THE SOLUBILITY OF PHENOLS I N PROTEINS E. A. COOPER
AND
MARJORIE TREADGOLD
Chemical Department, University of Birmingham, Birmingham, England Received July 7, 1933
The investigations so far carried out on this subject (1) have shown that the germicidal power of a disinfectant is not determined by the magnitude of the partition coefficient, which measures the ratio of its solubilities in a protein sol and water. Thus, p-chlorophenol is five times as efficacious in disinfecting pon-er as phenol, and yet the partition coefficients solubilitv in Drotein sols solubility in water
are of much the same order of magnitude, e.g., 4 and 3, respectively. In the case of proteins in the precipitated or coagulated state, however, it has been found that there is a general parallelism between the germicidal powers of the phenols and their partition coefficients between proteins and water. This result was unexpected, as the normal physiological state of proteins in the cell is not a coagulum, but a sol or gel. Furthermore, the partition coefficients are much higher with protein coagulae than with colloidal solutions, such as egg albumin sols, or gels, e.g., ordinary gelatin; in the case of phenol the partition coefficient is 3 with sols and gels, and 12 with albumin coagulated by heat or by phenol itself. Similar results were obtained with cresol and chlorophenols. The experimental work described in the present paper has been carried out with the object of discovering an explanation of these unexpected physico-chemical observations. I. DISTRIBUTION OF CHLORAL HYDRATE AND PICRIC ACID B E T W E E N PROTEINS AND WATER
In the first place, we have endeavored to ascertain whether such abnormalities apply to disinfectants and physiologically active substances other than phenols and cresols, and their halogen derivatives. For this purpose chloral hydrate and the nitrophenols were selected.
a. Chloral hydrate and gelatin Weighed amounts of gelatin (1 g.) were immersed in aqueous solutions of chloral hydrate varying from 5 to 20 per cent. The strengths of the solu259
260
-
E. A. COOPER AND MARJORIE TREADGOLD
WATER PHASE
U'EIQHT O F
CHLORAL HYDRATE
Initjal concentration per cc.
Final concentration per cc. A
T A K E N UP B Y 1 G. O F PROTEIN
grams
grams
grams
0.1920 0.1440 0.0960 0.0480
0.1853 0.1407 0.0960 0.0480
0.402 0.198 0 0
B
CONDITION OF PROTEIN
DISTRIBUTION RATIO
B/A
2.1
-
Precipitated
Unaffected 0
the water phase in the complete absence of the protein phase after equilibrium had been attained (three days). The results are set out in table 2. Parallel experiments were then set up with picric acid, the strengths of the aqueous solutions being estimated by titration with standard alkali (methyl red as indicator). Tables 3 and 4 give the results of these experiments. The following conclusions may be drawn from these results. (1) In the case of chloral hydrate with gelatin there is a slightly increased value of the distribution ratio a t precipitation, but with egg albumin no increase a t all is apparent. (2) With picric acid, the distribution ratio also increases slightly when the precipitation of the gelatin first commences, but as the concentration of the picric acid rises, the ratio again falls rapidly in magnitude, although the protein remains permanently in the coagulated state. The results are thus quite different from those observed in the case of phenol and cresol, and it may be concluded that the marked increase in the magnitude of the partition coefficient a t precipitation observed with these substances is not general for all hydroxy compounds.
26 1
SOLUBILITY O F PHENOLS I N PROTEINS
TABLE 2 Chloral hydrate and egg albumin 1 g. of albumin to 60 cc. of chloral hydrate solution WEIGHT O F CHLORAL HYDRATE T A K E N U P B Y 1 0. O F PROTEIN
Initial concentration per cc.
Final concentration per cc. A
grams
grams
grams
0.0834 0.0331 0.0081 0.0041
0.0810 0.0314 0.0079 0,0039
0.1380 0.1020 0.0120 0.0120
CONDITION OF PROTEIN
DISTRIBUTION RATIO
B
BIA
Precipitated Unaffected Unaffected Unaffected
1.7
3.0
TABLE 3 Gelatin and picric acid 0.5 g. of gelatin t o 40 cc. of picric acid solution WATER PHABE
Initial concentration per cc. grams
I-
0.0112 0.0056 0.0028 0.0019 0.0011 0.0006
Final concentration per cc. A
WEIGHT O F PICRIC ACID TAKEN UP BY 1 Q. OF PROTEIN
DISTRIBUTION RATIO
B
B/A
grams
grams
0.0085 0.0033 0.0009 0.0005 0.0004 0.0004
0.2154 0.1814 0.1527 0.1069 0.0534 0.0133
I
CONDITION OF PROTEIN
Precipitated Precipitated Precipitated Precipitated Unaffected Unaffected
25.3 55.0 158. ,5 214.0 133.6 33.2 1
I
I
TABLE 4 Gelatin and edestin with phenol I
I WATER PHABE
Initial concentration of phenol percc.
