studies in the physical chemistry of the proteins · 498 physical chemistry of proteins. viii it...

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS

VIII. THE SOLUBILITY OF HEMOGLOBIN IN CONCENTRATED SALT SOLUTIONS. A STUDY OF THE SALTING OUT OF

PROTEINS*

BY ARDA ALDEN GREENt

(From the Department of Physical Chemistry in the Laboratories of Physiology, Harvard Medical School, Boston)

(Received for publication, July 1, 1931)

Precipitation by concentrated salt solutions has for long proved the most satisfactory method for the separation of proteins. This procedure was first employed in the middle of the last century by Panum (18), by Virchow (28), and by Bernard (2). The diminu- tion in solubility of proteins in concentrated electrolyte solutions has ever since led to methods for their separation, purification, characterization, and occasionally, classification. The nature of the phenomenon was shown by Hofmeister (13) to depend upon the character of the neutral salt as well as on the protein.

Proteins may be “salted out” by a relatively small increase in electrolyte concentration. Moreover, the effectiveness of concentrated solutions of different salts in the precipitation of proteins varies greatly. It has often been the custom to determine the concentration of salt at which the protein first begins to precipitate, or the concentration at which no more can be induced to precipitate. The small difference in concentration of salt in which most proteins are on the one hand relatively soluble and on the other hand largely precipitated, yielded results which, although rather rough, led to a qualitative description of the phenomenon.

* A preliminary report of a portion of this investigation was read by Cohn and Green (7) at the Twenty-second Annual Meeting of the American Society of Biological Chemists.

t National Research Council Fellow in Medicine, 1927-29, during which time the experiments herein reported were performed.

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496 Physical Chemistry of Proteins. VIII

The decreased solubility of other types of substances in the presence of strong electrolytes must be considered analogous to, the precipitation of proteins. In 1892, Setschenow (22) described the decrease in solubility of gases in salt solutions in terms of an exponent,ial equation, empirically derived.

S = So eke, or In S = In SO + kc (1)

where SO is the solubility in water, S the solubility in the electrolyte solution, c the concentration of the electrolyte, and k a constant. The solubility of other non-electrolytes, as well as of gases, in electrolyte solutions has also been studied experimentally. Linder- Strom-Lang (16) has described the solubility of quinone, hydroqui- none, and succinic and boracic acids in terms of this equation.

The deviation in solubility in the presence of electrolytes from that expected on the basis of the ideal gas laws is one measure of the “activity coefficient” of the saturating body (15): Debye and MacAuley (9) described the activity coefficients of sugar solutions in the presence of salts in terms of a theoretical equation in which t,he activity coeficient of the non-electrolyte is, as a first approximation, proportional to the electrolyte concentration. Hiickel (14), working on the salting out of electrolytes in electrolyte solutions, added to the original Debye equation an expression in which the variation due to change in the dielectric constant of the solution is also considered a linear function of the concentration. Scatchard (21) has further considered the influence of both electrolytes and non-electrolytes on activity coefficients and Tammann (27) and Randall and Failey (19) have presented and discussed a large amount of bhe existing data on the solubility of various hon-electrolytes in terms of an equation similar to equation (1).

Following the theoretical derivation of the salting out equation for non-electrolytes by Debye and MacAuley and for electrolytes by Hiickel, its applicability to the proteins was demonstrated by Cohn (5) who suggested that equations of this type had been found to hold for both strong electrolytes and nonelectrolytes, and might be employed in considering the solubility of proteins. He used an equation of the type:

log S = ,9 - K,' p (2)


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A. A. Green 497

in which S is again the solubility, p is the ionic strength, and 6 is an intercept constant, which in the case of non-electrolytes is log So, and K,’ is the salting out constant. This constant must be considered apparent for certain proteins, since globulins, at least, increase in solubility in dilute salt solutions, and this solvent action considered in subsequent communications, must be considered to be effective to some extent, even in concentrated solutions.

The quantitative data of Chick and Martin (3) on the solubility of egg albumin and of Sorensen (23) on pseudoglobulin and of Sorensen and Hoyrup (24) on egg albumin were analyzed by Cohn employing the above relationship in which the logarithm of the solubility was found to be a linear function of the concentration of the ammonium sulfate used as precipitant. That this same exponential relation holds for other proteins in sufficiently concentrated salt solutions has since been reported for hemoglobin by Cohn and Green (7) and for fibrinogen by Florkin (11).

