I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
1298
Vol. 23, No. 11
Dilatometric Measurement of Protein Hydration’ K. H. Gustavson MAX
HERTZLEATIIER Co., NEWARK, N. J.
of the system. Theis a n d collaborators (117, who have studied the volume changes of such systems. The contraction or the expansion of the total volume has been assumed, without critical study or more profound investigations, to be a measurement of the degree of hydration or dehydration of the proteins. It was pointed out previously (15)in a review of the first of the hydration studies from Lehigh University, that it is questionable if any contraction of a system, such as hide-aqueous solutions, indicates simply the changed degree of hydration of the hide. Since the writing of this review, a paper on the hydration of gelatin as a function of H-ion concentration has appeared ( I d ) , and this article has been critically examined. This paper, as well as all other publications in this field by Theis, is lacking in exactness, in clear statements of experimental conditions, and in the terms of measurements. Therefore, they do not lend themselves readily to a quantitative examination. The few semi-quantitative data contained in the paper cited are, however, sufficient to establish the fact that the phenomenon measured and labeled as hydration of the proteins bears no relation to the actual hydration of the protein. Around this hydration concept, which it is the intention of the present paper to show to be a fallacy, a number of more or less systematic investigations of several tanning processes have been carried out by Theis and his collaborators.
is
change, which shown by his r e c e n t m o n o g r a p h ( 1 , 8). The unquestionable success of the Zwitter-ionic concept of Bjerrum ( 1 ) upon protein behavior has been the main factor in this conversion of Pauli’s views. Pauli’s opinion of the hydration of proteins, resulting in a compression of the water shell around the protein particles, is taken by Theis to imply “that if this is the correct view, it follow that a contraction of the system when gelatin is soaked or dissolved in water must indicate hydration” ( I d ) . From the graph in Figure 1 2 which gives the variation in the contraction of the system (or the hydration of gelatin) with variation in the final pH values of the solutions, the contraction shows a maximum value of 0.175 cc. in the pH range from 5 to 6; this corresponds to the isoelectric zone of 20 grams of gelatin in a total volume of 150 cc. A distinct minimum in contraction of 0.075 cc. occurs a t an equilibrium pH of 1.5-2.0. On the alkaline side of the isoelectric zone of the protein, the contraction is only slightly less than that observed in the isoelectric zone. But as the greatest pH value employed is only 9.0, the data do not permit a study of the very interesting behavior of proteins from the Zwitter-ionic standpoint in the pH range of 9 to 13. From a superficial examination, the authors draw the farreaching conclusion that the maximum hydration of gelatin
Value of the Technic
I n extending this volume-change technic from a relatively homogeneous system, as that of aqueous-gelatin solutions to pieces of raw hide in various solutions, the question of the experimental errors assumes a still more prominent place. The experimental difficulties and the experimental errors inherent in the measurements of the very small total volume changes in such heterogeneous systems (and with such wide variances in composition of the raw hide) as hide and aqueous solutions will not be considered in this discussion. A critical examination of the dilatometric technic under such difficult conditions must first be established by more precise measurements. It might, however, be in order to point out that a piece of hide with the hair on, and other materials, such as gases, occluded, probably includes, in itself, factors of a magnitude to invalidate this technic from a scientific standpoint. Furthermore, in the studies of the effect of time upon the hydration of raw hide, the bacterial action with its attendant gas formation adds another error. Therefore, it seems safe to conclude that the volume-change measurements, interpreted in terms of hydration of the hide protein, are devoid of any scientific meaning whatsoever. Kevertheless the dilatometric technic might have (and probably has, according to the experiences of critical observers) practical value as a sharp indicator of the point a t which less desirable changes, such as putrefaction of the hide, set in. Theis utilizes Pauli’s old views on the hydration of proteins without proper use of the literature on the subject. It may also be pointed out that Pauli’s ideas (6) on the hydra1
Received July 21, 1931.
