A CONTRIBUTIOX TO OUR KNOWLEDGE OF DISINFECTAKT ACTION. I1 The Relations of Phenols and Amines to Proteins BY E. ASHLEY COOPER AND JOHN MASON
I n the previous paper' it was shown that phenol and resorcinol were distributed between water and proteins in accordance with the partition-law, i.e. the phenols formed a true solution in the protein phase. This relationship is of an unusual type in the case of colloids, owing to surface phenomena such as adsorption, the amount of substance taken up or adsorbed by the colloid diminishing relatively with rise in concentration. It was also found that resorcinol was somewhat more soluble in dispersed or dissolved proteins than phenol, although the latter is considerably more potent as a disinfectant. The conclusion was therefore drawn that, although partition of the disinfectant between water-phase and cell proteins is an essential preliminary stage in disinfection, yet germicidal power is not merely determined by solubility, but must be associated with the subsequent denaturing action on the colloidal structure of the bacterial cell. In order to throw more light on the rBle of the preliminary process of solution in the mechanism of disinfection the work has been extended to other phenolic derivatives, e.g. picric acid and salicyl-sulphonic acid, and furthermore the relationships to proteins of disinfectants of a different type altogether, such as amines, has also been studied.
I. Picric Acid Walker and Appleyard* showed that when picric acid was taken up by silk from aqueous solution, the partition law did not apply, and the process was clearly one of adsorption. It seemed desirable t o extend this work to other proteins, and for this purpose serum albumin was employed. I grm. of powdered coagulated albumii was immersed in 30 cc. of each of a series of aqueous picric acid solutions, concentrations varying from 0 . 2 % to 1.5%. The experiment was conducted a t 37'C. The period required for the completion of adsorption was determined by preliminary experiment. The estimation of picric acid before and after adsorption was carried out by titration of the solutions with N/20 alkali, lacmoid being used as indicator. The results in Table I show that the amount of picric acid taken up by the protein increases with rise in concentration in the water, but not proportionately, so that the ratio of the concentration of picric acid in the protein to that in the water phase diminishes with increasing concentration. The results are similar to those obtained by WaUrer and Appleyard (loc. cit.) with Cooper and Sanders: J. Phys. Chem., 31, I (1927). 'Walker and Appleyard: J. Chem.,Soc., 69, 1349 (1896).
1
869
RELATIONS O F PHENOLS AND AMINES TO PROTEINS
silk. The uptake of picric acid by proteins is thus of the nature of adsorption or surface concentration, in contradistinction to the formation of a solution in the colloid phase. (cf phenol.) Phenol was previously found to be about 1 2 times as soluble in serum-albumin as in water, whereas the foregoing results show that the amount of picric acid adsorbed by the protein is from 2 7 to 922 times as great as the amount remaining in the aqueous solution at equilibrium. Picric acid is a much more efficacious disinfectant and proteinprecipitant than phenol, and we have therefore in this case some measure of correspondence between germicidal activity and uptake by proteins.
TABLE I Uptake of picric acid b I grm. aiibumin. A (I)
(2)
,2441 grm. .I785 .1840 . I454 ,0587 ,2788 ,2780 ,2678 ,2704
.2412 ,2081
,1756 ' I447 'I345 ,0612
Initial conc. of picric acid er cc. orwater phase
.or583 grm. .01246 .00992
Final conc. of picric acid per cc. of water phase.
B , 0 0 7 7 I grm.
Ratio
A /B 31.6 27.4 48.0
,0052 I
,00652 .00383 ,00039
,002 I I
,000I 7
374.0 345.0
,01475 .OI214 . O I196 .OIZI4 ,00894 ,007 44 ,00624
.Ow47 .00288 ,00304 .003 I3 .00090
96.6 88.2 86.3 268.7
,0005I ,0003 9
.00510
,00028
.0046 I .002 I8
.ooorg
.ooorg
51.0
401.9 451.9 517.8 922 ' 5 420.0
II. Salicylsulphonic Acid This substance is employed in medicine as a protein-precipitant, and has also been found to be moderately active as a germicide, being efficacious in concentrations of I in 300. A study of the nature of the distribution of salicylsulphonic acid between proteins and water was therefore undertaken. (a). Gelatin. Weighed amounts (I grm.) of gelatin in the form of small strips (4 by ins.) were immersed in aqueous solutions of salicylsulphonic acid ( j o cc.) at zo°C until equilibrium was attained (5 days). The strength of the initial and final solutions was determined by titration with N / I O alkali, using phenolphthalein as indicator. The results are set out in Table 11.
+
E. ASHLEY COOPER AND JOHN MASON
870
TABLE I1 Initial conc. of acid per cc. in water phase
Amount of acid taken up by I grm. gelatin. A
,00216grm,
.032 8 grm.