In 1888, Hofmeister (13) studied the effect of protein concentration on the amount of salt necessary for precipitation. Solutions containing a given concentration of protein but varying concentrations of salt were compared, and the salt concentration which would just induce precipitation was noted. This procedure was repeated with other concentrations of protein. When the protein just begins to separate out the solution may be regarded as saturated with respect to that component, and the concentration of the protein to yield its solubility. The protein used was egg white, and the salts, potassium acetate and ammonium sulfate. These data have, because of their historical interest, been recalculated, and are represented in Fig. 1. In the more careful investigations upon chemical individuals that have since been conducted, equilibrium has presumably been more nearly attained, and temperature and acidity more accurately controlled. None the less there can be no doubt that this early investigation beauti- fully reflects the relations that have subsequently been developed.

Horse hemoglobin has been chosen for the present series of investigations because it is a chemical individual, is readily separated by crystallization, and, being a globulin, can be studied both in dilute and in concentrated salt solutions. Its molecular weight has been determined both by osmotic pressure (1) and by ultracentrifugation (26) methods to be approximately 66,800.


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It therefore contains 4 iron atoms and four hematin groups in each molecule. The ultracentrifugation method of Svedberg has also demonstrated that aggregates of molecules are not formed in the presence of 0.019 M phosphate buffers from pH 6.0 to pH 9.05.

Further, Cohn and Prentiss (8) showed that the solubility of oxyhemoglobin was independent of the amount of protein in the solid phase. It dissolved in successive portions of a solvent to the same extent, until the saturating body had been completely dissolved. Carboxyhemoglobin, however, has been used at 25”

IONIC STRENGTH

FIG. 1. Curves plotted from the data of Hofmeister (1888)

instead of oxyhemoglobin because of its greater stability at the higher temperature.

The solubility of crystalline horse hemoglobin in concentrated solutions of various salts has now been studied at constant temperature and pH and also in a given salt under varying conditions. The experimental results may be thus conveniently divided into two parts. The first deals with the change of solubility of oxy- and carboxyhemoglobin in phosphate buffer mixtures at varying temperature and acidity. The salting out constant, K,‘, in a given salt appears to be independent of pH and also of temperature, and therefore to yield a physical constant characteristic only of the protein and the salt. The variation of K.’ with the


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A. A. Green

salt, that is to say, the quantitative description of the phenomenon noted by Hofmeister, constitutes the second part of this communication.

Experimental Method

Crystalline horse hemoglobin was used in this series of experiments. It was prepared by the method developed in this laboratory (10). After the cells in titrated horse blood had been allowed to settle the plasma was decanted. The cells, 3 or 4 liters in quantity, were then suspended in 12 liters of ice-cold hypertonic salt solution. This was either 1.5 per cent NaCl or 1.8 per cent KCl. The hemoglobin was separated from this solution by means of the Sharples centrifuge. If the filtrate was too concentrated and crystallization began immediately, water was added. The hemoglobin was always retained in the cold room as much as possible.

Crystallization was effected by the addition of 0.1 N acid which was added drop by drop, while the hemoglobin was thoroughly stirred mechanically in order to prevent a local excess. 0.1 N sulfuric acid was used in preparing hemoglobin for the sulfate experiments and 0.1 M phosphoric acid for the phosphate experiments. The hemoglobin for the citrate experiment was prepared with acetic acid since the solubility of hemoglobin in sodium acetate was being studied on the same preparation.

The strongly acid and alkaline solutions used in the preparation of hemoglobin must be employed with great circumspection. If added directly to the protein without precautions as to the method of addition, denaturation would promptly result. If, however, they are added very slowly, and the solution is at the same time effectively stirred so that no local excess of acid or base occurs, there is no denaturation.

The onset of crystallization could usually be detected by the development of a sheen in the solution, unless the solution was too dilute or the hemoglobin not completely oxygenated. It was generally necessary to add about 150 cc. of 0.1 N acid for every liter of the original cells. This brought the hemoglobin into the neighborhood of its isoelectric point where crystallization should take place. If crystallization appeared to be unsuccessful a drop of the solution was placed upon a microscope slide and allowed


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to dry slightly. If the crystals so formed were found to be needles, the solution was still too alkaline. Horse hemoglobin crystallizes in rhomboidal plates in the neighborhood of pH 6.6, in needles at more alkaline reactions. Care was always taken not to add too much acid since hemoglobin is so readily destroyed.

Recrystallization was effected at least once and usually twice. This was accomplished by first carefully dissolving the hemoglobin by the addition of the smallest requisite amount of normal alkali. The alkali was added in the same careful manner as the acid. The hemoglobin was then recrystallized by the addition of an equivalent amount of 0.1 N acid. Either sodium or potassium hydroxide was used to dissolve the protein, depending on whether the salt used was one of sodium or of potassium. Sodium hydroxide was used in preparing the material for the magnesium sulfate experiment. After each crystallization the hemoglobin crystals were washed two or three times in distilled water. This was accomplished by centrifuging in the ordinary centrifuge and decanting the supernatant solution, adding a quantity of water equal to about one-third the volume of the centrifuged crystals, stirring thoroughly, and again centrifuging. The crystals were then dissolved in alkali and the solution centrifuged to remove any remaining cell debris. At this point the hemoglobin was thoroughly saturated with carbon monoxide if carboxyhemoglobin was t,o be used in the experiment. Acid was then added to effect recrystallization.

