Principles of General Physiology

Hemmeter, however, (1913) was unable to find any difference in the potassium content of the ash of normal and inhibited hearts, but this would scarcely be expected to be the case. In the blood contained within the heart of the dog-fish, under both conditions, again no difference was found, but the amount diffusing into the blood might easily be within the limits of the experimental error of the method used, that of ordinary chemical analysis. Of more interest

is the fact that the blood passing by crossed circulation from the heart of one dog-H.-h to lli.it of another had no effect on the latter when the former was inhibited by the vagus nerve. But, in experiments of this kind, negative results are less convincing than positive out •>. Cfdarine. — Turning our attention to anions, perhaps the most striking action is that of chlorine on the central nervous system, according to the work of von Wyss (1906). When sodium bromide is given in large doses, the chlorine content of the blood can be reduced to one-third. The exact cause of this is disputed, but the interesting point is that, at this stage, characteristic symptoms of paralysis set in. According to von Wyss, these symptoms are not due to accumulation of bromine, but to loss of chlorine, since they are rapidly cured by giving sodium chloride. Moreover, while ammonium chloride is effective, sodium nitrate or sulphate or magnesium sulphate is without action. Grilnwald (1909) obtained similar results by depriving rabbits of chlorine in their food and administration of diuretics. The mechanism of this phenomenon cannot be said to be altogether clear.

Carbon Dioxide. — Whether carbon dioxide or CO3" ions have any special action on cell processes apart from that of the hydrogen ion also present in solutions of carbon dioxide, is doubtful. It is held by some, for example, Laqueur and Verzar (1912), that carbon dioxide as such has an exciting effect on the respiratory centre, but the experiments are not convincing (see Chapter XXI.). Rona (1912) stated that it has a similar one on the movements of the intestine. The addition of sodium bicarbonate to a saline solution containing neither bicarbonate nor phosphate, caused the movements of an excised intestine to change from an irregular character to a perfectly regular one. This was apparently not due to diminution in hydrogen ion concentration, since the addition of bicarbonate had the same effect if its solution were previously brought to the same hydrogen ion concentration as the solution to which it was added. Also the production, by sodium hydroxide, of the same degree of alkalinity as that caused by the bicarbonate, with glycine as "buffer," had no effect. The result is held to be due to CO3" ions or to H0CO3 itself.

When a strong acid is added to a strong base in dilute solution, there is a considerable fall in the electrical conductivity of the mixture as compared with the sum of those of the two reagents separately. Since the salt formed is dissociated to as great a degree as the acid or base, the diminution must be due to the disappearance of the fast moving ions H* and OH'. For example, the conductivity of a 0'05 molar hydrochloric acid at 21 '8° is 17,945 reciprocal megohms ; that of a similar concentration of sodium hydroxide is 9,695 reciprocal megohms ; together 27,640, whereas 0'05 molar sodium chloride is only 4,995. In the solution of the salt there are, per 20 litres, 1 molecule of Cl' ions and 1 molecule of Na' ions, together 2 moleculr>. very nearly ; in 20 litres of hydrochloric acid, 1 molecule of H' ions and 1 molecule of Cl' ions ; in 20 litres of sodium hydroxide, 1 molecule Na' ions and 1 molecule OH' ions ; so that, if uncombined when mixed, there would be in all 4 molecules. But, even if we double the value of the conductivity of the sodium chloride solution to allow for this, we only have 9,990, instead of 27,640. It is evident that the diminution is only partially due to the disappearance of H' and OH' ions in combination as water, but that the slower rate of migration of the Na' and Cl' ions also plays a part.

Again, if we neutralise a weak base, such as aniline, with a strong acid, we get a diminution of conductivity, or if a weak acid is neutralised with a strong base. On the other hand, if we take a weak base and a weak acid, the conductivity of the salt is higher than the sum of those of the base and acid together. This is because the salt is more highly dissociated, electrolytically, than either the base or the acid, so that there is an actual increase in the number of ions present.

