PHASE RULE STUDIES ON T H E PROTEINS. VI* Non-Aqueous Solutions BY WILDER D. BANCROFT AND S. LOUISA RIDGWSY**
Historical When proteins were found to be amphoteric and to contain amino acids, it was assumed without question that true compounds were formed with the acids and bases which the proteins “bound.” The later studies of colloid chemistry and adsorption suggested to some that the mechanism of the “binding” might be adsorption rather than compound formation. Van Slyke and Van Slykel were among the first to mention this possibility. They worked in water solution with casein and such dilute acids that no casein dissolved. They measured the acid left over by a conductivity method. After citing twelve references supporting compound formation between acids and proteins; and after carefully going over their own data with reference to the three possibilities of compound formation, solution of acid in protein, and adsorption, they decided that in their case they had adsorption. T. B. Robertson? immediately replied to this article to support the theory of compound formation. For each of the criteria of adsorption set up in the first article, he gave examples of chemical reactions which would fulfill it. Van Slyke and Van Slyke3answered Robertson to the effect that their differences were partly a matter of the definition of adsorption, and that their criteria determining adsorption could apply equally well to compound formation which is incomplete, reversible, and occurs between changing proportions of the mass of reactants. They said that proteins might form compounds with acids under other conditions, but that they did not believe they did in their case. Robertson in later works4 has continued to favor compound formation, and in the latest one cited says: “It is now admitted by all observers who have directed adequate attention to this question that the proteins accomplish the neutralization of acids and bases in stoichiometrical, that is, molecular or equivalentmolecular proportions.” In general, some stand is taken for one theory or the other by the various leaders of protein research and by many colloid chemists. Loebj is one of the * This work has been done under the programme now being carried out by Cornell
University and supported in part by a grant from the Heckscher Foundation for the Advancement of Research established by August Heckscher at Cornell University. * * Recipient of a grant in aid from the National Research Council. Am. Chem. J., 38, 383 (1907). * J. Biol. Chem., 4, 35 (1908). J. Biol. Chem., 4, 259 (1908). “Die physikalische Chemie der Proteine” (1912); “The Physical Chemistry of the Proteins” (1918); Chapter in Alexander’s “Colloid Chemistry”, 2 (1926). “Proteins and the Theory of Colloidal Behavior” (1924).
1286
WILDER D. BANCROFT AND 8. LOUISA RIDGWAY
strongest supporters of the compound theory. He studies proteins from the point of view that they are, at least partially, in true solution and follow the classical laws of stoichiometrical chemistry. He explains physical properties of proteins such as swelling, osmotic pressure, membrane potential, and viscosity on the basis of the Donnan membrane equilibrium, where the nondiffusible ion is a protein ion of a protein salt. This has been criticized by Donnanl who says that the Donnan theory rests only on the existence of equilibrium and the existence of certain restraints which restrict the free diffusion of one or more electrically charged or ionized constituents; and that protein with adsorbed acid or basic ions fulfills the second requirement as well as protein ions. Pauli2 criticized Loeb from a different point of view, namely that there is no restriction of the movement of the particles of protein in solution. He has also been criticized in reviews on his book by Bancroft’ and by Alexander‘ who feel that his experimental results point as much, or perhaps more, to adsorption than to compound formation. P r o ~ t e rProcter ,~ and Wilson: Wilson,’ and Wilson and Wilson: have developed a theory for the swelling of gelatine in dilute acid mathematically based on the Donnan membrane equilibrium and on the assumption that a highly ionized protein salt is formed. Their “chemical combination” would sound very much like adsorption to many chemists for it occurs on colloidal surfaces, is not stoichiometric, and is a function of the concentration of the acid present. They got formulae for both the concentration of the chemically bound electrolyte and the total quantity of electrolyte either combined with or present in the solution in contact with the colloid, as functions of the concentration of the electrolyte in solution. The curves for these functions are approximately the same as those obtained by the use of the ordinary empirical adsorption formula. Here again, Donnang has criticized them in the same way he did Loeb-that proteins with adsorbed ions would give the same results as protein ions. Atkinlo did work which he thought supported Procter and Wilson. Ghosh” confirmed the results of Procter and Wilson on the swelling of gelatine in acid. He derived an equation for the swelling of gelatine on the basis of the Donnan equilibrium, but he made fewer assumptions. He says that it is more likely that acid is adsorbed than combined. Some of the other workers who in various ways support compound formation are Schmidt, Greenberg, et a1.12 from the University of California, Chem. Rev., 1, 87 (1924). Alexander: “Colloid Chemiatry,”, 2, 223 (1926). J. Phys. Chem., 26, 687 (1922). ‘ Chem. Met. Eng., 27, 368 (1922). 3 J. Chem. SOC., 105, 313 (1914). E J. Chem. Soc., 109, 307 (1916). J. Am. Chem. Soc., 38, 1982 (1916). * J. Am. Chem. SOC., 40, 886 (1918). Chem. Rev., 1, 87 (1924). 10 J. SOC.Leather Traded Chem., 4, 248, 268 (1920). 11 J. Chem. Soc... 7 . 1 1 (1028). ., l2 J. Biol. Chem., 25, 63J1916); J. Gen. Physiol., 7,287,303,317(1924-25); 8,271 (192526); Univ. of Cal. Publications in Physlol., 5, 289, 307 (1926);7, 9 (1927). 1
I
PHASE RULE STUDIES ON THE PROTEINS
1287