I / E 0.05
2
,
,
,
,
,
,
T ,I 2 6 ,,s
,
,
0.00
0
a
2
3
8
4
e
7
B
s a o n
vum Figure 1 PB
occurs in its isoelectric range, thereby disclaiming Pauli’s statement that protein ionization connotes increased protein hydration, and, placing a maximum instead of a minimum of protein hydration in its isoelectric state. A critical study of the investigations of recent years leads to the view that there is neither a maximum nor minimum of hydration of the proteins in their isoelectric zones, but that the hydration of proteins shows no relationship to the H-ion concentration of the medium, or, in other words, to the degree of ionization of the protein. Previous Investigations
Only en passant, the present writer wishes to call to the attention of these authors a number of investigations which 2
This illustration is Theis’ Figure 4 in IND. ENG.CHBM.,22, 59 (1930)
IXD USTRIAL AXD EiYGIiVEERIiVG CHEMISTRY
November, 1931
clearly demonstrate the independence of protein hydration from H-ion concentration. Measurements of the hydration volume by Polanyi’s “nichtlosender Raum” technic (the water in an aqueous colloidal system, which is not available as a solvent for added crystalloids) in solutions of albumins from pH 1.5 to pH 8 by Weber (14), in his excellent series of investigations, substantiate this view. Sorensen (9) also demonstrates the independence of the weight of the protein hydrate of egg albumin as a function of its pH values. The independence of the hydration energy from the protein ionization in muscles has been demonstrated by Meyerhof ( 5 ) . The most striking contribution is Weber’s studies in the important paper on “The Bjerrum Zwitter-Ionic Theory and the Hydration of Proteins” ( I S ) , and the paper by Weber and Nachmannsohn on “The Independence of Protein Hydration from Protein Ionization” (14). In these investigations the dilatometer is used for measuring the volume changes in the simple systems of salts, acid, and bases upon their interaction; and for solutions of amino acids, polypeptides, and the more complicated systems of proteins in their reactions with acids and bases. From Tammann’s research (IO) it is known that aqueous sdutions of substances, with marked tendencies for hydration, behave, in regard to their compressibility, as water under increased pressure. The latter is a measure of the hydration forces for substances with a high degree ofhydration, such as inorganic ions ( 2 , 3 ) . This is the same view that Pauli applied to the polar aquo groups which take part in the hydration of proteins and which concept was uncritically annexed by Theis. The hydration capacity of proteins is relatively small compared to inorganic ions, and the change in internal pressure, due to the hydration forces, is dwarfed by other factors which influence the total volume of the system. In any neutralization process, resulting from the mixing of dilute solutions of strong acids and bases, such as 0.1 -V HC1 and 0.1 N NaOH in a quantity to form one gram equivalent of H20,a volume dilatation of -22 cc. is observed. Av in cc. = OH-) gram mole ( H +
+
+ -22
The dilatation, Aui, is in most cases controlled by the ionic reaction and the sum of the other factors which accompany the mixing of the solutions ( AVM A&) is negligible. In the systems of amino acids and proteins investigated by Weber, the conditions warrant an equal sign between Avi, the volume changes due to the ionization process, and Av, the total volume changes. In extensive series of measurements on solutions of simple organic acids, organic bases, and amino acids as the typical ampholytes, Weber reports the following volume changes in centimeters per mole of H+ or OH- combined. (Dilatation is indicated by the sign, and contraction is indicated by the - sign):
+
+
BASIC&oms-In
organic nitrogenous bases, as aliphatic and
aromatic amines.
+
R.NH2 H + +R.NHa+ R.NH,’+ O H - - - - t R . N H *
Av in cc. gram mole H+or OH- combined - 2 H20 24
+ H + + OH +22 CARBOXYL GROUPS-In aliphatic monocarboxylic acids, such +
-
as acetic, propionic, lactic, and butyric. R . COOR.COOH
++
Av in cc. gram mole H + or OH-combined H + +R . COOH 12 OH-+R.COO-+ Hz0 9
++
H+ + OH-
+21
1299
PHENOLIC GRouPs-Measured PhOPhOH
on phenol and dextrose. Av in cc. gram mole H+or OH- combined 19
+
+ H+ PhOH + OH- +PhO- + Hz0 H + + OH---f
+ 2 +21
Addition and removal of one gram equivalent of H ion (addition of one gram equivalent of OH-) results thus in a dilatation of 21 cc. in all instances. An important fact brought out by these data is that the values of Av for the fixation of H + or OH- by basic (amino) and acidic (carboxyl) groups show such wide differences that, by their means, an unequivocal decision is afforded to determine, in reactions of H+and OH- with ampholytes, which groups enter into the reactions with these ions. The simplest amino acid, the glycine, upon attachment of one mole of H and OH ions,.gives the following values of Av on one gram-equivalent basis. Au in cc. gram mole H+or OH- combined H++glycine cation 7.1 Glycine Glycine cation OH- +glycine H2O 15 2
+ +
+ +
+
H + + OH+ ++ glycine anion + H?O H + +glycine H + + OH-
+22.3 +23.6 - 1.8
Glycine OHGlycine anion
+21.8
Thus the addition of one mole of H + to the glycine and a subsequent de-ionization of the formed glycine cation by one mole of OH ions (resulting in the formation of electro-neutral glycine, the initial substance) shows the same value of Av as that found in the simple reactions between dilute solutions of strong acids and bases: H t f OH-
+HzO;
Av =
N
+22
CC.