It is not easy to see why, to take a specific case, the compound of the acetic anion with hydrogen ion is very little dissociated, whereas when it is combined with the cation of aniline there is considerable dissociation. The fact is probably of some importance in physiological processes. The organic acids and bases produced in cell metabolism are for the most part of the weak class, that is, very little electrolytically dissociated ; when they combine,

the salt is highly dissociated, so that a number of ions make their appearance. So far, -then, as the properties of a substance are those of its ions, the salts of weak acids with weak bases are more powerful agents than the substances from which they are formed. If the percentage dissociation of aniline acetate be calculated from measurements of the migration rates of its ions and of its degree of hydrolytic dissociation, it is foujid that, at a dilution of 1 molecule in 13 '75 litres, it is electrolytically dissociated to the extent of 45 per cent. , whereas hydrogen acetate is only 5 per cent, dissociated. Aniline acetate is hydrolytically dissociated to about 32 per cent. , so that about 25 per cent, is not dissociated in either way.

There are two practical points of interest in connection with this question. In the first place, the fact gives us a very convenient means of following the course of a tryptic digestion. The weak amino-acids produced, when they combine with the ammonia used to give the requisite degree of alkalinity, or with diaminoacids, acting as bases, give rise to a considerable increase in the conductivity of the mixture. The conductivity of leucine in O'Oo molar strength at 22° is only about 3 reciprocal megohms, that of ammonium hydroxide in the same conditions is 232 reciprocal megohms, together 235. When mixed, the salt formed is fairly highly dissociated and the solution has a conductivity of 1,548 reciprocal megohms. This may be compared with aniline acetate ; aniline in O'Oo molar solution has a value of 13, acetic acid in the same concentration is 330, together 343 ; while aniline acetate, O'Oo molar, is 1,518.

In the second place, we obtain some information as to the relative strengths of an acid and a base. An acid which is weak towards a strong base may be relatively strong towards a weaker base. For example, salicylic acid, which has a dissociation constant of 102 x 10~5, when combined with ammonium hydroxide, gives an increase of conductivity, that is, it is a weak acid towards the base ammonium hydroxide ; when combined with aniline, on the other hand, there is &Jall in conductivity, that is, it is .a relatively strong acid towards the very weak base, aniline. Maleic acid (dissociation constant = 1 170 x 10~5) is a strong acid to both bases and acetic acid (dissociation constant = 1 '8 x 10~5) is weak to both bases. The mono -amino-monocar boxy lie acids are too weak as bases to combine with acids as weak as acetic acid. On the other hand the diamino-mono-carboxylic acids are sufficiently strong as bases to combine with acids as strong as the mono-amino-dicarboxylic acids. For example, I found that diamino-propionic acid, 0'17 molar, had, at 40°, a conductivity of 1,672 reciprocal megohms, glutamic acid, 0'095 molar, had a conductivity of 950 on the same scale, together 2,622 ; a solution containing both in the same concentration as before had a conductivity of 5,142 reciprocal megohms, showing that combination had taken place (Bayliss, 1909, 2).

It is to be noted that the use of the words " weak " and " strong " in the above connection is to be taken only as referring to their relative power of combining with weak acids or bases respectively. It does not conflict with the expression as used in reference to the electrolytic dissociation of their solutions, which is an absolute measurement of their strength as compared with one another. There is an important class of substances, already referred to incidentally in connection with the colloidal properties of proteins, which can act either as acids or bases ; that is, they dissociate with the formation of H* and OH' ions. We have seen that water is a member of this class and we have now to turn our attention to a very important series of substances, the amino-acids. These owe their nature as both bases and acids to the fact that they contain one or more NH2 groups, together with one or more COOH groups.