Hitchcock,' Lloyd and Mayes; and Cohn? Others besides those already mentioned who favor adsorption arc Moeller,* de Izzaguirre: Shukoff and Stschoukareff,B and Fanselow.' Tolman and his co-workers* have a theory of the swelling of proteins in acid and alkali which is based on adsorption. This adsorption they suppose is of a chemical nature and occurs in the case of acids on the free amino groups and in the case of alkalies probably on enolized -COHNgroups. Gortner and his associatesg believe that between pH 2 . 5 and 10.5 there is true compound formation, and that a t a pH above or below these limits, there is true adsorption. They have performed numerous and careful experiments on which they base this belief. In an effort to distinguish between the two possibilities of compound formation and adsorption, many experiments have been made. Although the supporter of each theory can see proof for his own theory in his experiments, very often his opponent can see proof for the opposite one in the same experiments. Frequently the opponents appear to be separated further than is actually the case by a disagreement in the definition of terms, but there still remain two distinct possibilities, either of which, or both, may take place in a given case. The criteria which distinguish between the two ere not sufficiently clear-cut under most of the experimental conditions employed. Bancroft and Barnett,Ioand Beldenll performed some experiments of such a nature that the results show definitely whether there has been compound formation or adsorption, or both, under the conditions of the experiment in any given case. They treated solid proteins with gaseous HCl and NH3 in a special apparatus. If a compound formed, the pressure remained constant over the two solid phases as long as they both existed; if an adsorption complex formed, the pressure varied continuously. Casein, arachin, fibrin, gliadin, edestin, and gelatine were found to form compounds with HCl with subsequent adsorption of further acid on the compound formed. Zein simply adsorbed HCl. Casein, zein, arachin, fibrin, gliadin, and gelatine adsorbed NHa without any indication of compound formation. This method is obviously not applicable to caustic soda. People might claim that a strong base would react stoichiometrically with the proteins. Introduction The present work follows directly that of Bancroft, Barnett, and Belden. We have attempted to extend their method to apply to acids and bases, which J. Gen. Physiol., 4, 597, 733 (1921-22); 5,383(1922-23);6,95(1923-24);12,495 (192829); 14, 99 (1930-31). * Proc. Roy. Soc., 93B,69 (1922). a Physiol. Rev., 5, 349 (1925). 4 Collegium, 319,382 (1920). 5 Kolloid-Z., 32, 47 (1923). J. Phys. Chem., 29, 285 (1925). ' Colloid Symp. Mon., 6,237 (1928). * J. Am. Chem. SOC.,40, 264 (1918);41, 1503,1511 (1919). Colloid Symp. Mon., 2, 209 (1925);J. Phys. Chem., 34, 1071 (1930). 10 J. Phys. Chem., 34, 449, 753, 1217, 1930,2433 (1930). J. Phys. Chem., 35, 2164 (1931).
1288
WILDER D. BANCROFT AND S. LOUISA RIDGWAT
are not necessarily gases. The acid or base is dissolved in some solvent, chemically inert to the system, which does not dissolve the protein or the product formed. Varying amounts of the acid or basic solution are added to known weights of the protein and sufficient solvent added to make the volume a convenient and definite one. When equilibrium has been reached, some of the supernatant liquid is pipetted off and the excess acid or base determined by titration. From these data the amount of acid or base taken up per gram of protein may be calculated. These values are plotted against the acidity or basicity of the supernatant liquids. On the basis of the phase rule, we may predict the types of curves which will be obtained, and explain their significance. Suppose first that an adsorption complex forms and there is never more than one solid phase present. There are three components-protein, acid or base, and solvent. There are three phases-one solid, one liquid, one vapor. Then, since F = C - P z (where F = the number of degrees of freedom, C = the number of components, and P = the number of phases), 3 - 3 z = 2 . The temperature is fixed at room temperature, using up one degree of freedom. One variable is left-that of the concentration of the liquid phase. If a compound is formed instead of an adsorption complex, there are two solid phases until the protein is entirely used up, and therefore only one degree of freedom in that range. Since the temperature is fixed, there is no variable left. In the presence of two solid phases, then, the composition of the liquid must remain fixed. A smooth, continuously varying, curve indicates adsorption; one with a “flat” showing constant composition for the liquid phase indicates a compound. This method was used with success by Kawamura’ on stearic and humic acids with NaOH in water. Preliminary Experiments
+
+
Proteins are very complex bodies and there has been much controversy as to the character of their combination with acids and bases. In order to test thoroughly the method outlined above, it was first tried on some solid basic and acidic substances for which the results could be predicted accurately. I. Succinic Acid-It is difficult to find a fairly simple carboxylic acid which is not quite appreciably soluble in any solvent which could be used with proteins. Succinic acid and isobutyl alcohol were chosen as the best available pair although at 25’ the acid is soluble to the extent of nearly three percent in the alcohol. This is much greater than we should like for a test case, and affects considerably certain portions of the curve. The curve should show two flats corresponding to the mono- and di-sodium salts. There might or might not be adsorption on the latter. Experimental work on the acid immediately presented a second difficulty. The di-sodium salt formed in such a way as to coat the solids in the system almost completely and to hinder greatly the attainment of equilibrium. The first experiment was carried out with sodium isobutylate in absolute isobutyl alcohol in an effort to cut down the water content of the system and therefore
J. Phys. Chem., 30,
1364 (1926).
PHASE RULE STUDIES ON THE PROTEINS
1289
TABLE I Summary of the Runs on Succinic Bcid Substance No. treated I.
2
Base used
Succinic acid
Sodium Absolute isobutylate isobutyl alcohol
NaHsuccinate L
tf
Reacts with
HC1
Solvent
95% ethyl
alcohol NaOH HC1
,, ,
NaOH
Absolute alcohol 95% ethyl alcohol
48 days
285 hrs. hrs. 385 hrs.
14 days
__
t,
1,
,I
,,
1
7,
Univ. of Minnesota 9,
Eimer and Amend
,)
NaOH HCl NaOH HCl NaOH HCl NaOH
3 17 hrs.
,
,,
,,
Time of shaking 343 hrs.
53 days 3 0 days
I,
HC1
,
I
,,
,,
Conn. Agr. Expt. Sta.
Length of run 52 days
,,
j!
290
__
25 days 53 days
2 70 hrs. 285 hrs.
days
190 hrs.
35 days 85y0acetone 35 days
3 50 hrs.
3 0 days
345 hrs.
with 52% benzene 95% ethyl alcohol
21
,9
,>
95% ethyl alcohol
,, 7,
1,
,,
,, 3 1 days >l
450
hrs. ,v
I,
330 hrs. 1,
PHASE RCLE STUDIES ON THE PROTEINS
I305
TABLE IX Summary of the Runs on Proteins SO.
Amount of peptization
None Negligible 3. Considerable in high nos. I.
2.
4.
5. 6.
7. 8. 9. IO.
11.
12.
13. 15. Ij.
16. 1;.
