The Organism as a Whole, from a Physicochemical Viewpoint

4. Fifty or sixty years ago surgeons did not hesitate to transfuse the blood of animals into human beings. The practice was a failure, and Landois1 showed by experiment that if blood of a foreign species was introduced into an animal the blood corpuscles of the transfused blood were rapidly dissolved and the animal into which the transfusion wras made was rendered ill and often died. The result was different when the animals whose blood was used for the purpose of transfusion belonged to the same species or a species closely related to the animal into which the blood was transfused. Thus when blood was exchanged between horse and donkey or between wolf and dog or between hare and rabbit no hemoglobin appeared in the urine and the animal into which the blood was transfused remained well.2 This was the beginning of the investigations in the field of serum specificity which were destined to play such a prominent role in the development of medicine. Friedenthal was able to show later that if to

1 Landois, L., Zur Lehre von der Bluttransfusion. Leipzig, 1875. 3 This is probably true only within the limits of exactness used in these experiments. 10 c.c. of serum of a mammal three drops of defibrinated blood of a foreign species are added and the whole is exposed in a test tube to a temperature of 38°C. for fifteen minutes the blood cells contained in the added blood are all cytolyzed; that this, however, does not occur so rapidly when the blood of a related species is used. He could thus show that human blood serum dissolves the erythrocytes of the eel, the frog, pigeon, hen, horse, cat, and even that of the lower monkeys but not that of the anthropoid apes. The blood of the chimpanzee and of the human are no longer incompatible, and this discovery was justly considered by Friedenthal as a confirmation of the idea of the evolutionists that the anthropoid apes and the human are blood relations. I

This line of investigation had in the meanwhile entered upon a new stage when Kraus, Tchistowitch, and Bordet discovered and developed the precipitin reaction, which consists in the fact that if a foreign serum (or a foreign protein) is introduced into an animal the blood serum of the latter acquired after some time the power of causing a precipitate when mixed with the antigen, i. e., with the foreign substance originally introduced into the animal for the purpose of causing the production of antibodies in the latter; while, of course, no such precipitation occurs if the serum of a

non-treated rabbit is mixed with the serum of the blood of the foreign species. In 1897 Kraus discovered that if the filtrates from cultures of bacteria (e. g., typhoid bacillus) are mixed with the serum of an animal immunized with the same serum (e. g., typhoid serum) it causes a precipitate; and that this precipitin reaction is specific. This fact was confirmed and has been extended by the work of many authors. Tchistowitch in 1899 observed that the serum of rabbits which had received injections of horse or eel serum caused a precipitate when mixed with the serum of these latter animals.

Bordet found in 1899 that if milk is injected into a rabbit the serum of such a rabbit acquires the power of precipitating casein, and Fish found that this reaction is specific inasmuch as the lactoserum from cow's milk can precipitate only the casein of cow's milk but not that of human or goat milk. Wassermann and Schutze reached the same result independently of each other. Myers and later Uhlenhuth showed that if white of egg from a hen's egg is injected into a rabbit, precipitins for white of egg are found in the serum of the latter, and Uhlenhuth1 found, by trying the white of egg of different species of birds, that the precipitin reaction

called forth by the blood of the immunized animal is specific, inasmuch as the proteins from a hen's egg will call forth the formation of precipitins in the blood of the rabbit which will precipitate only the white of egg of the hen or of closely related birds. To Nuttall1 belongs the credit of having worked out a quantitative method for measuring the amount of precipitate formed, and in this way he made it possible to draw more valid conclusions concerning the degree of specificity of the precipitin reaction. He found by this method that when the immune serum is mixed with the serum or the protein solution used for the immunization a maximum precipitate is formed, but if it is mixed with the serum of related forms a quantitatively smaller precipitate is produced. In this way the degree of blood relationship could be ascertained. He thus was able to show that when the blood of one species, e. g., the human, was injected into the blood of a rabbit, after some time the serum of the rabbit was able to cause a precipitate not only with the serum of man, or chimpanzee, but also of some lower monkeys; with this difference, however, that the precipitate was much heavier when the immune serum was added to the serum of man. The method thus shows the existence of not an absolute but of a strong quantitative specificity of blood serum. This statement may

'Nuttall, George H. P., Blood Immunity and Blood Relationship, Cambridge Univ, Press, 1904, be illustrated by the following table from Nuttall. The antiserum used for the precipitin reaction was obtained by treating a rabbit with human blood serum. The forty-five bloods tested had been preserved for various lengths of time in the refrigerator with the addition of a small amount of chloroform. Marsupialia Petrogale xanthopus ' Petrogale penicillata Onychogale frenata