In most cases the last precipitation was accomplished in concentrated solutions of the electrolyte with which the experiment was concerned. The last crystallization was watched carefully, and if the concentration of hemoglobin was not too high, crystallization did not take place immediately upon neutralization of the alkali used in dissolving the crystals, and it was possible to transfer portions of the solution to vessels containing an amount of very concentrated electrolyte solution sufficient to yield the approximate salt concentration desired in the experiment. If the hemoglobin did crystallize in the dilute salt solution, the concentrated solution was added as soon as possible. Crystallization of the hemoglobin in an electrolyte solution of the approximate composition of that in which solubility was to be determined facilitated the attainment of equilibrium.


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A. A. Green 501

The solubility determinations were made in the manner previously described from this laboratory (8). The crystals were repeatedly equilibrated with a given salt solution at constant temperature, filtered, and the filtrate analyzed for nitrogen. All determinations were made as near pH 6.6 as possible, since this is the point of minimum solubility. The relative amounts of the mono- and dihydrogen phosphates necessary were calculated by the equation of Cohn (6). Since concentrations greater than those previously studied were also employed, the pH of the mixtures was checked with the hydrogen electrode. The pH of the saturated solutions of carboxyhemoglobin in the other electrolytes used was controlled by adjusting the pH of both the hemoglobin and the salt solution. When the solution was well saturated with carbon monoxide reproducible E.M.F. measurements could be made with the hydrogen electrode. The satisfactory use of the hydrogen electrode for carboxyhemoglobin has also been reported by Hastings et al. (12) and by Stadie and Hawes (25). The pH of the saturated solutions is recorded in Table V. In general it remains between pH 6.5 and 6.7, a range in which solubility changes but little.

In determining the solubility of carboxyhemoglobin at 25’ the crystals were placed in closed 250 cc. centrifuge bottles. The container was originally about half filled with crystals. The appropriate electrolyte solution was added and the mixture repeatedly and thoroughly saturated with carbon monoxide, since reduced hemoglobin is much more soluble than either oxyhemoglobin or carboxyhemoglobin. The containers were gently shaken for at least 5 hours in a constant temperature water bath at 25”. This period had been previously determined to be sufficient in which to attain equilibrium. The shaking apparatus was that previously described (4). After the solution had become saturated it was filtered at 25” through a Whatman No. 42 filter paper and the filtrate was analyzed. The crystals were returned to their container, more electrolyte solution was added, the mixture was resaturated with carbon monoxide and again equilibrated at 25”.

The oxyhemoglobin was brought into equilibrium with the phosphate buffer solutions, with use of the method of Cohn and Prentiss (8), by placing the hemoglobin in open vessels immersed


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in an ice water bath and stirring by rotating rods projecting into the cups. The use of open containers insures constant saturation of the hemoglobin with oxygen from the air. These solutions were also filtered through funnels immersed in an ice water bath. The carboxyhemoglobin solutions at 0’ were, however, equilibrated in closed vessels and a different shaking machine was used. These solutions were also filtered at 0”.

In all cases the crystals were washed with the salt solutions until the solubility of successive filtrates became constant. Solu- bility was calculated from the nitrogen analyses, by assuming horse hemoglobin to contain 17.7 per cent nitrogen.

Part I. Solubility of Hemoglobin in Concentrated Solutions of Phosphate

In order adequately to characterize solubility in concentrated electrolyte solutions the variation in p and K,‘, the constants in equation (2), have been estimated under different conditions.

The solubility of carboxyhemoglobin in potassium phosphate buffers at 25” and pH 6.6 is described by the data given in Table I. The results are given in det,ail in order to illustrate the constancy of solubility in successive filtrates and in different preparations, provided temperature and acidity are maintained constant. The solubility of carboxyhemoglobin and of oxyhemoglobin at 0”, and at the same pH is given in Table II. In order to conserve space the number of successive equilibrations and the average of the measurements in each experiment are published. The de- tailed protocols comparable to Table I for all other experiments reported in these papers are filed in the laboratory. In all tables the experiment number is given in the first column, experiments of the same number being upon the same preparation of hemoglobin. The experiments on oxyhemoglobin and on carboxyhemoglobin belong to different series.