For convenience, we may call the radicle which is combined with H arid OH, R, which is : — in the case of glycine. Then, according to the investigations of Bredig (1899) and of J. Walker (1904), the solution contains the following molecules and ions: — H-, OH', HR', ROH', HROH, and R. Whether R is to be looked upon as an ion with both a negative and a positive charge is doubtful. If so, it is formed by giving off both H' and OH' ions and would be represented thus : —

in the case of glycine and is sometimes known as a "hermaphrodite" ion. In Bredig's scheme, however, it is represented as devoid of charges and is probably, in fact, an internal anhydride: — As such, we must suppose that the two groups combined have opposite charges, so that it is not impossible that they might exist as such on a single ion. An interesting suggestion is made by Bredig (1894, p. 323), as to the length of the chains which can exist without self-neutralisation. If a sufficiently long chain could be formed, having opposite charges at the ends, it should be possible by optical means to detect an orientation to an electrical current passed through the solution. Bredig, himself, was unable to detect any sign of this in the case of betaine.

It is unnecessary to remark that an ion with two opposite charges moves to neither electrode, being equally attracted to both, so that it can take no part in the conduction of a current. In this aspect, it is not, in any case, entitled to the name of an ion, in Faraday's sense. As we have seen above (page 105), there is no evidence that an amino-acid can combine with the positive and negative ions of a neutral salt simultaneously. A "hermaphrodite" ion should be able to do this.

The various ions enumerated above as present in solutions of the amino-acids exist in very small concentrations, so that their electrical conductivity is very low, especially in the case of the mono-amino-monocarboxylic series. The acidic and basic groups are mutually antagonistic, so that both dissociation constants are very small. The mono-amino-monocarboxylic acids are very weak indeed, both as acids and as bases. The carboxyl group is a little stronger as acid than the NH., group is as base, so that the acid properties very slightly preponderate.

When we have another COOH or another NH0 group added on, as in aspartic acid or lysine respectively, the acidic function is considerably increased in the first case and the basic function in the second. From Winkelblech's investigations (1901) it is interesting to note that, when the strength of the acid group considerably exceeds that of the basic one, as in taurine (amino-ethylsulphonic acid), salts are formed only with bases, not even with acids as strong as hydrochloric acid. Conversely, if the basic group is considerably stronger than the acid one, as in betaine (tri-methyl-glycine), then salts are formed only with acids. It is also somewhat unexpected to find that, comparing glycine, alanine, leucine, sarcosine and betaine, the stronger acid is at the same time the stronger base, but the fact appears to hold only for the mono-amino-monocarboxylic series.

As to the methods of determining the two dissociation constants, one of these is that of conductivity measurements of their salts with hydrochloric acid and with sodium hydroxide, and another is that of hydrolytic dissociation. The papers by Lunden (1908) and by Winkelblech (1901) may be consulted. I insert here the values of the dissociation constants of a few amphoteric electrolytes, at 25°, taken from Lunden's work (1908, p. 81). With regard to proteins, we have seen in dealing with them from the colloidal point of view how the effect of acid and alkali on the sign of their electrical charges is explained by their nature as amphoteric electrolytes. A further proof of this fact is afforded by the measurements of the freezing points of their salts with acid and alkali, as obtained by Bugarszky and Liebermann (1898, p. 72). In the table below, the first column gives the number of grams of egg albumin added to 100 c.c. of the acid, base, or salt in 0'05 molar concentration, and the three remaining columns give the depressions of the freezing point in each of these cases.

It will be seen that there is a considerable diminution of A in the cases of acid and base, due to formation of salts with the protein. In the case of the neutral salt there is no such effect. The contrary effect, a rise of A with the sodium chloride, is, in fact, due to the albumin itself, since 6-4 g. of the protein in 100 c.c. of water gave a freezing point depression of 0-022 ; this, added to 0-183, gives 0-205, as in the table. The powerful effect of hydrogen and hydroxyl ions in traces has been exemplified in the case of the heart. Further instances will occur in the course of this book.