Among the Primate bloods that of the Chimpanzee gave too high a figure, owing to the precipitum being flocculent and not settling well, for some reason which could not be determined. The figure given by the Ourang is somewhat too low, and the difference between Cynocephalus sphinx and Ateles is not as marked as might have been expected in view of the qualitative tests and the series following. The possibilities of error must be taken into account in judging of these figures; repeated tests should be made to obtain something like a constant. Other bloods than those of Primates give small reactions or no reactions at all. The high figures (10%) obtained with two Carnivore bloods can be explained by the fact that one gave a loose precipitum, and the other was a somewhat concentrated serum.1

We have mentioned that even the proteins of the egg are specific according to Uhlenhuth. Graham Smith, one of Nuttall's collaborators, applied the lat- 1 Nuttall, Blood Immunity and Blood Relationship, pp. 319 and 320. ter's quantitative method to this problem and confirmed the results of Nuttall. A few examples may serve as an illustration. Duck's Pheasant's Fowl's Silver Pheasant Blackbird's Crane's Moorhen's Thrush's Emu's Hedge-Sparrow ' Chaffinch's Tortoise serum Turtle serum Alligator serum

Frog, Amphiuma, and Dogfish sera, as well as Tortoise and Dogfish egg-albumins, were also tested, with negative results. Fowl's Fowl's Silver Pheas Pheasant's Crane's Blackbird's Duck's Moorhen's Thrush, Emu, Greenfinch, and Hedge-sparrow egg-albumins were tested and gave traces of precipita, as also did Tortoise and Turtle sera. The egg-albumins of the Tortoise, Frog, Skate, and two species of Dogfish did not react. Alligator, Frog, Amphiuma, and Dogfish sera also yielded no results.1

By improving the quantitative method in various ways, Welsh and Chapman2 were able to explain why the precipitin reaction with egg-white was not strictly specific but gave also, though quantitatively weaker, results with the egg-white of related birds. They found that by a new method devised by them "it is possible to indicate in an avian egg-white antiserum the presence of a general avian antisubstance (precipitin) together with the specific antisubstance."

The Bordet reaction was not only useful in indicating the specificity and blood relationship for animals but also among plants. Thus Magnus and Friedenthal3 were able to demonstrate with Bordet 's method the relationship between yeast (Saccharomyces cerevisicz) and truffle (Tuber brumale). 5. We must not forget, while under the spell of the problem of immunity, that we are interested at the moment in the question of the nature of the specificity of living organisms. It is only logical to conclude

that the fossil forms of invertebrate animals and of algae and bacteria, which Walcott found in the Cambrian and which may be two hundred million years old, must have had the same specificity at that time as they or their close relatives have today; and this raises the question: What is the nature of the substances which are responsible for and transmit this specificity? It is obvious that a definite answer to this question brings us also to the very problem of evolution as well as that of the constitution of living matter.

There can be no doubt that on the basis of our present knowledge proteins are in most or practically all cases the bearers of this specificity. This has been found out not only with the aid of the precipitin reaction but also with the anaphylaxis reaction, by which, as the reader may know, is meant that when a small dose of a foreign substance is introduced into an animal a hypersensitiveness develops after a number of days or weeks, so that a new injection of the same substance produces serious and in some cases fatal effects. This hypersensitiveness, which was first analysed by Richet, * is specific for the substance which has been injected. Now all these specific reactions, the precipitin reaction as well as the anaphylactic reaction, can be called forth by proteins. Thus Richet, in his earliest experiments, showed that only the protein-containing part of the extract of actinians, by which he called forth anaphy-

laxis, was able to produce this phenomenon, and later he showed that it was generally impossible to produce anything resembling anaphylaxis by non-protein substances, e. g., cocain or apomorphin. I Wells isolated from egg-white four different proteins (three coagulable proteins and one non-coagulable) which can be distinguished from each other by the anaphylaxis reaction, although all come from the same biological object.2 Michaelis as well as Wells found that the split products of the protein molecule are no longer able to call forth the anaphylaxis reaction. Since peptic digestion has the effect of annihilating the power of proteins to call forth anaphylaxis, we are forced to the conclusion that the first cleavage products of proteins have already lost the power of calling forth immunity reactions.

A pretty experiment by Gay and Robertson3 should be mentioned in this connection. Robertson had shown that a substance closely resembling paranucleins both in its properties and its C, H, and N content can be formed from the filtered products of the complete peptic hydrolysis of an approximately four per cent, neutral solution of potassium caseinate by the action of pure pepsin at He considered this a case of a real synthesis of proteins from the products of its hydrolytic cleavage. This

interpretation was not generally accepted and received a different interpretation by Bayliss and other workers. Gay and Robertson were able to show that paranuclein when injected into an animal will sensitize guinea-pigs for anaphylactic intoxication for either paranuclein or casein and apparently indiscriminately. The products of complete peptic digestion of casein had no such effect, but the synthetic product of this digestion obtained by Robertson's method has the same specific antigenic properties as paranuclein, thus making it appear that Robertson had indeed succeeded in causing a synthesis bf paranuclein with the aid of pepsin from the products of digestion of casein by pepsin.

