2. The acids studied may be divided into two groups, the acids of one of which liberated CO, and appeared to penetrate more slowly than they actually do. This group includes hydrochloric, nitric, sulphuric, arsenic, phosphoric, oxalic, citric, tartaric, and mono-, di-, and trichloracetic acids.

The second group of acids includes acetic, salicylic, butyric, and benzoic acids, which are unable to replace CO, (except very slightly in the case of salicylic acid) and penetrate with great rapidity.

3. Evidence is submitted to show that living protoplasm is not the only agency regulating the rate of penetration of acids, since dead cells behave somewhat like those which are alive.

Acknowledgments.-The writer takes pleasure in acknowledging the courtesies afforded by the Miami Aquarium Association, where this work was done, and in expressing much gratitude to the authori ties of the Carnegie Institution of Washington, D. C., who made arrangements for collecting plants.

REFERENCES.

(1) Pfeffer, W.: Osmotische Untersuchungen. 1877.

(2) Ruhland, W.: Jahr. f. Wiss. Bot., xlvi. 1908.

(3) Harvey, E. N.: Publicat. No. 212, Carnegie Instit., Wash. 1915.

(4) Crozier, W. J.

(5) Crozier, W. J.:

Jour. Biol. Chem., xxiv, No. 3. 1916.

Science, N. S., xlii, No. 1090, 1915.

(6) Crozier, W. J.: Jour. Gen. Physiol., v, No. 1, 65.

1922.

(7) Osterhout, W. J. V.: Jour. Biol. Chem. xix, 493. 1914.

(8) Haas, A. R. C.: Jour. Biol. Chem., xxvii, 225. 1916.

(9) Loeb, J.: Jour. Gen. Physiol., v, 231. 1922-23.

(10) Loeb, J.: Proteins and the theory of colloidal behavior. MacGraw-Hill Book Co., New York City. 1922.

(11) Crozier, W. J.: Jour. Gen. Physiol., i, 581. 1919.

(12) Osterhout, W. J. V.: Jour. Gen. Physiol., v, 225. 1922.

(13) Brooks, M. M.: Proc. Soc. Exp. Biol. and Med., xx, 39. 1922.

(14) Conklin, E. G.: Jour. Morphol., xxiii, 159. 1912.

(15) Wodehouse, R. P.: Jour. Biol. Chem., xxix, 453. 1917.

(16) Harvey, E. N.: Internat. Z. phys.-chem. Biol., i, 463. 1914.

(17) Crozier, W. J.: Jour. Biol. Chem., xxvi, 217. 1916.

(18) Loeb, J.: Jour. Gen. Physiol., v, No. 2, 255. 1922.

STUDIES ON THE PERMEABILITY OF LIVING AND DEAD CELLS. H. OBSERVATIONS ON THE PENETRATION OF ALKALI BICARBONATES INTO LIVING AND DEAD CELLS.

By MATILDA MOLDENHAUER BROOKS, Assistant Biologist, Division of Pharmacology, Hygienic Laboratory United States Public Health Service.

In the previous paper, dealing with the effects of acids upon the protoplasm of living and dead cells, carbonic acid was not included because of the characteristic changes which it produces in the cellsap of Valonia. In the case of other acids there is a progressive

increase in the acidity of the sap until its pH is equal to that of the solution in which the plants are immersed, whereas in the case of carbonic acid the increase in acidity is only temporary and is followed by a progressive increase of alkalinity. It was thought of interest to study the pH of the cell sap of Valonia when it is immersed in a solution containing carbonic acid or its salts.

Among the acids used, carbonic acid is peculiar in yielding alkali metal salts capable of hydrolytic dissociation which thus furnish an opportunity for studying the penetration of their two ions separately, and for determining whether either of them affects the permeability of protoplasm to other ions. Carbonic acid is also normally present in the cell. A description of the method used for determining the pH of the sap of Valonia was given in the preceding paper and will not be repeated here. Suffice it to say that two sets of pH determinations were made one set upon freshly extracted sap containing all its free CO2, and the other set upon the same samples of sap after the CO, had been removed by aeration with CO2-free air.