One or two striking cases of the action of inorganic salts in minute quantities may be referred to here. Elissafoff (1912) showed that the effect of the quadrivalent thorium ion on the surface charge of quartz was such as to lower it by 50 per cent., when the solution contained only one gram ion in a thousand million grams of water. The extraordinary effect of zinc in traces on the growth of moulds was discovered by Raulin (1870, 1 and 2), as also that of manganese. This observer was doubtful whether the effect of manganese salts was not due to traces of zinc, and the matter was further worked out by Bertrand and Javillier (1912). They found that manganese itself actually has an effect of this kind. One part of manganese in one million of the culture solution raises the crop of Aspergillus from 0*610 to 0-631 and the effect continues to increase even up to one part in 100. In further work it was found that the combination of zinc with manganese was more effective than either alone. To take an example : —

The data also show the really astonishing effect of zinc alone. In another experiment, indeed, we find that the addition of one part of zinc to twenty-five millions of solution increases the crop from 3'00 to 4'54, that is, by more than 50 per cent., and one part in ten millions nearly doubles it. The authors point out how important is this function of elements present only in traces ; they regard it as being of a catalytic nature. We shall have occasion later to return to the question of the effect of substances, not only inorganic ones, which, although present only in infinitesimal amount, are, as it seems, absolutely indispensable to the normal functional capacity of protoplasm.

From the work of Raulin it appeared also that iron in traces had a great effect on the normal production of the fructification (conidia) of the mould. Bertrand (1912), having been able to prepare solutions in a great state of purity, found that, although iron and zinc might both be present, there were no conidia formed unless manganese was also present. If any one of these three elements is wanting, or present in too small a quantity, complete normal growth is impossible. But whereas vigorous growth of mycelium takes place with iron and zinc alone, no conidia are formed in the absence of manganese.

The opposite phenomenon to the favourable action by traces of zinc on Aspergillus are to be found in the toxic action of certain metals, especially copper, more particularly to the higher organisms. In a posthumous paper by Nageli (1893), some very important results are described in relation to this question. It was noticed that ordinary distilled water was rapidly fatal to Spirogyra, just as Ringer and Phear at a later date (1895) found that it was to tadpoles.

Nageli discovered that the toxic action was due to the presence of minute traces of compounds of various heavy metals in the water. Tap water, which originally did not show this property, became poisonous after being in contact with metallic copper, mercury, lead, tin, iron, or silver. It was also found that the addition of various insoluble solids, such as paper, wool, paraffin, or of certain colloids, such as gum or gelatine, deprived the water so treated of its toxic character. From what has been said in previous chapters of this book, when dealing with the colloidal state and the phenomena of adsorption, the explanation of this neutralising power of surfaces will be obvious. The toxic metal is present either as hydroxide or carbonate in the colloidal state ; this, as an electro-positive colloid, will be strongly adsorbed by electro-negative surfaces, such as those used by Nageli. The fact noticed by Ringer (1886, p. 292) that calcium phosphate is more effective in neutralising the toxic properties of distilled water than calcium chloride is, is easily explained by the greater precipitating action of the trivalent PO4'" ion on an electro-positive colloid than that of the univalent Cl' ion.

Nageli estimated the amount of copper present in 1 2 litres of distilled water, which had been for four days in contact with 12 two-pfennig pieces. It contained one part in seventy-seven millions. This water was powerfully toxic to Spirogyra, killing it in one minute. On account of the very small quantity of copper in the water, Niigeli gave the name of " oliyodynamic " to .the action in question. Locke (1895), in repeating these experiments, found that, of the various metals tested, copper was by far the most toxic. A strip of bright copper, 4 "5 by 1*5 cm. in dimensions, placed for twenty hours in 200 c c. of distilled water, made the water toxic to tadpoles and to the river worm, Tubifex. Brass had the same effect as copper, but zinc, although toxic, was not so powerfully active, while tin appeared to be innocuous.