CONDITION OF PROTEIN
1 ~
-
Final concentration of phenol p i y .
I
I AMOUNT OF PHENOL DI~SOLYED BY 1 G . OF
PARTITION COEFFICIENT
BIA
PROTEIN
B
(a) Gelatin. 1 g. of gelatin to 40 cc. of phenol solution
5
grams
Precipitated by dialyzed iron. ......... i:4iii “Sol” ................................ (b) Edestin.
grams
0.0124 0.0090
6.2
4.3
1 g. of edestin to 40 cc. of phenol solution
“Sol”. ............................... Insoluble edestin chloride.. . . . . . . . . . . .
.I
0.0032 0.0032
1
0.0031 0.0031
~
0.0040 0.0040
1.3 ~
1.3
262
E. A. COOPER AND MARJORIE TREADGOLD
11. SOLVENT POWER O F COAGULATED PROTEINS FOR PHENOL
Experimental work has also been carried out with the object of ascertaining whether the increased solvent power for phenol, which is observed when proteins are coagulated by heat, or by the action of phenol itself, also takes place when precipitation of the protein is effected by other means. In the first place, a comparison has been instituted between the solvent powers for phenol of a gelatin sol and gelatin precipitated by the addition of a colloid of opposite electric charge, e.g., dialyzed iron. And, in the second place a similar comparison has been made in the case of edestin dissolved in salt solution and the insoluble edestin chloride formed by adding a drop of hydrochloric acid to edestin solution.
a. Gelatin sols Gelatin (1 g.) was introduced into viscose dialyzers, containing 15 cc. of a dilute phenol solution and immersed in 25 cc. of the same phenol solution. The experiment was carried out at 37”C.,so that the gelatin dissolved and formed a ‘(sol.” Into one of the dialyzers sufficient dialyzed iron was added to precipitate the ‘(sol,” and after three days for equilibrium to be attained the phenol concentration in the protein-free water phase (outside the dialyzers) was estimated by Lloyd’s method (3), which depends on the formation of tribromophenol from the interaction of bromine and phenol. In the experiment with edestin 10 cc. of a 5 per cent salt solution in which 1 g. of the protein had been dissolved was introduced into dialyzers, which stood in 30 cc. of a phenol solution of known strength. To one of the dialyzers one drop of concentrated hydrochloric acid was added. After three days the phenol in the water phase was estimated as before. The results of these experiments are shown in table 4. The observations Show that in the case of gelatin the partition coefficient is slightly raised by precipitation with dialyzed iron, but the increase is small compared with that caused by precipitation of the gelatin by phenol itself (Cooper, 1912). In this case the partition coe@cient rises as high as 30. The results obtained in the edestin experiment indicate that the solvent power of an edestin “sol” for phenol is not increased at all by conversion into the precipitated chloride. The increased solvent power is thus only manifested under certain experimental conditions, and is restricted to the particular cases of coagulation by heat and the action of phenols, Le., it is associated strictly with the specific change induced in proteins by the process of ‘(denaturation.” 111. T H E SIGNIFICANCE O F T H E WATER FACTOR
Early in these researches (Cooper, 1923) it was realized that in a colloidal solution of a protein a portion of the water present is probably associated or imbibed with the protein phase, and this question was considered
SOLUBILITY O F PHENOLS I N PROTEINS
263
in relation to the problems under investigation. Possibly the imbibed water might possess less marked solvent properties than normal water. Hence when the protein was dehydrated by coagulation, the associated water thrown out would dilute the water phase, and as a result the increased solvent power for phenol observed when a protein is coagulated might be merely apparent, and the calculations fictitious. Experiments carried out a t the time, however, suggested that this was not the case, and the results supported the view that the calculated partition coefficients were real. Since then considerable progress has been made in the field of colloidal chemistry, and in view of the new data now available further experimental work has been carried out.