When equilibrium was established it was assumed that the hemoglobin-salt solution contained the same amount of salt as the original electrolyte solution. Thus, in estimating the ionic strength the amount of water present per liter of solution saturated with hemoglobin was calculated by subtracting the weight of the hemoglobin, plus the weight of the salt in the original electrolyte solution, from the total weight of the solution. Calculations in


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A. A. Green

TABLE 1

lobin at 25’ in Phosphate Solutions at

503

H 6.6 Solubility of Carbox: -

.-

--

- -

- -

--

--

--

--

--

--

-

Solubility

gm. 6.72 6.61 6.44 6.61

m

2.32 6.59 6.94 1.84 0.841

2.69 2.53 2.56 2.58

2.76 2.59 2.74 0.72: 0.438

2.50 2.57 2.57 2.60

2.76 2.56 2.71 0.71; 0.433

1.75 1.58 1.63 1.60

2.97 1.64

1.07 1.05 1.02 1.015

1.04

1.74 0.451

0.29:

0.240

3.20 1.11 0.045

1.07: 0.52t Average..

3 3 4 5 6

! .1239 2.20 --

--

1.1453 2.60 1.26:

1.26:

0.525

0.525

Average..

5 4 5 6 7

Average..

-- 1.1434 2.60

3 3 4 5 6

0,525

-- 1.1574 2.80 --

--

..1658 3.00

Average.. 1.36;

3 3 4 5 6

Average.. 1.46: 0.527


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TABLE I-Concluded

3.20

-

-

1 -_

-

7 _-

T -

-

8”.

1.09 1.06 1.00

1.05 1.12 -

0.441 0.367 0.367 0.378

3.68 0.388 0.41E 1 1

0.446 0.463 0.429 0.429 0.423

3.68 0.438 -

0.475 i I -

0.294 I I

- 0.109 ,

- D.124

-

--

1.1646 3.00 ~-

Average . 1.46:

Average.. 1.65;

4 3 4 5 6 7

Average.. 1.65;

0.525

0.527

1.621

1.677

1.1830 3.40 --

-- ..I843 3.40

which the weight of hemoglobin was subtracted from the total weight of the solution and the remainder assumed to contain the same proportion of salt to water as in the original buffer yielded the same result, Analysis of the composition of salt solutions of varying nature and concentration containing hemoglobin, in- dicated that 1.4 gm. of hemoglobin displaced 1 gm. of water. Calculations could have been made using this value, but because of the relatively low amount of hemoglobin, all the methods of calculation yield approximately the same result. The symbol T is that introduced by Debye for the “ionic strength” per liter; while /I is rigorously employed as defined by Lewis ((15) p. 373), being calculated as the ionic strength per 1000 gm. of water. The solubility of the hemoglobin was experimentally determined in


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A. A. Green 505

gm. per liter. The results have been calculated in terms of gm. of solute per 1000 gm. of water and also in terms of the mol fraction, N, the molecular weight of horse hemoglobin being assumed to be 66,800. Both of these functions of solubility should .be independent of volume changes due to temperature.

TABLE II

Solubi& of Hemoglobin in Concentrated Phosphate Solutions at pH 6.6

Solubility of carboxyhemoglobin at 0”

nozs per 1.

0.879 1.072 1.267 1.267 1.462 1.462 1.657

0.524 0.526 0.527 0.527 0.527 0.527 0.527

1.1093 1.1257 1.1444 1.1427 1.1629 1.1627 1.1826

-

1.98 2.41 2.81 2.81 3.24 3.24 3.69

om. om.

68.7 75.5 52.8 57.9 22.6 24.3 22.8 24.5

9.56 10.3 12.08 13.0 3.62 3.9:

-

16 16 16 17 17 17 17 17

-

-

2.00 1.878 1.53 1.762 0.640 1.386 0.645 1.389 0.271 1.012 0.342 1.114 0.103 0.594

Solubility of oxyhemoglobin at 0”

0.524 1.1063 0.526 I, 1240 0.527 1.1426 0.527 1.1429 0.527 1.1526 0.527 1.1726 0.527 1.1815 0.527 1.1925

1.95 2.38 2.80 2.80 3.01 3.46 3.70 3.94

52.2 56.6 1.498 1.753 34.7 37.4 0.990 1.573 18.8 20.3 0.534 1.307 19.3 20.8 0.547 1.318 12.8 13.77 0.362 1.139 5.07 5.48 0.144 0.739 3.41 3.71 0.097 0.569 1.84 2.01 0.052 0.303

-

Variation of Solubility with Temperature-The values of p and K,’ in equation (2), at constant pH, namely 6.6, have been calculated, by means of simultaneous equations, for carboxyhemoglobin at 0” and at 25” and are given in Table III. It is to be noted that the calculated values of K,’ are remarkably constant for the different salt concentrations in both cases and have the same


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value, 0.88, at both temperatures. In Fig. 2, where the logarithm of the solubility is plotted against the ionic strength, the con- formity of the experimental points with the straight line demanded by the equation is evident. Moreover the lines are drawn parallel.