In this cyclical process the amino acid acts as an acceptor for H and OH ions. By removal of H and OH ions in the neutralization process, the dilatation is considered due to the release of aquo groups held by these highly hydrated ions, upon their transformation into H20. The volume change observed in the solution of glycine is, by analogy, due to the discharge of H+ and OH- in their combination with the amphoteric glycine molecule. For isoelectric gelatin the Av values are of the same order as those reported for glycine. Av in cc. gram mole H+or OH- combined Gelatin H + +gelatin cation 9.9 Gelatin cation OH - +gelatin HzO 11,6
+ +
+
+
Gelatin OHGelatin anion
H+ OHgelatin anion H20 H + +gelatin
+
+21.5 17.2 3.8
+ OH-
SE1.0
---t
+
++
+
H+
+ +
The very important point in Weber’s clarifying researches is the method it affords for the differentiation between the different protein groups involved in reactions with H and OH ions, because, as before mentioned, the values of Av differ widely. Considering glycine as a prototype in reactivity for proteins, the classical concept formulates the addition of one mole of HC1, as HOCO.CHz.NH2
+ H + + C1-+
HOCO.CHz.NH3+
+ C1-
The volume change in the interaction of H ions with primary amino groups, as in methylamine and aniline, shows a value of Av = -2 cc. For glycine, a Av value of +7.1 cc. is obtained. Accordingly, the reaction cannot be formulated as previously. Applying the concept of the glycine molecule as an amphoteric ion,
INDUSTRIAL A N D ENGINEERING-CHEMISTRY
1300
OCO-.CHa.NHs++ H + + Cl-+HOCO.CHZ.NHs+
+ C1-
Thus, H ions are attached to carboxyl ions, which are comThe reaction pletely discharged a t a p H value of -2. between carboxyl ions in the simple monocarboxylic acids and H ions showed a Av of + I 2 cc., which value agrees reasonably well with that of +7.1 cc. for glycine. These measurements support the view of the amphoteric ionic nature of amino acids in their isoelectric state. I n the reaction between glycine and hydroxyl ions, the classical concept gives HOC0 . CHn. NH2 N a + OH- + Na+ OCO-.CH2.KH2 H20 Instead of the expected value of Av of $9.0 cc. for the reaction of OH- with the carboxyl group, according to the previous scheme, the value for glycine is $23.6 cc. The values vary far too much. According to the Zwitter-ionic concept the reaction is formulated as OCO-. CH1.NH3+ N a + OH- + Na+ OCO-. CHz.NH2 Ha0 Thus, addition of OH ions brings about a discharge of the NH3 ions in the Zwitter-ionic glycine structure, transforming the same into NH2 groups by a process of hydrolysis, which is complete a t a pH value of about 12.75. The simple amines in cationic form show a Av value of $24 cc., in close agreement with the +23.6 cc. observed for glycine. Weber’s logical researches form the very strongest evidence for the existence of proteins in their isoelectric state as amphoteric ions. Harris’ important contributions (4) in his studies of the titration curves of ampholytes in solutions, with or without the addition of formaldehyde, further substantiate the ideas of Bjerrum that proteins in electro neutral state exist, to a great extent, as polyvalent Zwitter ions. The important bearing of this concept upon the neutral salt effect on hide proteins, the theory of chrome tanning and several other problems of great theoretical and practical importance in the manufacture of leather, such as the difference between formaldehyde and quinone tannages, on one hand, and the vegetable tanning process, on the other, in regard to their pH function, will be discussed in a coming paper.
-
+
+
+
+
+
+
-
+
+
Conclusions Accordingly, it is evident from this discussion that the dilatation of solutions of gelatin upon their interaction with H and OH ions is not caused by changes in the hydration of the gelatin, but is due to the removal of highly hydrated hydrogen ions (oxonium ions, OH$+)and hydroxyl ions, which, upon their discharge by the combination, donate their aquo holdings to the system which accordingly must expand, as the pressure on the HzO, which has been associated with the H ion, is released: HIO+ protein +H.protein+ H20
+
+
A simple dissection of the values obtained for the hydration of gelatin as a function of the final pH of the medium by the authors of the paper under examination, as given in their graph in Figure 4 ( 1 2 ) , 3 is illuminating, as it brings out the fact that the volume dilatation observed upon decreasing the final p H values of the aqueous gelatin system from a value of 5, the H-ion concentration corresponding to the isoelectric zone, to the point of complete interaction of the 2 , is solely due to the gelatin with H ions, or a pH of removal of H ions from the solution. As 20-gram portions of flake gelatin were used by the authors in most of their experiments, it is assumed to be the quantity used in their experiments graphically illustrated in their Figure 4. On a
-
8
Figure 1 of this text.