a. Experiments with red cells According to Krevisky ( 2 ) the amount of free water in the red cell is equal to one-third of the cell volume, whilst an equal proportion of water is bound water, i.e., associated with the protein and hence forming part of the disperse phase. By carrying out distribution experiments with red cells and phenol on the lines of those already described in this paper, it has therefore been possible to calculate the partition coefficients by two different methods: (1) assuming that the water imbibed in the protein phase is normal water and thus for purposes of calculation may be regarded as part of the water phase; ( 2 ) assuming that the association with the proteins renders the bound water abnormal in solvent properties and such that it must be regarded as an intimate constituent of the protein phase and not to be calculated as an integral part of the water phase. Red cells were obtained by centrifuging ox and pig blood. Fifteen cubic centimeters were introduced into a series of viscose dialyzers, which were immersed in varying concentrations of phenol (25 cc.), and after three days for equilibrium the phenol solutions outside the dialyzers were again analyzed. The percentage of protein in red cells is 30 per cent. The results have been calculated by the two different methods already mentioned, the respective data being as follows: (1) Protein 4.5 g. (30 per 10 cc. (two-thirds of red cells) = cent of red cells); water phase 25 cc. 35 cc. ( 2 ) Protein 4.5 g.; water phase 25 cc. 5 cc. (one-third of red cells) = 30 cc. (See tables 5A and 5B, respectively.)
+
+
b. Experiments with egg white St. John (4) has shown that the amount of water bound with the proteins of egg white is equal to 20 per cent of the total water content. Similar experiments to those carried out with red cells have therefore been made with egg white and phenol, and the partition coefficients have been calculated as before in two ways, (1) assuming the whole water content
264
E. A. COOPER AND M A R J O R I E TREADGOLD
TABLE 5A Experiments with red cells 1
I WATER PHASE
Initial phenol concentration per cc.
l
AMOUNT OF PHENOL
Final phenol concentration per cc.
A
DIBSOLVED BY 1 G. OE PROTEINS
B
1
DISTRIBUTION RATIO
B'A
ox grams
oram8
grams
0.0043 0.0064 0.0071 0.0107 0.0150 0.0164 0.0236 0.0257
0.0031 0.0040 0.0044 0.0074 0.0097 0.0103 0.0159 0.0162
0.0093 0.0187 0 0209 0.0256 0.0412 0.0475 0.0599 0.0739
3 4.7 4.7 3.4 4.2 4.6 3.6 4.6
0.0088 0.0267 0.0381
0.0055 0.0178 0,0198
0.0256 0.0692 0.1423
4.6 3.8 7.2
I
TABLE 5B Experiments with red cells
I
WATER PHASE
Initial phenol concentration per cc.
A
AMOUNT OF PHENOL DISSOLVED BY 1 G. OF PROTEINS
Final phenol concentration per cc.
B
DISTRIBUTION RATIO
BIA
ox grams
grams
grams
0.0050 0.0075 0.0083 0.0125 0.0175 0.0192 0.0275
0.0031 0.0040 0.0044 0.0074 0.0097 0.0103 0.0159 0.0162
0.0126 0.0233 0.0260 0.0340 0.0520 0.0593 0.0773 0.0920
4.1 5.8 5.9 4.6 5.4 5.7 4.8 5.7
0.0320 0.0893 0.1646
5.8 5.0 8.3
0.0300
Pig
0.0103 0.0312 0.0445
0.0055 0.0178 0.0198
265
SOLUBILITY O F PHENOLS I N PROTEINS
of egg white is normal water and thus counted as water phase, and (2) assuming the bound water to be abnormal and thus regarded as part of the protein phase. Calculated in the ordinary way, the partition coefficients amount of phenol dissolved in motein sol amount of phenol dissolvod in water
were on the average 3.7. Calculated in the alternative way by regarding the proportion of bound water as a constituent of the disperse phase, the mean coefficient was 5.0. In the case of the coagulated protein, the partition coefficient was, however, 12. From the experimental results with red cells and egg white it is seen that the particular method of calculation makes very little difference in the actual value of the distribution ratios. By introducing the correction for the amount of imbibed water, the distribution ratios only increase by about one-third. Yet the partition coefficient in the case of a coagulated protein is approximately four times as great as that for the protein in a state of colloidal solution. The increased solvent power for phenol noticed when a protein is coagulated is thus a reality, and is not due to fictitious calculations arising out of movements of water from one phase to the other. A comparison was next made of the uptake of phenol by the moist and dry clot of egg white. Water is imbibed to a certain degree in a moist clot obtained by boiling a solution of egg white, but not by the clot after it has been dried a t 100°C. Thirty-five cubic centimeters of phenol solution were mixed with 10 cc. of half-strength eggwhite (Le.,egg white diluted with an equal volume of water and containing 6.0 per cent protein) for the moist clot experiment, the egg white then being coagulated in situ in the bottle by heat. For the dry clot experiment, 35 cc. of solution was used with 10 cc. of water and the dried and powdered clot from 10 cc. of diluted egg white was then added. This experiment thus afforded a further simple method of studying the significance of the water factor. WATER PHASE
Initial concentration per cc.