J>
None Complete JI
Some Considerable Considerable Complete Considerable Considerable None Some Negligible Negligible
App. amt. of Compound Mg. per gm. hydrolysis formation protein 0.8-0.97~
+
Equiv. X
10-5
61
16;
75
18;
Comhining weight
600 53 5
25 68 I470 might be considered to be 31 86 I 160
30
75
1330
-
62 124
1613 806
I08
925
I t will be well, before taking up the specific proteins, to consider briefly some of the difficulties encountered in interpreting the curves obtained. Theoretically there are none, but practically they are met. There is first the possibility of such complete adsorption that the amount of substance left in the supernatant liquid is too small to be detected experimentally. The curve then appears to show compound formation. Examples of this are the adsorption of dyes by charcoal, and some of our own work on succinic acid. Then a lack of equilibrium in compound formation, due to its slowness, may appear as a n adsorption curve. This situation was met several times during the experiments on uric acid. Hydrolysis of the protein must profoundly affect the shape of the curve. In the proteins used, there is roughly from 15 to 70 times as much amino nitrogen in the completely hydrolyzed as in the unhydrolyzed material; so that even one per cent hydrolysis would mean a t least a fifteen per cent increase in the amount of amino nitrogen. This makes the protein in the higher numbers of each run, where there is most hydrolysis, appear to take up more acid or especially more alkali, than is really the case. In the lower numbers of each run, particularly in acid, this is cut down t o negligible proportions. Fortunately for us, it is these lower numbers which determine whether or not a compound is formed. The amount of adsorption on the original protein or on any compound formed, as determined from the upper part of the curve, is unreliable.
1306
WILDER D. BANCROFT AND 5. LOUISA RIDGWAY
We do not claim that our results will be applicable to water solution. Too often results have been compared which have been obtained under radically different experimental conditions. It would take a vast amount of work to harmonize them. Our results are simply those under the conditions of our experiments. Some agree well with values obtained in water solution. We will give the results of other workers for the different proteins. Many times, they give the maximum amount bound, while we give that present in the compound. I n order to facilitate comparisons between the different proteins, all the results on them are given in Table IX. I. Casein-The casein was a technical product purchased from Kahlbaum and used without further purification. The runs in both acid and alkali seemed perfectly normal in every way. I n acid, the appearance of the casein did not change during the run. I n alkali, it became somewhat yellow and swollen, but there was apparently no peptization. The hydrolysis of the casein in each case was less than one percent. Casein formed a hydrochloride (Table FIQ.7 X and Fig. 7), as Bancroft and Barnett’ Casein and HC1 in 95% found, and there was then adsorption to Alcohol a slight extent on the compound. It contained 61 mg. HCl per gm. of casein or 167 X IO-6 equivalents. This gives casein a combining weight with the acid of about 600. Bancroft and Barnett’s compound contained 234 X IO-^ equivalents. Other results reported are: go X IO-^ equivalents a t a pH of 2.5 and 600 X
IO-+ equivalents a t the maximum acid concentration by Hoffman and Gortnerl
85 X IO+ equivalents at a pH of 2.5 by Sandstroms 33 X IO-^ equivalents when acid is “saturated” with protein by Robertson4 60 X IO-^ equivalents a t neutrality to phenolphthalein by Bracewells 59 X IO+
equivalents by Hitchcock6
72 X IO-^ equivalents by Loeb7 as recalculated by Cohn.* 1
J. Phys. Chem., 34, 449 (1930).
* Colloid Symp. Mon., 2, 209 (1925). a 5
J. Phys. Chem., 34, 1071 (1930). J. Phys. Chem., 13, 469 (1909). J. Am. Chem. SOC., 41, 1511 (1919). J. Gen. Physiol., 5, 383 (1922-1923). J. Gen. Physiol. 3, 547 (1920-1921). Physiol. Rev., 5, 349 (1925).
PHASE RULE STUDIES ON THE PROTEINS
I307
TABLEX Casein and HCI in 95% Ethyl Alcohol I
gm. casein, equivalent to ,901gm. dry casein, used in each number Volume of each number-2o cc. Length of run-52 days a No.
cc. N HCl added
b cc. N base for I O cc. supernat. liquid
C
cc. N acid in super. liquid b X2
d e cc. N acid mg.HC1 per used per gm. casein gm.casein d X 36.46 equiv. X IO-^ (a - c)/.901
I.
0.50
0.00
0.00
0.56
20.5
2.
I .OI
0.00
0.00
1.13
3. 4. 5. 6.
I.52
0.01
0.01
I ,67
2.03
0.25
0.51
I .69
41.o 61.o 61.5
7.
2.54 4.05 4.56
0.38 I .os I .30
8.
5.07
1.50
0.76 2.09 2.60 3 .OI
I .97 2.18 2.18 2.29
71.7 79.3 79.4 83.5
TABLEXI Casein and NaOH in 95% Ethyl Alcohol I
gm. casein, equivalent to ,901 gm. dry casein, used in each number Volume of each number-20 cc. Length of run-52 days a
b cc. N acid for I O cc. supernat. liquid
c . d e ~ cN. base cc. N bme mg. NaOH per m super. gm. casein used per gm. casein d X 40.008 liquid equiv. X 10-2 b X 2 (a - c)/.901
No.
cc. N NaOH added
I.
0.50
0.00
0.00
0.56
22.2
2.
I .oo
0.00
0.00
1.11
3.
1.50
0.00
0.00
I .66
4.
2.00
0.00
0.01
2.21
5. 6.
2.50
0.01
0.01
2.76
3 .oo
0.05
0.IO
3.21
44.4 66.6 88.4 110.4 128.6
7. 8.
3.50 4.00
0.23
0.46 0.38
9.
134.9 160.9 166.3
IO.
4.50 5 .oo
3.37 4.02 4.16
I I.