Immersion of normal cells in acids such as HCl and HNO, lowers the pH of the sap to about 5.2, at which point the acidity remains fixed for a considerable time, only ultimately going on to a higher acidity and death. The curves representing as a function of time the pH of the sap of cells placed in these acids, show a general tendency to "flatten out" at a pH, of 5.2. This is probably due to a steady decomposition of bicarbonates with liberation of CO2, but other substances which have a buffer action at pH 5.2 may play a part. The buffer effect of the bicarbonate-carbonic acid system lies at a pH between 7.0 and 8.0 when the system is in equilibrium with ordinary air, but increased CO2 tension would cause this range to lie at a lower pH. If the intracellular CO, tension were raised to that of air containing about 3 to 5 per cent of CO2, the pH of the buffer range would be about that actually observed (5.2). At this pH an accumulation of acid would therefore be needed before further change in reaction occurred. Even when sea water is saturated at atmospheric pressure with CO, so that its pH becomes 5.4, the pH of cell sap of plants placed in this solution does not exceed 5.2.

There is undoubtedly a balance between the production of respiratory CO, and its escape from the cell; and under ordinary conditions this mobile equilibrium keeps the H-ion concentration of the sap approximately constant. There seems to be an intracellular CO2 tension normal for Valonia and responsible for the observed differences between the pH of sap with and without free CO2. These differences are normally about 0.6 of a pH unit (6.2 to 6.8). When the balance

is upset, changes in the permeability of the protoplasm or alterations in the distribution of ions between the sap and protoplasm take place. This is nicely illustrated in the following simple experiment: By

allowing cells to remain in sea water containing enough CO, to produce a pH of 6.8 to 7.0, an abnormally large amount of CO, was made to accumulate in the sap, which became acid, attaining a pH of 5.2 to 5.3. After a time the pH of the sap when free CO, was removed began to increase in spite of the fact that the cells were in a solution the pH of which was 7.0 until the alkalinity approached pH 8.0 in three hours.

Observations on the effects of sodium and potassium bicarbonates dissolved in sea water, upon the pH of the cell sap show that, as in the case of sea water containing free CO,, there is at first a rapid increase of acidity and of free CO, in the sap. After a time the acidity decreases gradually and the pH finally approaches or even exceeds

FIG. 1.-Rate of penetration of free CO2 into the cell sap of Valonia from solutions of sea water containing either free CO alone or KHCO3 or NaHCO3 (0.03 M) (curves "A"). Curves "B" show the changes in alkalinity of the sap. The ordinates represent the pH values, the abscissæ the time in minutes.

that at the beginning of the experiment. This is connected with an increased alkalinity of the CO,-free sap, which begins immediately and proceeds until the pH approaches that of the external solution when the latter is freed from CO, (9.0 to 9.2). The curves in Figure 1 show the effects of placing Valonia in solutions consisting of 200 c. c. of sea water containing KHCO,, 0.03 M, NaHCO, in the same molecular concentration, or enough free CO, to produce a pH of 6.8 to 7.0. The pH of the potassium and sodium solutions was about 7.9 in sea water. When freed from CO2 their pH was 9.0 to 9.2. Curves “A” show the pH of the sap before and curves "B" after the free CO, has been removed. The concentration of the CO, in the cell sap is increased most rapidly when the cells are placed in solutions contain

ing CO, only, but the data representing penetration of CO, and other ions from such a solution are not quantitatively comparable to those of the other curves (KHCO, and NaHCO,), inasmuch as CO2 was present in a much higher concentration. The curves for sodium and potassium bicarbonate solutions are comparable, and show that CO2 penetrated more rapidly from the latter. Curves "B" also show differences in the rate of the changes producing alkalinity. Here again the change is more rapid when KHCO, has been used than when NaHCO, is present. Increased alkalinity might be due to substances given off by the protoplasm, but is more probably due to entrance of ions from the external solution.