Vol. 23, No. 11
15 per cent moisture basis, the weight of dry gelatin is 17 grams. The maximum H-ion fixation by gelatin is reached a t a p H of about 2, where 1 gram of gelatin binds 0.84 millimole of H ions. Thus 17 grams of gelatin have fixed 17 X 0.84 = 14.28 millimoles or milligrams H’. The observed dilatation of the system, as going from a pH of 5 to a pH of 2 , was estimated from the graph to be from 0.175 to 0.075 cc., or 0.100 cc. Accordingly, Av in cc. per gram of H ion combined is 0.100 divided by 0.01428, or $7.4 cc. Weber’s corresponding value for gelatin is +9.9 cc. The agreement between these values is satisfactory, in view of the approximations necessary in this graphical estimate and of the incomplete and less precise statements of the authors. From the author’s own studies of the reactivity of hide proteins, it is indicated that uncharged acidic and basic protein groups are present in these particular proteins and thus that the extent of Zwitter-ion formation is not a t its maximum value. As the reaction between H ions and NHz groups shows a slight contraction, the presence of uncharged groups in gelatin would tend to lower the value of Av, derived under the assumption of a complete amphoteric ionic structure of the electro-neutral protein. On the alkaline side of the electro-neutral zone of the gelatin up to a pH value of 9 (the most alkaline point covered by the authors) the curve shows only a slight deviation toward ryl increase in Av, which general trend is to be expected, as the extent of t’he fixation of OH ions up to this p H value is very small. Weber has shown that the de-ionization of NH3 ions first begins at p H values greater than 9, and that the interaction is complete a t a p H value of 12.75. The removal of amino groups by deaminization does not interfere with the alkali-binding capacity of the protein in the p H range from 5 to 9, which demonstrates the fact that NHS ions do not come into play. I n the pH range from 9 to 12.75, the deaminization results in a 0.4-millimole decrease in the amount of OH ions being fixed by the deaminized gelatin compared to gelatin proper. A differentiation of the reactivity of various groups in the proteins is also feasible by means of the Linderstrom-Lang and Willstatter titration methods in acetone and alcoholic solutions, in which solvents the activity of the acidic and basic protein groups is repressed. An extension of the hydration curve in the authors’ graph by measurements up to a p H of 13 (the point of maximum hydroxyl-ion fixation) would have given an approximate figure of 0.257 cc. from the considerations given previously. Instead of the dotted line with a slight curvature, which simply has been added to the graph, as no value a t p H 10 is plotted (for what reason it is.not clear t2 the writer) a sharp drop of the curve from p H 9 to a p H of 12.75 should result, reaching a value corresponding to about 0.08 cc. on the negative side of the y-axis. Literature Cited (1) Bjerrum, Z . physik. Chem., 104, 147 (1923). (2) Fajans, Nafurwissenschuffen, 9, 727 (1921). (3) Fricke, Z . Elektrochem., 28, 161 (1922). (4) Harris, Biochem. J., 24, 1080 (1930). (5) Meyerhof, PAiigers Arch., 195, 22 (1922); 204, 295 (1924). (6) Pauli, “The Colloid Chemistry of the Proteins,” p. 10, Blakiston, 1922. (7) Pauli, Kolloid-Z., 63, 51 (1930). ( 8 ) Pauli and Valk6, “Elektrochemie der Kolloide,” p. 391, Springer, 1929. (9) SBrensen, Z . physiol. Chem., 106, l ( 1 9 1 9 ) . (10) Tammann, “i’ber die Beziehungen zwischen den inneren Kraften und Eigenschaften der Loslingen,” Voss. Leipzig, 1907. (11) Theis and Neville, IND.ENG.CHEM.,21, 377 (1929); 22, 64; 22, 66 (1930). (12) Theis et al., Ibid.. 22, 57 (1930). (13) Weber, Biochem. Z., 218, 1 (1930). (14) Weber and Nachmannsohn, Ibid., 204, 215 (1929). (15) West, “Annual Survey of American Chemistry,” Vol. IV, chapter on “Leather,” Chemical Catalog, 1930.