10.00
4.47 6.62
178.9 264.7
0.19 0.38 0.49 2.02
0.75 0.97 4.05
1308
WILDER D. BASCROFT A S D S. LOUISA RIDGWAY
Casein also formed a compound with NaOH (Table XI and Fig. 8) as was expected from its acidic character and much previous work done on it. There was considerable adsorption on this. It contained about 7 5 mg. NaOH per gm. casein or 187 X IO-5 equivalents, and gives casein a combining weight with strong base of 535. This is much more base than is contained
FIG 8 Casein and SaOH in 9 5 6 Ethyl A4kohol
in the “caseinates” often mentioned. This result may be compared with others reported: I I X IO-5 equivalents when baseis‘lsaturated” withprotein by Robertson’ and L. L. Van Slyke and Bosworth? 50-5 j X IO-^ equivalents at neutrality to litmus by R ~ b e r t s o n ,L. ~ L. Van Slyke and Hart,4 and Soldnerj 9 0 x IO-^ equivalents at neutrality to phenolphthalein by L. L. \’an Slyke and Hart: Soldner,j Bosworth and L. L. VanSlyke,6 Laqueur and Sackur,’ and Courant8 1 1 . 2 , 21.4, 58.4 and 8 7 . 2 X IO-^ equivalents in Mg. compounds of casein by L. L. Van Slyke and Winterg 90 x IO-^ equivalents at a pH of 1 0 . 2 , I j 5 x IO+ at a pH of 10.5,and a maximum of 1400 x IO-5 by Hoffman and Gortner‘O J. Phys. Chem., 13, 469 (1939). J. Biol. Chem., 14, 211 (1913). a J. Phys. Chem., 14, 528 (1910). Am. Chem. J., 33, 461 (1905). 5 Z. angew. Chem., 1895, 370. J. Biol. Chem., 14, 207 (1913). Beitrage Z. chem. Physiol., 3, 193 (1902). 8 Archiv ges. Physiol., 50, 109 (1891). J. Biol. Chem., 17, 287 (1914). l o Colloid Symp. Mon., 2, 209 (1925).
*
PHASE RULE STUDIES ON THE PROTEINS
I309
146 X IO-^ equivalents a t a pH of 10.5 by Sandstroml 136-40X IO-^ equivalents “under some conditions” by Cohnz 155-60 X IO-^ equivalents by Greenberg and Schmidt8 and by Cohn and Berggren4 if casein is not “nach Hammarsten” 180 X I O + equivalents by Robertson5 and Cohn and Berggren4
.VG. H C l P E R G M GEL.ITI.\
E
FIQ.g Gelatine and HCl in g j R Ethyl Alcohol 2. Gelatine-The gelatine was from the Eastman Kodak Company, and was not further treated. I t was from pig-skin, had a pH of 5.0, and was in the form of a powder. Three runs with HCl were made. (See Tables XII-XIV and Fig. 9.) Both 95% and absolute ethyl alcohol were used. I n the 95y0 alcohol, there I 2
a 4
J. Phys. Chem., 34, 1071 (1930). Physiol. Rev., (2) 5 , 349 (Igzj). J. Gen. Physiol., 7,317 (1924-5). J. Gen. Physiol., 7, 4 j (1924-j). J. Phys. Chem., 14, 528 (1910).
1310
WILDER D. BANCROFT AND S. LOUISA RIDGWAY
was considerable peptization in the higher numbers. I n the absolute alcohol, there was practically none. Hydrolysis of the gelatine was less than 2.5% in each. The time of the runs was from a month to two months. The results of all of them fall very nicely on the same curve, showing that equilibrium is reached in the time allowed. A compound was formed and a little adsorption took place on it. It contained 2 5 mg. HCI per gm. of gelatine or 68 X IO-^ equivalents, and gelatine therefore has a combining weight of 1470 for strong acid. If point B on the curve is taken to represent the composition of the hydrochloride (and this does not seem unreasonable), 86 X IO-^ equivalents are bound. This value agrees better with those obtained by other worken. Some of these are:
TABLE XI1 I
Gelatine and HCI in 95% Ethyl Alcohol gm. gelatine, equivalent to .886 gm. dry gelatine, used in each number Volume of each number-20 cc. Length of run-53 days No.
a cc. N HC1 added
b cc. N base for IO cc. supernat. liquid
C .0 :
N acid
in super. liquid b X 2
I.
0.76
0.00
0.00
2.
I .52
3. 4. 5. 6.
2.03 2.54 3.04 3.30 3.55 4.06 4.56 5.07
0.34 0.54 0.74 I .OI
0.68 I .08 1.49 2.03
I . 13
2.25
7. 8. 9. IO.
I .25
2.50
1.45 I .71
.g1 3.41 3.65
I .82
2
d cc. N acid used per grn. gelatine equiv. X 1 0 - 2 (a - c)/.886
mg. HC1 per gm.gelatine d X 36.46
0.86 0.95 I .07 1.18 1.15 I .IS 1.18 I .30 I .30 I .61
31.3 34.8 39 .o 43.2 41.8 43 .O 43.2 47.4 47.4 58.5
e
TABLEXI11 Gelatine and HCI in 95y0 Ethyl Alcohol A continuation of Table XIII, except that the length of the run is 30 days I. 0 .oo 0.00 0.00 0.00 00.0 2.
0.12
0.00
0.00
3. 4. 5. 6. 7. 8. 9.
0.24 0.35 0.47 0.59 0.71 I .88 3.29 4.71
0.00
0.00
0.00
0.00
0.13 0.27 0.40
0 00
0.00
0.53
0 .oo
0.00
0.66
0.03
0.07 0.93
0.72
IO.
0.47 I .os 1.73
2 . IO
3.46
I .08 1.35 I .41
04.9 09.7 14.6 19.4 24.2 26.3 39.1 49.3 51.3
1311
PHASE RULE STUDIES ON THE PROTEINS
TABLEXIV I
Gelatine and HC1 in Absolute Ethyl Alcohol gm. gelatine, equivalent to ,886 gm. dry gelatine, used in each number Volume of each number--90 cc. Length of run-48 days 8
No.
cc. N HC1 added
b cc. N base for I O cc. supernat. ltquid
C d e c ~ N. acid cc. N acid mg. HCI per used gm. gelatme m super. liquld gm. g e E m e d X 36.46 b X 2 equiv. X 10-3 (a - ~ ) / . 8 8 6 0.004 0.46 16.6
0.41 0.81
0.002
2.
0 .os
0.10
0.81
3.
;. 2 2
0.38 0.76
0.95
0.98
I . 13
I
I .SI
I
I.
4.
I
5.
2.03
0.19 0.38 0.56
6.
2.44
0.76
7.
2.85
0.91
8.