.0051 7
,0474 ,0453 '0993
,00805
,01998 ,03488 ,04889
,
I502
,
I645
Final conc. of acid per cc. in water phase.
B
Ratio
Physical state of protein
A/B
.ooro6grm. ,00360 ,00654 ,01669 ,02989 ,04376
30.8
Swollen
13.2
6.9 5.9 5.0 3.5
,I 97
Opalescent Solid
(b). Albumin. This protein is soluble in water, and it was therefore necessary to use a different technique, viz. the dialysis method. Into a number of bottles (widemouthed), viscous dialysers were placed, and within each dialyser were introduced 2 0 cc. of a 67, egg-albumin solution. Outside the dialysers 3 0 cc. of salicylsulphonic acid solutions, of different strengths, were placed. Control bottles were set up, containing 20 cc. of water within the dialyser and 30 cc. of the acid solutions outside. By titrating the solutions outside the dialysers after standing for equilibrium to be attained, the amount of salicylsulphonic acid taken up by the protein in equilibrium with the water phase could be ascertained. The results are given in Table 111. TABLE
111
Initial conc. of acid per cc. of water-phase
Amount of salicylsulphonic acid taken up by I grm. of protein
,00309grm. ,00614 ,01239
. 0 704 grm.
,00133 grm.
"344 ,1972 ,2266 ,2266
,002 76 ,00743 ,01534
A
.02102
,02890 .os575 .o81og
Final conc. per cc. of water phase.
B
Ratio
A/B
53 ' 1 48.7 26.5
,02322
14.7 9.7
,2113
,05045
4.2
,2207
,07553
2.9
The results are again similar in type to those obtained in the case of picric acid, the amount of salicylsulphonic acid taken up by the proteins diminishing relatively with rise in concentration in water-phase, thus causing a marked fall in the magnitude of the distribution ratio A/B from 53 to 3. It is also of interest to note that the distribution ratio is lowest when the protein is in the precipitated condition (Table 11.). I n the case of phenol, however, it had previously been found' that the partition-coefficient was much higher (I2) for precipitated proteins than for proteins in colloidal solution ( 2 . 5 ) . ~
~
Cooper and Sanders: loc. a t .
RELATIONS OF PHENOLS AND AMINES TO PROTEINS
87 1
111. Ethylamine Although organic bases are known to be efficacious germicides, (ethylamine, for example, being antiseptic' in concentrations of I in 1500,and thus more powerful than phenol), their relations to proteins have not apparently been previously investigated. Weighed amounts of gelatin (I grm.) as before were therefore immersed in 50 cc. of various concentrations of ethylamine in water (.3%-37c), and by titrating the initial and final concentrations with standard acid, the amount of amine taken up by the proteins could be estimated. The period allowed for equilibrium was 2 days. The results are given in Table 117.
TABLE IV Amount of ethylamine taken up by I grm. of protein A
Final conc. of ethylamine per cc. of water phase E
.00329 grm. ,00650 ,01307
.0198grm. .OI99 ,0234
.0029 grm.
BIB 6.8
.006I
3.2
,0126
1.9
.o1981 ,02651
,0254
,0193
,0307
,0259
1.3 1.2
Initial conc. of ethylamine per cc. of water phase
Ratio
Again the adsorption factor predominates, the distribution ratio falling from 6.8to 1.2 as the concentration in water-phase rises.
IV. Aniline Aniline can be estimated in aqueous solution by titration with standard hydrochloric acid, using either bromphenol-blue or Congo Red as indicator. The former was found more sensitive and therefore employed. I grm. of gelatin was introduced into 50 cc. of solutions of aniline (.2%2%), and the experiment carried out as before. The results are given in Table V.
TABLE V Initial conc. of aniline per cc. of water-phase
Uptake of aniline per grm. of protein
Final conc. per cc. of waterphase B
Ratio
. oj49 grm.
0.178grm.
3.0
. 0 1 50
2.7
.0120g
,0397 ,0274
.OII 5
2.4
.009 I 2
,041 I
,00630 ,00358
. 0000
,0083 ,0063
4.9 -
,0034
,0029
1.2
,002I 2
.OIO8
,0019
5.7
.01890grm. . O I580
A
Cooper and Mason: J. Hygiene, 26, 48 (1927).
A/B
E. ASHLEY COOPER AND JOHN MASON
872
The analytical method was less precise in the case of aniline than the foregoing experimental work, and it was not surprising therefore to find irregularities in the value of the distribution ratio A/B. This ratio however tends to fall with rising concentration, suggesting again an adsorption process.