Whereas K,’ thus appears to be independent of temperature the intercept constant, p, varies widely; for carboxyhemoglobin at 0” it is3.86, and at 25’ 2.87, a difference of 1.0. Since /3 is a logarith- mic function this means that in concentrated salt solutions of the same ionic strength horse hemoglobin is 10 times more soluble at 0” than at 25”. This is an inversion of the solubility relations in dilute solutions where increase in temperature increases the solu-

TABLE III

Values of fl and K,’ for Hemoglobin in Concentrated Phosphate Solutions at pH 6.6

Oxyhemoglobin at 0” Carboxyhemoglobin at 0 Cartuxyhemoglobin at 25’

II KS’ B P KS’ B P KS B

2.80 0.864 3.78 2.41 0.864 3.85 2.32 0.908 2.88 3.01 0.867 3.79 2.81 0.878 3.89 2.76 0.901 2.87 3.46 0.846 3.78 3.24 0.891 3.86 2.97 0.886 2.85 3.70 0.869 3.83 3.69 0.906 3.84 3.20 0.869 2.88 3.94 0.953 3.77 3.68 0.854 2.87

__-

Average.. 0.879 3.79 0.885 3.86 0.884 2.87

bility. This increased solubility of proteins in concentrated solutions of salt in the cold has been used by Redfield’ as a means of crystallizing hemocyanin, and by the author in crystallizing human hemoglobin. The effect of temperature changes on solubility of proteins had of course been used before in their crystallization. Ritthausen (20), Osborne (17), and subsequent investigators have crystallized vegetable globulins by heating them gently in salt solutions and then gradually cooling. At the concentrations of salt employed in this general method of crystallization, edestin, like hemoglobin, is in the range of maximum solubility. Here temperature has little effect upon hemoglobin,

1 Personal communication.


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A. A. Green 507

whereas solubility is increased by temperature at lower, but decreased at higher concentrations of salt.

The decreased solubility of certain non-electrolytes, for instance gases, at higher temperatures has long been known. The data for the solubility of oxygen and nitrous oxide recalculated by Randall and Failey (19) yields the same value for K,’ in a given salt solution at different temperatures.

Id

I 3.0 410/u 20 3.0

P,

FIG. 2. The solubility of hemoglobin in concentrated phosphate buffers, of varying temperature and pH.

The numerical values of K,' are, of course, dependent upon the units in which the concentration of hemoglobin and of salt is defined. If solubility is calculated in gm. of protein per liter of

solution and the salt concentration as i; that is, if equation (2)

has the form

log ‘(gm. per liter) = B - Ka’ i

K,' is 1.00 for carboxyhemoglobin at 25” and pH 6.6 and P is 3.01. If, on the other hand, solubility is calculated as mol fraction, N, and the equation is written


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log N = B - K.’ 2’ (4)

K,’ is 1.01. All of these equations describe the experimental findings over the range of salt concentration investigated. The convention has been adopted generally of describing solubility in terms of gm. of protein per 1000 gm. of water and ionic strength as p, in characterizing the solubility of protein in concentrated salt solutions.

TABLE IV

Shensen and H$yrup’s* Experiments upon the Solubility of Egg Albumin in Ammonium Sulfate at Various Temperatures

Experi- ment No.

1 6 8 9

11 12

4 5 7 8

10 5

11

-

E

.-

-

Gltrate No.

6 7 6 7 6 7 4 4 6 8 6 6 4

-

Tempera- ture

“C.

0 0 0 0 0 0

12 12 20 20 29 29 29

Ir Volubility Jh3s B -_

6.408 3.18 0.502 6.333 5.457 21.81 1.339 6.305 5.457 22.02 1.343 6.309 5.457 19.90 1.299 6.265 5.457 21.30 1.328 6.294 5.457 20.71 1.316 6.282 6.408 2.09 0.320 6.151 5.457 14.89 1.173 6.139 6.408 1.81 0.258 6.089 5.457 13.70 1.137 6.103 6.408 2.24 0.350 6.185 5.457 16.80 1.225 6.191 5.457 16.00 1.204 6.170

- * See (24) p. 234, Table 43.

The values of K,' and /3 for oxyhemoglobin at 0” also have been calculated and are recorded in Table III. K,' has the same value as for carboxyhemoglobin at the same temperature and pH. The difference in fl is slight, being 3.79 rather than 3.86. The solubility curves are therefore almost superimposable in concentrated salt solutions as may also be seen in Fig. 2. This is not the case in more dilute salt solutions where carboxyhemoglobin is less soluble than oxyhemoglobin.