3.25 4.07 4.88
I
9. IO.
.63
.06
I
.81
.03 .os
I . 17
.28
2.12
I
1.44
2.87
1.76
3.52
1.35 1.53
29.5 34.6 35.8 37.4 38.2 42.5 46.8 49.2
55.9
80 X IO-^ equivalents by Bugarszky and Liebermam' as recalculated by
Cohn2
85
x x
IO+
equivalents by Atkin and Douglas3
89 IO-^ equivalents by A. E. Stearn' 92 X IO-^ equivalents from viscosity measurements by Bacon5 9 4 x 10-5 equivalents as the best value of many determinations by Loeb
and by Hitchcocks 104 X IO-^ equivalents (of acid dye) by Chapman, Greenberg, and
Schmidt' 10-5 equivalents by Wintgen and his associates8 10-6 equivalents by Procter and Wilsong IO-^ equivalents by Manabe and MatulalO IO-^ equivalents by Lloyd and Mayesll 300 X IO-^ equivalents by Belden12 using solid protein and gaseous HCl.
x 130 x 150 x 300 x I 13
1
Pfliiger's Archiv, 72, 51 (1898).
* Physiol. Rev., 5, 349 (1925).
J. SOO. Leather Trades' Chem., 8, 359, 528 (1924). J. Gen. Physiol., 11, 377 (1927-28). 6 Ferguson and Bacon: J. Am. Chem. Soc., 49, 1921, 1934 (1927); Bacon: J. Phya. Chem., 33, 1843 (1929). J. Gen. Physiol., 4, 733 (1921-22); 6,95, 201 (1923-24); 12, 495 (1928-29). ' J. Biol. Chem., 72, 707 (1927). 8 Wintgen and Kriiger: Kolloid-Z., 28, 81 (1921); Wintgen and Vogel: 30, 45 (1922). 0 J. Chem. Soc., 109, 307 (1916). Biochem. Z., 52, 369 (1913). 11 Proc. Roy. SOC.,93B, 69 (1922). 11 J. Phys. Chem., 35,2164 (1931). a
1312
WILDER D. BANCROFT AND S. LOUISA RIDGWAY
I n an attempt to duplicate the work of Belden who obtained a much higher value for the amount of acid bound by gelatine, the run in absolute alcohol was made. It was thought that perhaps the presence of water hindered the binding of the acid, but as previously noted, the result was the same as in 95% ethyl alcohol. In a further effort to duplicate Belden’s results much stronger HCl (up to 4 N) in absolute alcohol was used. Even this relatively high concentration of HCl caused no more compound formation. There are some indications that alcohol is adsorbed by the protein, and for the present we must postulate this as the most likely explanation of the different results.
FIG.I O Gelatine and NaOH in 9570 Ethyl Alcohol
Three runs with NaOH in 95% ethyl alcohol were made. (See Tables IO.) Peptization was considerable and became nearly complete in the more alkaline numbers. Two of the runs contained a large excess of alkali. Hydrolysis of the gelatine in the most alkaline numbers of these was about 4 0 % ~ and was 17-18% in the most alkaline number of the third. The first two runs lasted two weeks and the other lasted nearly four. One of the first two was on a different sample of gelatine. The curves for all three coincide fairly well in the lower ranges, but the third soon shows the result of greater hydrolysis due to its longer standing. There is indication of the formation of a compound, but it is hard to judge accurately the amount of base bound in it due to the high hydrolysis. I t contains about 30 mg. NaOH or 7 j x IO-^ equivalents per gm. of gelatine, thus giving it a combining weight with strong alkalies of 1330. Another run with NaOH was made using about a 50% mixture of benzene and 95% ethyl alcohol as solvent. In all numbers of this run, all the NaOH was used up. This must be due to hydrolysis, which was very high-36%-even though the concentration of the alkali was no higher than usual, or we would be faced with the necessity of explaining a compound containing more than 530 x IO-6 equivalents of NaOH.
XV-XVII and Fig.
PHASE RULE STUDIES ON THE PROTEINS
I313
TABLE XV Gelatine and NaOH in 95% Ethyl Alcohol I
gm. gelatine, equivalent to .886 gm. dry gelatine, used in each number Volume of each number-zo cc. Length of run-14 days a
No.
cc. N NaOH
added
b cc. N acid for I O cc. supernat. liquid
C
cc. K base in super. liquid b X
2
d e cc. N base mg. NaOH per used per gm. gelatine d X 40.008 gm. gelatine equiv. X I O O (a - c)/.886
I.
0.00
0.00
0.00
0.00
00.0
2.
I
.oo
0.04
0.09
I
.03
3. 4.
2
.oo
0.28
I
.63
3.00 4.00 j .oo 6.01
0.52
=55 I .03
2 22
0.82
1.65
2.66
41.2 65.2 89.0 1 0 6 ,I
1.11
2.22
1.65
3.30
3.14 3 .os
125.4 121.9
7.01
1.59 I .89
4.32 4.78 5.26
172.9 180.9 210.5
5.20
207.8
7.43
296.8
5. 6. 7.
8. 9. IO.
8.01 9.01
11.
10.01
2.70
3,I7 3.77 4.34 5.40
12.
1 5 .OI
4.21
8.43
2.17
TABLE XVI Gelatine and NaOH in 9 5% Ethyl Alcohol The same as Table XVI except on slightly different sample of gelatine .31 2.62
0.12
2.
3. 4. 5. 6.
3.93 5.24 6.55 7.86
0.74
7. 8.
9.17 10.48
2.52
9.
11.79 1 3 .I O
3.37 3.83
I.
IO.
I
0.40 I.
24
I .67 2.04
2.96
0.24 0.80 1.47 2.47 3.34 4.09 5.04 5.91 6.75 7.65
23 2.06
I .
2.77
3.I2 3.62 4.27 4.66 5.16 5.69 6.15
49.1 82.2 110.9 .o
125
144.8 170.3 186.5 206.3 227.6 246.0
WILDER D. BAKCROFT AND S. LOUISA RIDGWAT
1314
I
TABLE XVII Gelatine and NaOH in 95y0 Ethyl Alcohol gm. gelatine, equivalent to ,886 gm. dry gelatine] used in each number Volume of each number-20 cc. Length of run-25 days No.
a cc. N NaOH added
b cc. N acid for I O cc. supernat. liquid
C
cc. N base in super. liquid bX2
d e cc. N base mg. NaOH per used per gm. gelatine d X 40.008 gm. gelatine equiv. X 1 0 - 2 (a - c)/.886
0.00
0.00
0.65
26 .o
0.00
0.00
I
1.73 2.30 2.88
0.16
0.32
0.29 0.42 0.57 0.69 0.82
51.9 63.3 78.1 91.8 104.2 119.1
8. 9.