V. Hydrazine Hydrate Hydrazine hydrate was found to bel a very powerful germicide, being active in concentrations of I in 20,000. Its behaviour towards proteins was therefore of interest. Distribution experiments, using gelatin, were carried out in the usual way, the hydrazine being estimated by an iodimetric method. It was found however, that hydrazine was not adsorbed by the protein; in fact, the solutions appeared to become slightly stronger, suggesting therefore a condition of “negative” adsorption. The solute was more concentrated in the aqueous phase than a t the surface of the colloid.
VI. Hydroxylamine Hydrochloride This substance was also found to be active against bacteria, being inhibitory in concentrations of I in 10,000. Hydroxylamine hydrochloride is also a protein precipitant in dilutions of I in 400 to I in 1500. Stronger solutions however have no precipitating action, the protein being soluble in excess. The uptake of the hydrochloride by egg-albumin was therefore next studied. The estimation was carried out by the methods:(I) Titration of the hydrochloride with standard alkali, using phenolphthalein as indicator. (2) Raschig’s method, consisting in the oxidation of the hydroxylamine with ferric salts, and estimation of the equivalent amount of ferrous compound produced by titration with permanganate. 2 0 cc. of a solution of egg-albumin (.47 grm.) were placed in a series of viscose thimbles, surrounded in stoppered bottles by 30 cc. of solutions of hydroxylamine hydrochloride of known concentrations. Control solutions without albumin were set up a t the same time. After allowing a week for equilibrium to be attained, the strength of the external solutions was again estimated by titration with alkali; and by difference the amount of hydroxylamine hydrochloride taken up by the protein could be ascertained. The reresults were tabulated in Table VI. Initial conc. of NHsOH.HC1 per cc. of water phase 0297
TABLE __ VIWeight of XHZOH.HC1 taken up by ~ g malbumin . A
grm.
01196
B
. 0000 grm.
.0297
,0170
.OII8
*
00802 .0060
.0239 .0316
,00302
, 0 1 3I
Cooper and Mason: loc. cit.
Final conc. of NH*OH.HCl per cc. of water phase
Ratio A/B
grm.
0.0
I.4
RELATIONS O F PHENOLS AND AMINES TO PROTEINS
873
I n this experiment i t is possible that the adsorption of the acidic ion is being measured as well as that of the hydroxylamine. The uptake in fact is greater than that observed when the hydroxylamine was directly estimated by Raschig's method (Table VII), although the general relationship of adsorption to concentration is the same in the two cases.
TABLE VI1 Initial conc. of NHzOH.HC1 per cc. of water-phase
Amount of NH20H.HCl taken up by I gm.protein A
.ozg7
grm.
Final conc. of NH*OH.HCl per cc. of water-phase B
Ratio A/B
Negative adsorption
.OI21
.0078 ,00584
.0000
,0078
0.0
.0044
,0058
.00293
.0026
,0029
0.77 0.90
The type of distribution is quite different from that obtaining with the foregoing phenols, acids, and amines. In the case of hydroxylamine hydrochloride the amount taken up by the albumin a t first slightly increases with ascending concentration, reaching a maximum, and with further concentration rise the uptake diminishes to zero. I n experiment z (Table VII) at the highest concentrations negative adsorption was actually observed. Similar results were obtained by Cooper and Nicholas' in the distribution of acetone between water and proteins. I n the case of acetone a chemical explanation of the phenomena is improbable as the ketone only reacts slightly and with extreme slowness with amino-acids.* I n the course of the study of the uptake of phenols, acids, and bases, by proteins, four types of distribution have thus been observed. ( I ) Simple partition or solution, e.g. phenol, resorcinol. ( z ) Adsorption, e.g. picric acid, ethylamine. ( 3 ) Negative adsorption, e.g. hydrazine hydrate. (4) Maximum uptake a t low concentrations, e.g. hydroxylamine hydrochloride.
VII. Correlation of the Results with Observations on Germicidal Power In the following table the distribution-ratios of the foregoing and other substances between proteins and water are coordinated with their germicidal and inhibitory powers. 'Cooper and Nicholas: Biochem. J., 19, 533 (1925). *Cooper and Maaon: loc. cit.
874
E. ASHLEY COOPER AND JOHN MASON
TABLEVI11 Distribution Ratios Protein Water
Substance
Protein in colloidal solution
Phenol m-Cresol *o-chlorphenol p-chlorophenol Resorcinol Picric acid Salicylsulphonic acid E t h y lamine Aniline Hydrazine hydrate Hydroxylamine HCl.
Protein in insoluble state (or pptd.) 12.0
3.0 3.0
18.0
3.2
28.8
4.2
36.0
4.0
5.9-30.8 I .2-6.8 I , 2-