Sorensen and Hoyrup studied the rate of precipitation of egg albumin at various temperatures (24). Equilibrium was reached


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A. A. Green 509

more rapidly in systems which were changed from a condition of lower to one of higher solubility. The lowest solubilities recorded, representing the most complete crystallization of the protein are reproduced in Table IV. Two concentrations of ammonium sulfate were employed. On the assumption that K,’ is independent of temperature, values of /3 should be independent of salt concentration, but dependent upon temperature precisely as they have been shown to be dependent upon pH (5). K,’ for egg albumin in ammonium sulfate when the salt concentration is employed as ionic strength per liter, is 1.19 (7); when mols per

t I I 5.0 6.0 d

IONIC STRENGTH

3

FIG. 3. The solubility of egg albumin at various temperatures from the experiments of Sorensen and Hgyrup (24).

1000 gm. of water, 0.91 (11). Values of 8, calculated on the latter basis, are given in Table IV. These appear to be consistent with the hypothesis of a constant value of K,’ a,s may be seen in the accompanying Fig. 3, where the lines through the experimental points are again drawn parallel. A series of experiments carried out at room temperature by the same authors is represented in Fig. 3 and recorded in their Table 40 ((24) p. 224). As in hemoglobin, solubility at 0” is greater than solubility at room temperature in concentrated solutions of electrolytes, but egg albumin exhibits a minimum solubility at about 25”. Solubility determi-


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nations have not been carried out, on hemoglobin at enough different temperatures to ascertain if this is also the case for that protein. However, in egg albumin as in carboxyhemoglobin K8!, in contrast to 8, is apparently independent of temperature.

Variation of Solubility with pH-The solubility of carboxyhemoglobin at 25” in phosphate buffers at various reactions on both sides of the pH of minimum solubility, is also represented in Fig. 2. Here also the lines are parallel to that at, pH 6.6 under the same conditions. Data defining the variation of solubility in different concentrations of salt over a considerable pH range are considered in the next communication of this series. Here it seems necessary merely to indicate the constancy of K,‘. Since there is such a change in /3 with pH, and since the solubility is increased ten-fold in changing from pH 6.6 to 7.4, the necessity of controlling pH in the salting out of proteins is evident.

SIdrensen and Hoyrup’s data on the solubility of egg albumin in (NH&S04 at varying pH values, and in two concentrations of salt, have previously been shown by Cohn (5) to yield constant values for /? at a given pH when K,’ is taken as a constant. The constancy of K,’ was also demonstrated by Florkin in a study from this laboratory upon the solubility of fibrinogen. On the other hand, 0 varies with pH as in egg albumin and in hemoglobin.

The investigations on these three proteins may be considered to have demonstrated that although the solubility of a protein in a given salt solution varies with temperature and pH, K,’ remains constant. Accordingly in the next section of this paper these conditions are maintained constant and the variation of K,’ with the nature of the salt is considered.

Part II. Solubility of Hemoglobin in Concentrated Solutions of Di$erent Salts. The Hofmeister Series

The relative effectiveness of the various ions in the precipitation of a protein under constant conditions can be quantitatively expressed by the value of the apparent, salting out constant, K,‘. The value of 8, the intercept constant, appears to be approximately the same for a given protein at, a given pH and temperature. K,’ on the other hand is the slope constant, and varies widely with the electrolyte. Evaluation of 0 and K,’ for different electrolytes should be an accurate description of the precipitating power of these electrolytes on the given protein.


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A. A. Green

TABLE V

Solubility of Carbozyhemoglobin at 25’

511

No. of Concen- Exwri- deter- merit min*-

tration NO.

elect,.,,- Density P tions lYb

PH

fhlubility

11 5 15 3 11 5 11 4 15 1 24 1 11 4 11 4

6 4 21 1 21 1

6 5 6 5 6 6 6 6 6 5

mOf8

pd.

0.675 0.80 0.809 0.944 1.00 1.065 1.079 1.214 I 1.0816 2.12 6.60

2.46 6.71 1.0962 2.48 6.50

2.91 6.28 3.09 6.70

1.1243 3.30 6.57 3.34 6.61

1.1405 3.76 6.48 --

Solubility in ammonium sulfate

am. am.