3.45 4.03 4.60 5.18
0.57 0.84 I . 14
.30 1.58 1.95 2.30 2.61 3 .oo
IO.
5.75
3.34 3.60 3.91
133.6 144.1 156.6
I.
2.
3. 4.
5. 6. 7.
0.58 1.15
0.99 I . 14
1.39 I .64 I .98 2.28
Some results recorded for gelatine' and NaOH are: 56-7 X IO-^ equivalents by Loeb and by Hitchcock' as recalculated by Cohn2 6 0 X IO-^ equivalents a t a pH of 11 by Greenberg and Schmidt3 70-1 X IO-^ equivalents (of basic dye) by A. E. Steam4and Rawlins and Schmidt5 74 X IO-^ equivalents by A. E. S t e a d 8s X IO-^ equivalents by Atkin and Douglas' 130 X IO-^ equivalents by Procter and Wilson8 2 0 0 0 X IO-^ equivalents by Lloyd and Mayesg 3. Zein-The zein was very kindly sent to this department by Mr. H. B. Vickery of the Connecticut Agricultural Experiment Station. With HCI in 95% ethyl alcohol, zein showed no compound formation, but considerable adsorption. (See Table XVIII and Fig. 11.) This result was also obtained by Bancroft and Barnett.lo It is to be expected from the fact that zein is acidic, since it contains dicarboxylic amino acids and no free amino groups. Hoffman and Gortner" who say that it combines with J. Gen. Physiol., 6 , 457 (1923-24).
* Physiol. Rev., 5, 349 (1925).
Proc. Soc. Exp. Biol. Med., 21, 281 (1923-24). J. Biol. Chem., 91,325 (1931). 6 J. Biol. Chem., 82, 709 (1929). 0 J. Gen. Physiol., 11, 377 (1927-28). J. Soc. Leather Trades' Chem., 8, 359, 528 (1924). * J. Chem. Soc., 109,307 (1916). Proc. Roy. Soc., 93B,69 (1922). 10 J. Phys. Chem., 34, 449 (1930). I1 Colloid Symp. Mon.,2, 209 (1925). a
4
PHASE RULE STUDIES ON THE PROTEINS
1315
2 0 x 10-5 equivalents of HCl a t a pH of 2.5 and a maximum of 65 x 10-5 equivalents, are the only authors who have mentioned a compound with acid. There was very considerable solution of the zein. It is a prolamine which is soluble in solutions with a higher percentage of alcohol than most of them. The hydrolysis of the zein was about two percent. With NaOH in 95% ethyl alcohol, there was complete solution or peptization and 25-3070 hydrolysis of the zein. Although the upper part of the curve is undoubtedly displaced far to the right, the first few points where hydrolysis was very much less, show that a compound was formed. (See
FIQ.1 1 Zein and HC1 in 9570 Ethyl Alcohol
TABLEXVIII I
Zein and HCl in 95% Ethyl Alcohol gm. zein, equivalent to .963 gm. dry zein, used in each number Volume of each number--lo cc. Length of run-21 days No.
I. 2.
3. 4. 5. 6. 7. 8. 9. IO. 11.
a cc. N HC1 added
0.43 0.86 I .28 1.71 2.14 2.57 3.08 3.42 4.28 5.13 5.13
b cc. N base for I O cc. supernat. liquid
0.03 0.17 0.25
0.44 0.56 0.73 0.78
C
cc. N acid in super. liquid b X z 0.07
0.34 0.51 0.88 1.12
I .OI
1.45 1.55 2.03
1.35 1.53 I .66
3 .os 3.33
2.70
d e cc. N acid mg. HC! per gm. zein used per gm. zein d X 36.46 equiv. X IO-^ (a - c)/.963
0.37 0.54 0.81 0.86 I .06 1.15 1.58 1.45 I .63 2.16 I .87
13.6 19.6 29.3 31.5 38.7 42. I 57.7 52.7 59.5 78.6 68.3
1316
WILDER D. BANCROFT AND S. LOUISA RIDGWAI'
Table XIX and Fig. 12.) It contained approximately 2 8 mg. NaOH per gm. of zein, or 7 0 X IO-^ equivalents, and gives zein a combining weight with NaOH of 1430. Cohn, Berggren, and Hendry' found less NaOH bound30 X IO-^ equivalents. Hoffman and Gortnerl report 2 0 X 10-5 equivalents bound at pH values of 1 0 . 2 and 10.5, and a maximum binding of 1400 X IO+ equivalents.
MG
N O O H i/5€/J
/-€eGM
Z€/N
FIG.1 2 Zein and NaOH in 95% Ethyl Alcohol
TABLEXIX I
Zein and NaOH in 95% Ethyl Alcohol gm. zein, equivalent to .963 gm. dry zein, used in each number Volume of each number--90 cc. Length of run-35 days
No.
I. 2.
3. 4. 5. 6. 7. 8. 9. IO. 11.