The objection might be raised that this increase of alkalinity was due not to entrance of bases but to exosmosis of acids presumably other than carbonic. However, it is very improbable that carbonic acid should displace any stronger acid, and anions of weaker acids have not yet been found in the sap of Valonia.

In order to find direct evidence for the penetration of Li, LiCO, (0.03 M) was added to sea water and enough CO, added to produce a pH of 7.0; the sap of Valonia became pH 5.3 in a few minutes, and the CO2-free sap became alkaline gradually as in the case of Na and K bicarbonates. When cells of Valonia were allowed to remain in this solution for four hours, and their sap then collected and evaporated nearly to dryness, it was not possible to demonstrate the presence of Li by spectroscopic analysis. This is of interest because in the case of Nitella (1), a fresh-water alga, the writer found Li in the sap of plants which had been in a 0.05 M solution for 24 hours. The time element may account for this difference, but the penetration of Li in the case of Nitella was much slower from a balanced solution than from an unbalanced one. As the salts of balanced solutions affect the penetration of other salts into living cells, it is possible that the concentration of the salts of sea water in the case of Valonia prevented the entrance of more than a trace of Li; whereas in the case of Nitella the Li penetrated readily because of the low salt concentration of the surrounding medium.

Then, too, the change in the pH of the CO2-free sap of Valonia was from 6.6 to 8.0. If this increase in alkalinity were due entirely to the penetration of Li compounds, its concentration could not be more than about 1x 10 N. Since it was possible to detect solutions of LiCl of 1×10 but not 1x10 N, it is quite probable that Li entered the cell of Valonia, but in amounts too slight to be detected by the spectroscope.

-3

The length of survival of plants treated with the above solutions was also determined. It was found that normal cells lived under laboratory conditions in running sea water from 10 days to 1 month, whereas most of the plants which had been obviously injured during

the process of experimentation cytolyzed before 10 days. Therefore, cells which remained in good condition 10 days in sea water after having been in the test solutions were considered not to have been irreversibly injured. In all of the experiments represented in Figure 1, the plants apparently suffered no permanent injury when allowed to remain in the solutions one hour before being transferred to sea water. All the cells survived at least 10 days and some almost 1 month.

Some of the plants which had been in the bicarbonate solutions for one hour and were then transferred to sea water, were tested after six days to determine whether the sap still had the same pH that it had when the cells were replaced in sea water. It was found that the pH had returned to normal. This appears to have been due to an exosmosis of ions, but a study of this point has been left for future. investigation.

It was thought that perhaps the pH 8.0 was responsible for the rapid entrance of basic ions in the case of K and Na bicarbonates rather PH

FIG. 2.-Rate of penetration of free CO, into the sap of dead cells (curve "A") and the basicity of the sap when the free CO2 has been removed (curve "B").

than pH 7.0, that of sea water containing CO2. For this reason, to the solutions containing K and Na-bicarbonates CO, was added until pH 7.0 was obtained. The results were as follows: Cell-sap of plants attained a pH of 5.2 (with CO2) and a gradual alkalinity of the CO,-free sap which was slower in rate of attaining a higher alkalinity than when pH 8.0 was used. It would seem from these data that much CO, present hinders the entrance of basic ions into the interior of the cell, or that a more alkaline reaction of the surrounding medium is more favorable to the entrance of certain basic ions.

Figure 2 shows the effects of placing dead cells in sea water containing CO2 and having a pH of 6.8 to 7.0. The pH of the sap, which was originally 8.6, drops to 7.4 in 30 minutes (curve “A"'). When the CO2 is removed, it is found that the sap has a pH of 8.8 (which is the same as that of the surrounding medium without CO). Its basicity is unaltered (curve "B"). Therefore, the initial increase