9.56 9.78 5.82 5.94 4.74 4.87 2.39 2.46 1.92 1.98 1.305 1.345 1.15 1.29 0.552 0.577

Solubility in sodium sulfate

2.609 0.990 1.576 0.774 1.296 0.688 0.651 0.391 0.524 0.297 0.356 0.129 0.343 0.111 0.152 I.761

1.20 1.0821 3.90 6.63 3.30 3.59 0.946 1.24 1.0904 4.03 6.60 2.26 2.45 0.645 1.36 4.48 6.40 1.20 1.32 0.346 1.40 1.0950 4.64 1.41 1.565 0.410 1.48 1.0992 4.92 6.50 0.922 1.023 0.268 1.60 1.1074 5.38 6.52 0.535 0.599 0.157 1.80 1.1202 6.12 6.48 0.193 0.2185 0.0568 2.00 1.1320 6.92 6.56 0.063 0.0727 0.0188

Solubility in magnesium sulfate

0.555 0.389 0.121 0.194 0.009 1.777 1.3395 2.8615

11 4 1.940 1.2175 7.93 6.34 4.75 4.86 1.267 0.687 21 1 2.04 8.43 6.30 4.63 4.75 1.240 0.677 11 3 2.183 8.99 6.28 1.98 2.03 0.526 0.308 11 4 2.304 1.2529 9.47 6.33 1.55 1.595 0.405 0.203 11 3 2.425 1.2607 10.00 6.24 1.00 1.03 0.266 0.013

Solubility in sodium citrate

7 4 0.404 1.0852 2.50 6.67 8.38 8.62 2.30 0.935 7 4 0.505 1.1033 3.13 3.33 3.44 0.918 0.536 7 4 0.606 1.1227 3.77 1.28 1.33 0.352 0.124 7 4 0.808 1.1615 5.09 6.54 0.172 0.181 0.048 I.256


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512 Physical Chemistry of Proteins, VIII

In Table V are recorded solubility determinations on carboxyhemoglobin at 25” and pH 6.6 in concentrated solutions of sodium, ammonium, and magnesium sulfates and in sodium citrate. The column headings have the same significance as those in Table I. These results are graphically represented (Fig. 4) in order to show that the above linear relation holds, and that K,’ varies for the different salts.

The values for K,’ and /3 have been calculated by means of simultaneous equations and the average values reported in

FIG. 4. The solubility of carboxyhemoglobin at 25” and pH 6.6 in concentrated solutions of various electrolytes.

Table VI on the basis of equations (2), (3), and (4). The order of decreasing precipitating ability and, also, the order of decreasing K,’ is KHZ04 + KzHPO,, Na&04, NaJ3J&,O~, (NH&S04, and MgS04. The chlorides studied, and considered elsewhere, have even less precipitating ability, but may be added in the order KCl, NaCl. If one calculated K,’ in terms of equivalents rather than on an ionic strength basis, the following values are obtained: Na3CBH607, 1.29; KH2P0, + KJIPOI, 1.15; Na2S04, 1.08; (NH&SO~, 0.84; MgSO,, 0.62. In the series developed by


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A. A. Green 513

Hofmeister the above salts, on the basis of equivalents per liter, are given in the following order: Na+SOd, K2HP04, and NaG,H607, (NH&SOs, MgSO+ NaCl, and KC1 (13). This was, as we have seen, determined as the lowest concentration of salt necessary to cause precipitation of the protein. The agreement is striking.

The values of 8, recorded in Table VI, are not constant. Since j3 is a hypothetical extrapolated quantity, this is not surprising. Hemoglobin is a globulin and the linear relation holds only in concentrated solutions of salts. The varitions in /3 may thus be

TABLE VI

Values of j3 and K,’ joor Carboxyhemoglobin in Concentrated Salt Solutions at pH 6.6

Electrolyte / “z;;&;’ 1 NarSO, / N.&OH&~ 1 (NH.)&304 1 MgSOa

Defined by the equation, log s,,, 1ooo gm, &o) = 6 - Ks’r

Defined by the equation, log Scwr ,iterl = fl - Ke’ i

BK,, / ;:i / zi 1 ~:~ 1 1:;: 1 I Defined by the equation, log N = j3 - KS’ i

explained by the increased solubility in the presence of lower concentrations of electrolytes. This effect will be further considered in a later communication.

Fibrinogen has been quantitatively studied in sodium chloride, ammonium sulfate, and in phosphate buffers by Florkin (11). Fibrinogen is completely precipitated by sodium chloride whereas the solubility of hemoglobin is not at all decreased in a saturated solution of the same salt. The salts are, however, effective as precipitants in the same order in fibrinogen as in hemoglobin.


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The role of the protein in this relation is graphically represented in Fig. 5 in which is plotted the solubility of a number of proteins in ammonium sulfate. The curves for pseudoglobulin and egg albumin are from the data of Sorensen (23) and of Sorensen and Hoyrup (24) also recalculated by Cohn (5). The curves for fibrinogen are Florkin’s.

The great difference in the slopes of the straight lines and therefore in the values of K,’ for different proteins is to be stressed rather than their position, for neither was the pH of the systems

FIG. 5. The solubility of various proteins in concentrated ammonium sulfate solutions.

studied always the same, nor were all the proteins near their isoelectric points, where solubility might be expected to be minimal.