a cc. N NaOH added
0.66 I .31 1.97 2.62
3.28 3.93 4.59 5.24 5.90 6.55 6.33
b cc. N acid for I O cc. su mat. &uid 0 .oo
0.00
0.09
0.19 0.42 0.66 0.88 I ,32 1.59 1.73 2.35
0.21 0.33 0.44 0.66 0.79 0.87 I . I8 I .41 1.44
J. Gen. Physiol., 7, 81 (1924-5). q (1925).
* Colloid Symp. Mon., 2, z
d e C cc. N base mg. NaOH per c,c. N base gm.zem in super. liquid &?zg d X40.008 equiv. X IO-' b x a (a - cY.963
2.82
2.88
0.68 1.17 I .60 2.04 2.49 2.71
3.11 3.64 3.67 3.86 3.58
27.2
46.6 64.0 81.5 99.5 108.5 124.5 145.7 147 ' 1 154.8 I43 .o
PHASE RULE STVDIES ON THE PROTEINS
1317
Because zein went into solution almost completely in the 95% ethyl alcohol, runs were tried in 85 % acetone. This seemed to be an ideal solvent for Foreman1 says that it may be substituted for 95% alcohol in titrating amino and carboxyl groyps, and Galeotti and Giampalmoz say that zein is insoluble in water-acetone mixtures. But there was still considerable peptization of the zein in the acid and alkaline acetone. The hydrolysis of the
FIQ.13 Gliadin and HC1 in 95% Ethyl Alcohol
zein was 15% in acid and 2 0 % in alkali. The run with NaOH got very dark brown, so that the end-points were very poor; and the run with HC1 had poor end-points, although the solutions were clear. With HC1 the first part of the curve was the same as the one in 95yo alcohol. Then the increased hydrolysis of the zein in acetone became apparent in a break in the curve which veered far to the right. With alkali, apparently much more was taken up in the form of a compound than in alcohol. We are inclined to doubt this. These runs in acetone were far from satisfactory. 4. Gliadin-The gliadin was a gift to this department from Professor R. A. Gortner of the University of Minnesota. With HCl in 95% ethyl alcohol, gliadin did not peptize and hydrolyzed to the extent of about 1 . 4 7 ~ . Contrary to expectations, it showed absolutely no indication of compound formation. (See Table XX and Fig. 13.) Bancroft and Barnett3 obtained a curve with three flats and therefore showing three compounds, or at least three different pressures of HC1 necessary for the formation of one or more compounds. In order to show whether our curve represented the true result under the conditions of our experiment, or whether it was lack of equilibrium, we made gliadin hydrochloride by the method of Bancoft and Barnett. This was put into 9jY0ethyl alcohol. I t lost HCl until it reached a point (A-Fig. 13) on our curve, which must, therefore, be correct. Biochem. J., 14, 4
I
(1920).
* Kolloid-Z., 3, I18 f1908)
J. Phys. Chem., 34, 449 '(1930).
1318
WILDER D. BANCROFT AND 8. LOUISA RIDGWAY
TABLE XX Gliadin and HCl in 95% Ethyl Alcohol I
gm. gliadin, equivalent to .946 gm. dry gliadin, used in each number Volume in each number-zo cc. Length of run-30 days a
No.
cc. N HC1 added
b cc. N base for Io cc. supernat. liquid
d
C
c?. N acid m super. liquid b X 2
e
cc. N acid mg. HC1 used per gm. g l i a g gm. gliadin d X 36.46
equiv. X IO-' (8 - ~)/.946 0.44
16.2
I.
0.43
0.003
0.007
2.
0.86
0.10
0.20
0.69
25.3
3.
1.28
0.24
0.48
0.86
31.4
4.
I
.71
0.79
0.98
35.7
5.
2.14
0.39 0.60
I .20
0.99
6.
I
.46
1.17
36.3 42.5
I
.60
1.57
57.2
2
.oo
I
.SI
7.
2.57 3 .OS
8.
3.42
0.73 0.80 I .oo
9.
4.28
1.33
2.66
1.71
54.9 62.2
IO.
5.13
I
.61
3.23
2 .OI
73.3
Compounds of gliadin with acid have been reported to contain: 28 x 10-6 equivalents by Bracewell' 34 X 10-6 equivalents by Cohn2 40 X 10-6 equivalents at pH 2 . 5 by Hoffman and Gortne? and to bind a maximum of 600 X IO-^ equivalents 184, 288, and 387 X IO+ equivalents in three compounds by Bancroft and Barnett.' With NaOH in 95% ethyl alcohol, there was some peptization and 14% hydrolysis of the gliadin. A compound (see Table XXI and Fig. 14) was formed which had 25 mg. or 62 X IO-) equivalents of base per gm. gliadin. The combining weight with NaOH is then 1600. Some values given for the binding of alkali by gliadin are: 2 0 X IO-^ equivalents by Woodman6 3 0 X IO-^ equivalents by Greenberg and Schmidt6 and Cohn2 2 0 X IO+ equivalents a t pH values of 10.2 and 10.5 and a maximum of 1300 X IO-^ equivalents by Hoffman and Gortner.' J. Am. Chem. Soc., 41,
1511 (1919).
* Phyaiol. Rev., 5, 349 (1925). a
Colloid Symp. Mon., 2, zog (1925).
6
J. Agri. Science, 12, 231 (1922). Proc. 9oc. Expt. Biol. Med., 21, 281 (1924). Colloid Symp. Mon., 2, zog (1925).
' J. Phys. Chem., 34, 449 (1930).
TABLE XXI Gliadin and NaOH in 95% Ethyl Alcohol I
gm. gliadin, equivalent to .946 gm. dry gliadin, used in each number Volume in each number-20 cc. Length of run-30 days No.
B
b
C
cc. N NaOH added
cc. N acid for I O cc. supernat. liquid
c c N baae in super. liquid
bX2
I.
0.58
0 .oo
0.00
2.
1.15
0.07
0.15
3. 4.
1.73 2.30 2.88 3.45 4.03 4.60 5.18 5.75
0.18 0.28 0.39 0.44
0.37
5.
6. 7. 8.
9. IO.
0.71 0.88
0.96 0.97
d baee
cc. N
per.
gm. gliadin equiv. x 1 0 - 3 (a - c)/.946
0.61 I .06 1.44 .85
0.55
I
0.78 0.88 I .41 1.77
2.22
I .92
1.93
e
mg. NaOH per
gm.gliadin d X 40.008
24.3 42.5 57.5 74.0 88.8 108.7
2.72 2.76
110.6
3.w 3.44 4.04
119.9 137.7 161.7
5 . Edeslin-Edestin was purchased from Eimer and Amend and used as it was bought. With HCl in 95% ethyl alcohol, there was no peptization and about 1.470 hydrolysis of the edestin. A compound (see Table XXII and Fig. 15) was formed with 45 mg. or 124 X IO-^ equivalents of HCl per gm. of edestin; or edestin has a combining weight of 810. Other values for the binding of acid by edestin are :
1320
WILDER D. BANCROFT AND 6 . LOUISA RIDGWAY
18 X IO-^ equivalents as the amount of strong monobasic acid required to
dissolve
I
gm. edestin by Hardy'
14 X IO-^ equivalents necessary to "saturate"
acid with edestin by
OsborneZb 115 X IO-^ equivalents at pH 127
2.5
by Sandstrom3
X IO-^ equivalents by Cohn4 as the best value recalculated from H i t c h c ~ c k , ~ Kodama,'j "~ and Osborne."