Raulin, in the course of the work referred to in the preceding section, had also noticed that one part of silver nitrate in 1,600,000 of the culture medium was sufficient to prevent germination of the spores of Aspergillus ; in fact, if the medium is contained in a silver vessel, sufficient metal is dissolved to prevent growth therein. Ringer and Phear did not attribute the toxic action of their distilled water to " oligodynamic action," but Locke, in the paper quoted above, showed clearly that the explanation lay in this fact, since distilled water condensed in glass had no injurious action.

It has been found that certain bright metals pass readily into the colloidal state when placed in contact with pure distilled water (see Traube-Mengarini and Scala, 1912). Thus lead, zinc, iron, tin, aluminium, copper, and nickel form, in this way, colloidal solutions in which the dispersed phase is, at first, in the metallic state, but subsequently becomes hydroxide. There is a group of substances, which, investigated in various methods, are found to show, in solution in water, a higher osmotic pressure than that corresponding to their molar concentration. All these substances are found to be conductors of electrical currents, that is, they are electrolytes, to use the name introduced by Faraday.

It is clear, therefore, that the molecules of electrolytes are split up, dissociated, in solution in water, so that there are more osmotically-active elements in their solutions than in those of non-electrolytes in the same molar concentration. Since electrolytes conduct electricity by means of their " ions," which appear at the two electrodes (Faraday), the view was put forward by Arrhenius that these ions exist in solutions of electrolytes in ordinary conditions, independently of the passage of electrical currents.

Evidence of various kinds has been brought to show that this is the case. Hydrochloric acid, for example, is more or less completely split up into hydrogen ions, each carrying a unit positive charge, and chlorine ions, each carrying a unit negative charge. This is known as " electrolytic dissociation." The more dilute the solution, the more complete is the dissociation. The power of conducting a current depends both on the actual number of ions engaged in the carriage of the charges and also on the rate at which they move. The rate has considerably different values for different ions and is in relation not only to the atomic or molecular weight of the ion, but to the number of molecules of water which are attached to it (Hydration of Ions). The value is constant for each ion under similar conditions. The absolute rate of movement is slow. Hydrogen ions, the most rapid, have a velocity, under a potential fall of one volt per centimetre, of only 0-0033 cm. per second ; but the rate is, of course, dependent on the force producing the motion.

The reason why it is impossible to separate the oppositely charged ions by diffusion, or other means except an electrical one, is the enormous electrosfcft&e- attraction between them, which prevents a positive ion from being separated ^ from its fellow negative one beyond infinitesimal distances. When, however, one of the ions moves faster than the oppositely charged one, it does actually form a layer in front of that of the more slowly moving ions, at a very minute distance. This phenomenon is known as the " Helmholtz doublelayer" and is the cause of the appearance of an electromotive force at the boundary surface between solutions containing ions of differing mobility.

The source of the energy required to dissociate the molecules of electrolytes when dissolved in water is discussed in the text, as also the relation of the process to the dielectric constant of the solvent. While the equilibrium between non-dissociated molecules and ions in the cases of weak acids and bases obeys the law of mass action, as shown by their behaviour on dilution (Ostwald's Dilution Law), that of strong acids, strong bases and salts obeys a different law. The explanation of this fact has not yet been given. It has been suggested by Noyes and his co-workers that there may be two different kinds of combination between ion,s to form molecules, one rather of an electrical nature and somewhat loose, the other more strictly chemical and more stable. The former would be the case with the strong electrolytes.

^JVi f.Vip.ir intervention in physio^og^a! processes, electrolytes may be said to act "mainly in three ways. By the electrical charges on their ions, as in colloidal phenomena: by their effect on the properties of the solvent, " lyotropic " action ; and by the purely chemical properties of their ions or molecules. The important part played by acidity and alkalinity shows the value of the electrolytic dissociation theory in an especially striking way. These properties of solutions can be expressed by the numerical values of their concentration in