Studies of the variation of B under different conditions may be expected to indicate procedures for the separation of proteins from each other that have not yet been apprehended, but the value of K,’ will presumably not be found to vary widely with physicochemical conditions, but rather to reflect a characteristic of the protein more deep seated than its state. In this connection it should be noted that the solubility of human and horse hemoglobin under identical conditions differs enormously. This


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A. A. Green 515

suggests both the lack of wisdom of inferences as to solubility relationships of analogous ‘proteins in different species, and the delicacy of the methods of analyses employed in this method of characterization.

I am indebted to Professor Edwin J. Cohn for suggesting these problems on the solubility of hemoglobin and for his continued interest during the progress of the investigation.

SUMMARY

1. The solubility of carboxyhemoglobin and of oxyhemoglobin has been studied in concentrated potassium phosphate buffers, and of carboxyhemoglobin in concentrated solutions of sodium, ammonium, and magnesium sulfates and sodium citrate.

2. The solubility in concentrated salt solutions at constant pH and temperature is described by equations of the type :

log 5’ = p - K: p

3. p is a variable dependent upon the nature of the protein and the pH. Increase in temperature from 0” to 25” decreases ten- fold the solubility of carboxyhemoglobin in concentrated phosphate solutions.

4. K,’ appears to be independent of temperature and of pH. 5. The value of K,’ varies with the salt, and decreases in the

order: KHZPOI + K2HPOI, Na&04, NasCsHa07, (NH&SO+ and MgS04. This is essentially the same order as that given by Hofmeister in 1888.

BIBLIOGRAPHY

1. Adair, G. S., Proc. Roy. Sot. London, Series A, 120, 573 (1928). 2. Bernard, C., in Robin, C., and Verdeil, F., Trait.6 de chimie Anat-

omique, Paris, 299 (1853). 3. Chick, H., andMartin, C. J., Biochem. J., 7,380 (1913). 4. Cohn, E. J., J. Gen. Physiol., 4,697 (1922). 5. Cohn, E. J., Physiol. Rev., 6,349 (1925). 6. Cohn, E. J., J. Am. Chem. Sot., 49,173 (1927). 7. Cohn, E. J., and Green, A. A., J. Biol. Chem., 78, p. xxxii (1928). 8. Cohn, E. J., and Prentiss, A. M., J. Gen. Physiol., 8,619 (1927). 9. Debye, P., and MacAuley, J., Physik. Z., 26,22 (1925).

10. Ferry, R. M., and Green, A. A., J. Biol. Chem., 81,175 (1929). II. Florkin, M., J. Biol. Chem., 87,629 (1930).


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12. Hastings, A. B., Sendroy, J., Jr., Murray, C. D., and Heidelberger, M., J. Biol. Chem., 61,317 (1924).

13. Hofmeister, T., Arch. exp. Path. u. Pharmakol., 24, 247 (1887-88). 14. Hiickel, E., Physik. Z., 26,93 (1925). 15. Lewis, G. N., and Randall, M., Thermodynamics and the free energy

of chemical substances, New York and London (1923). 16. Linderstrom-Lang, K., Compt. rend. trau. Lab. Curlsberg, 16, No. 4

(1924). 17. Osborne, T. B., Am. Chem. J., 14,662 (1892). 18. Panum, P., Virchows Arch. path. Anat., 4, 419 (1852). 19. Randall, M., and Faiiey, C., Chem. Rev., 4,271 (1927). 20. Ritthausen, H., J. prakt. Chem., 23,481 (1881). 21. Scatchard, G., Tr. Faraday Sot., 23, 454 (1927); Chem. Rev., 3, 383

(1927). 22. Setschenow, Ann. chim. et physique, series 6, 26,226 (1892). 23. Serensen, S. P. L., J. Am. Chem. Sot., 47,457 (1925); Compt. rend. trav.

Lab. Curlsberg, 16, No. 11 (1924). 24. Sereneen, S. P. L., and Hoyrup, M., Compt. rend. trav. Lab. Curlsberg,

12,213 (1915-17). 25. Stadie, W. C., and Hawes, E. R., J. Biol. Chem., 77,241 (1928). 26. Svedberg, T., and Nichols, J. B., J. Am. Chem. Sot., 49,292O (1927). 27. Tammann, G., Z. anorg. u. allg. Chem., 166, 25 (1926). 28. Virchow, R., Virchows Arch. path. Anat., 6,572 (1854).


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Arda Alden GreenSALTING OUT OF PROTEINS

SOLUTIONS. A STUDY OF THEIN CONCENTRATED SALT

THE SOLUBILITY OF HEMOGLOBINCHEMISTRY OF THE PROTEINS: VIII.

STUDIES IN THE PHYSICAL

1931, 93:495-516.J. Biol. Chem.

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