130 X IO-^ equivalents using tropaeolin as indicator by Osborne." 134 X
IO+
equivalents as his best value by H i t c h c o ~ k . ~ " ~ ~ ~ '
250 X IO-^ equivalents by Bancroft and Barnett.'
TABLEX X I I
I
Edestin and HCl in 95% Ethyl 'Alcohol gm. edestin, equivalent to .928 gm. dry edestin, used in each number Volume in each number-zo cc. Length of run-3 I days a
d
added
b cc. N base for I O cc. supernat. liquid
I.
0.43
0.00
0.00
0.46
16.8
2.
0.00
0.00
0.92
3.
0.86 I. 28
0.04
0.08
1.27
33.6 46.2
4.
1.71
0.23
0.46
1.35
49
I
.46
53.1
1.58
57.6
No.
cc. N HC1
C
cc.
N acid
in super. liquid b X 2
cc.
N acid
used per gm. edestin equiv. X 10-8 (a - c)/.928
e
mg. HCI per gm. edestin d X 36.46
'
1
5.
2.14
0.39
0.79
6.
2.57
0,55
1.10
7.
3.08
0.80
I
.60
I
8.
3.42
I
.oo
2
.oo
1.53
55.9
9.
4.28
I
.36
2.73
I
.67
60.8
IO.
5 . I3
I
.76
3.53
1.73
63 . o
.60
58.2
J. Physiol., 33, 2 5 1 (190j).
* (a) J. Am. Chem. SOC., 21, 486 (1899); (b) 24, 39 (1902). J. Phys. Chem., 34, 1071 (1930). Physiol. Rev., 5, 349 (1925). 6 (a) J. Gen. Physiol., 4, 597 (1921-22);(b) 5, 383 (1922-3); (c) 14, 99 (1930-31). 6 J. Biochem. (Japan), 1, 419 (1922). 7 J. Phys. Chern., 34, 449 (1930). 3 4
1321
PHASE RULE STUDIES ON THE PROTEINS
FIG.1 5 Edestin and HC1 in 95% Ethyl Alcohol
TABLEXXIII Edestin and NaOH in g 5% Ethyl Alcohol I
gm. edestin, equivalent to ,928 gm. dry edestin, used in each number Volume in each number--no cc. Length of run-31 days added
b cc. N acid for I O cc. supernat. liquid
1.
0.58
0.00
0.00
0.62
24.8
2.
1.15
0.02
3. 4.
1.73 2.30
0.04 0.24
1.19 I .60
47.7 64.0
0.23
5.
2.88 3.45 4.03 4.60 5 . I8
1.99 2.37 3.04
79.7 94.6
6. 7. 8. 9.
0.45 0.68 0.63
121.7
1.25
2.99
119.6
.48 .87
134.6 142.5
IO.
5.75
3.36 3.56 3.80
a
No.
cc. N NaOH
C
e!. N base
in super. liquid b x z
d cc. N base used per gm. edestin equiv. X 1 0 - 3 (a
0.12
0.34 0.31 0.63 0 ' 74
I
0.93
I
1.11
2.22
e
mg. NaOH per gm. edestin d X 40.008
- c)/.928
152
.o
With NaOH in 95% ethyl alcohol, there was no peptization. There was hydrolysis of the edestin. A compound was Pormed (see Table XXIII and Fig. 16) on which the hydrolysis made the curve appear to represent a large amount of adsorption. The compound had 43 mg. or 108 X IO& 11%
1322
WILDER D. BANCROFT AND S. LOUISA RIDGWAP
0
50 100 MC. NOOH US€/J PER C N €LJ€St/N
FIG.16 Edeatin an< NsOH in 95% Ethyl Alcohol
equivalents of NaOH per gm. edestin, and therefore gives the protein a combining weight of 925. Other values for edestin and alkali are: I O X IO-^ equivalents as necessary to dissolve edestin by Hardy' 7 X IO-^ equivalents to "saturate" alkali with edestin by Osbornel 36 X IO+ equivalents a t pH 10.5 by Sandstrom' 7 5 x 10-5 equivalents by Kodama' as recalculated by Cohns 90 X IO-^ equivalents by Hitchcock' as recalculated by Cohn5 summary
The phase rule method of studying proteins, developed by Bancroft and Barnett, has been extended to apply to acids and bases dissolved in a solvent, chemically inert to the system, which does not dissolve the protein or its product with acid or base. 2. The method of titrating excess strong acid or base in alcoholic solution in the presence of weak acids or bases, using thymolphthalein and thymol blue, has been applied to peptized proteins and their hydrolysis products. 3. Succinic acid, used as a test of the method, was found to form a monoand a di-sodium salt. 4. Uric acid formed a mono- and a di-sodium salt; and in the presence of excess alkali, formed a tri- and a tetra-sodium salt. These formed much more readily in 95% isobutyl than in 95% ethyl alcohol. The presence of a small percent of water in the alcohol hastened the formation of the compounds but was not necessary to it. I.
-
J. Physiol., 33, 251 (1905). J. Am. Chem. SOC.,24, 39 (1902). a J. Phys. Chem., 34, 1071 (1930). 6 J. Biochem. (Japan), 1, 419 (1922). 5 Physiol. Rev., 5, 349 (1925). 6 J. Gen. Physiol., 4,597 (1921-22).
*
PHASE RULE STUDIES ON THE PROTEINS
'323
5 . Uracil formed a compound with one equivalent of NaOH in 95% ethyl alcohol. With HCI, no compound formed, and there was very little adsorption. 6 . Alanine formed a compound with one equivalent of NaOH and another with one equivalent of HC1. 7. Casein, gelatine, and edestin showed compound formation with HC1 in ethyl alcohol. Zein and gliadin showed only adsorption with HC1. 8. Casein, gelatine, zein, gliadin, and edestin showed possible compound formation with NaOH in ethyl alcohol plus marked adsorption, or adsorption alone with the adsorption practically complete a t the lower concentrations. 9. In general, less HCI was taken up from contact with an alcoholic solution than from contact with gaseous HCl. cornell UniVer&.