___
Final concentration per GC. A
AMOUNT OF PHENOL
BIA
-
@rams
grams
grams
0.01027 0 01027
0.00729 0.00699
0.2233 0.2460
30.6 Moist clot 35.2 Dry clot
The dry clot of egg white absorbs slightly more phenol than the moist clot from solutions of the same str6ngth. Thus, in the case of phenol, it makes very little difference whether the water in the system is entirely in
266
E . A . COOPER A N D MARJORIE TREADGOLD
one phase or whether it is present in the protein phase as well. The manner of distribution of the water hence does not affect the value of the partition coefficients to any appreciable extent, and the results are confirmatory of those already described in this section. Although the rise in the value of the partition coefficient at coagulation is not due to errors in calculatioh introduced by transfer of water from one phase to another, yet this water factor may be of real significance in another way. The solubility of phenol in proteins will probably be diminished by the presence of bound or imbibed water, and the normal solvent power of proteins for phenol will hence not be manifested until the phenol replaces the associated water, which it will drive out through its dehydrating action, thus causing coagulation. According to this view the partition coefficients obtained with coagulae measure the true solubility of phenols in proteins, and an explanation is offered for the abnormally low coefficients observed with colloidal solutions. The interpretation of the observations that the germicidal powers of phenol, cresol, and chlorophenols run parallel with their partition coefficients with protein coagulae, whilst the coefficients with protein sols are all of the same order of magnitude (about 3) is however not so clear. If the solubility of phenols in coagulae is regarded as their normal solubility in proteins, then germicidal power in the case of phenols is proportional to the true partition coefficients, and the formation of a solution of the disinfectant in the bacterial proteins may be considered the first stage in the process of disinfection. No explanation is then given, however, of the absence of proportionality between germicidal power and the partition coefficients measured with proteins in the natural state, in which they occur in the cell (sols and gels). At the present time it can only be suggested that the bound water diminishes the solubility of cresol and chlorophenol in proteins to a greater extent than it does that of phenol itself, thus tending to maintain the partition coefficients at approximately the same value in all cases. When, however, the protein becomes coagulated through the dehydrating effect of the phenols, the associated water is expelled and transferred to the continuous phase, and the true partition coefficient specific and characteristic of each individual phenol reveals itself. The first phase in the process of disinfection by phenols is thus the formation of a solution in the bacterial proteins followed by coagulation, and cresol and chlorophenol are superior to ordinary phenol in germicidal power, because they induce coagulation, i.e., expulsion of bound water, a t a lower concentration (Cooper, 1913, 1923). SUMMARY
1. Coagulated proteins possess a much higher solvent power for phenols than proteins in a natural condition, e.g., sols and gels. This is true also
SOLUBILITY O F PHENOLS I N PROTEINS
267
of picric acid, but to a lesser degree. Chloral hydrate, however, is exceptional, the uptake of which by proteins is not materially affected by the physical state of the protein. 2. Proteins precipitated from colloidal solution by the addition of a colloid of opposite electric charge or by formation of an insoluble salt do not exhibit this increased solvent power for phenol. 3. By means of experiments with red cells and egg white, in which due allowance has been made for the proportion of water bound to the protein phase, it has been found that the increased partition coefficients observed at coagulation are realities, and not due to calculation errors introduced by movement of water from one phase to another. The imbibed water may however diminish the solubility of phenol in proteins, thus accounting in the case of sols and gels for the low partition coefficients, which rise to the normal figure when the water is expelled at coagulation. REFERENCES (1) COOPER:Biochem. J. 6, 362 (1912); 7, 175 (1913). COOPERA N D MASON:J. Phys. Chem. 32,868 (1928). COOPER AND SAXDERS: J. Phys. Chem. 3 1 , l (1927). Biochem. J. 17,600 (1923). COOPERAXD WOODHOUSE: (2) KREVISKY:Biochem. J. 24, 815 (1930). (3) LLOYD:J. Am. Chem. Soc. 27,16 (1905). (4) ST. JOHN: J. Am. Chem. Soc. 63, 4014 (1931).
