev., 19, 241 (1936). have been determined. That the observed phe(16) I t has been suggested t h a t the results may be interpreted in nomena can be ascribed to the globulins and not terms of electrokinetic potential changes. However, in the simplest application of such an explanation, i t would be expected t h a t a divato other constituents of the meal is shown in exlent anion (sulfate) would be a more effective precipitant than a periments with isolated globulin preparations. monovalent ion (chloride), which is contrary to the observations.
June, 1940
EFFECTS OF PHENOLS AND ALCOHOLS ON ACIDSTRENGTH
Analogous results have been obtained with hempseed meal. The data of Smith and co-workers“ have been interpreted to indicate that the influence of salts on the solubility of soybean proteins in
[CONTRIBUTION FROM THE CONVERSE
1339
the neutral PH range is a parallel phenomenon. The observations cited are exceptions to the hitherto-accepted generalization that the solvent action on proteins of salts and alkali is additive. RECEIVED JANUARY 22. 1940
P I T T S B ~ G H , pA.
MEMORIAL LABORATORY OF HARVARD UNIVERSITY]
Increases in the Acid Strength of Hydrogen Chloride in Dioxane Brought about by Phenols and Alcohols BY PAULD. BARTLETT AND HYPJ. DAUBEN,
JR.
/=-.
/CI-H
Some years ago Meerwein and his co-workers’ showed that the metallic chlorides which catalyze the rearrangement of camphene hydrochloride into isobornyl chloride are all capable of forming complexes with hydrogen chloride which are stronger acids than hydrogen chloride itself. This property was demonstrated by a method introduced by Hantzsch.* A solution of hydrogen chloride in dry ether was made just too dilute (0.02 N ) to redden the indicator butter yellow (pdimethylaminoazobenzene) . The addition of equivalent stannic chloride (which alone a t this concentration did not redden the indicator) now turned the indicator red, and a dilution of 120fold was necessary to restore the yellow color. This was clearly due to the fact that in ether an acid of the type HzSnC16 is stronger than hydrochloric acid. Meerwein concluded that the catalytic activity of the chloride and its acid-strengthening power were closely related manifestations of its complex-forming ability. By Hantzsch’s method it can be shown that the phenols, which also catalyze the rearrangement of camphene hydrochloride, conform to this generalization in that they, too, increase the acid strength of hydrogen chloride in ethereal solution. The effect is much less than with stannic chloride, but quite definite.. We are forced to the conclusion that phenols, like the complex-forming inorganic chlorides, can form complexes with hydrogen chloride in which the phenol is attached to the chlorine and encourages the separation of this as an ion. The only probable manner in which a phenol can form such an attachment is through hydrogen-bond formation at its hydroxyl group
X:CI:-, .. is functioning as an “acceptor)) center, in the terminology of S i d g w i ~ kwhile ,~ the chloride ion functions as a “donor.” Lewis prefers to substitute the terms “acid’) and “base” for “acceptor” and “donor,” and says of the Sidgwick notation : “This expressive nomenclature I should be glad to adopt if I did not hope to show that his classification coincides absolutely with the classification of acids and bases, so that the need for new names disappear^."^,^ Now in the present instance we have a substance, a phenol, which can function as an acid in the Bronsted sense by donating a proton to some acceptor; it can also function as an acid in the Lewis sense, as an acceptor, by forming a complex with the chloride ion or with hydrogen chloride through its chlorine. If these two kinds of acidity run completely parallel in a series of phenols, being governed by exactly the same structural features, then we have no need to differentiate between them. However, as we shall show, the Lewis
(1) Meerwein, Hammel, Serini and Vorster, Ann., 46S, 16 (1927); Meerwein, ibid., 46S, 227 (1927). (2) Hantzsch, 2. Elektrochem.. 09, 226 (1923); Be?., 68, 627, 631 (1925).
(3) G. N . Lewis, J . Franklin Inst., 206, 293 (1938). (4) Lewis and Seaborg, THISJOURNAL, 61, 1886,1894 (1939). (5) Sidgwick, “The Electronic Theory of Valency,” Oxford University Press, New York, N.Y., 1929.
/H
and we thus have a method of making some quantitative measurements on the relative tendencies to hydrogen bonding by a series of hydroxyl compounds. This subject is of particular interest a t the present time in view of the attention directed by G. N. Lewis3s4to the general aspects of the acid-base relationship. Any molecular species X which can form a coordinate link with the chloride ion,
..