There are a few statements in the literature to the effect that the specificity of organisms might be due to other substances than proteins. Thus Bang and Forssmann claimed that the substances (antigens) responsible for the production of hemolysis were of a lipoid nature, but their statements have not been confirmed, and Fitzgerald and Leathes1 reached the conclusion that lipoids are non-antigenic. Ford claims to have obtained proof that a glucoside contained in the poisonous mushroom Amanita phalloides can act as an antigen. But aside from this one fact we know that proteins and only proteins can act as antigens and

are therefore the bearers of the specificity of living organisms. Bradley and Sansum1 found that guinea-pigs sensitized to beef or dog hemoglobin fail to react or react but slightly to hemoglobin of other origin. The hemoglobins tried were dog, beef, cat, rabbit, rat, turtle, pig, horse, calf, goat, sheep, pigeon, chicken, and man. 6. It would be of the greatest importance to show directly that the homologous proteins of different species are different. This has been done for hemoglobins of the blood by Reichert and Brown, 2 who have shown by crystallographic measurements that the hemoglobins of any species are definite substances for that species.

The crystals obtained from different species of a genus are characteristic of that species, but differ from those of other species of the genus in angles or axial ratio, in optical characters, and especially in those characters comprised under the general term of crystal habit, so that one species can usually be distinguished from another by its hemoglobin crystals. But these differences are not such as to preclude the crystals from all species of a genus being placed in an isomorphous series (p. 327).

2 Reichert, E. T., and Brown, A. P., "The Differentiation and Specificity of Corresponding Proteins and other Vital Substances in Relation to Biological Classification and Organic Evolution." Carnegie Institution Publication No. 116, Washington, 1909. As far as the genus is concerned it was found that the hemoglobin crystals of any genus are isomorphous. In some cases this isomorphism may be extended to include several genera, but this is not usually the case, unless as in the case of dogs and foxes, for example, the genera are very closely related.

The most important question for us is the following: Are the differences between the corresponding hemoglobin crystals of different species of the same genus such as to warrant the statement that they indicate chemical differences? If this were the case we might say that blood reactions as well as hemoglobin crystals indicate that differences in the constitution of proteins determine the species specificity and, perhaps, also species heredity. The following sentences by Reichert and Brown seem to indicate that this may be true for the crystals of hemoglobin.

The hemoglobins of any species are definite substances for that species. But upon comparing the corresponding substances (hemoglobins) in different species of a genus it is generally found that they differ the one from the other to a greater or less degree ; the differences being such that when complete crystallographic data are available the different species can be distinguished by these differences in their he aoglobins. As the hemoglobins crystallize in isomorphous series the differences between the angles of the crystals of the species of a genus are not, as a rule, great; but they are as great as is usually found to be the case with

minerals or chemical salts that belong to an isomorphous group (p. 326). As Professor Brown writes me, the difficulty in answering the question definitely, whether or not the hemoglobins of different species are chemically different, lies in the fact that there is as yet no criterion which allows us to discriminate between a species and a Mendelian mutation except the morphological differences. It is not impossible that while species differ by the constitution of some or most of their proteins, Mendelian heredity has a different chemical basis.

It is regrettable that work like that of Reichert and Brown cannot be extended to other proteins, but it seems from anaphylaxis reactions that we might expect results similar to those in the case of the hemoglobins. The proteins of the lens are an exception inasmuch as, according to Uhlenhuth, the proteins of the lens of mammals, birds, and amphibians cannot be discriminated from each other by the precipitin reaction.1 7. The serum of certain humans may cause the destruction or agglutination of blood corpuscles of certain other humans. This fact of the existence of "isoagglutinins" seems to have been established foi man, but Hektoen states that he has not been able to find any isoagglutinins in the serum of rabbits, guineapigs, dogs, horses, and cattle. Landsteiner found the

1 Uhlenhuth, Das liologische Verfaliren zur Erkennung und Unterscheidung von Menschen und Tierblut, Jena, 1905, p. 102. remarkable fact that the sera of certain individuals of humans could hemolyze the corpuscles of certain other individuals, but not those of all individuals. A systematic investigation of this variability led him to the discovery of three distinct groups of individuals, the sera of each group acting in a definite way towards the corpuscles of the representatives of each other group. Later observers, for example Jansky and Moss, established four groups. These groups are, according to Moss, x as follows :

Corpuscles agglutinated by sera of Groups 2, 3, 4. Group 2. Sera agglutinate corpuscles of Groups I, 3. Corpuscles agglutinated by sera of Groups 3, 4. Group 3. Sera agglutinate corpuscles of Groups 1,2. Corpuscles agglutinated by sera of Groups 2, 4. Group 4. Sera agglutinate corpuscles of Groups i, 2, 3. The relative frequency of the four groups follows from the following figures. Of one hundred bloods tested by Moss in series of twenty there were found :

Groups 2 and 4 are in the majority and in overwhelming numbers, which indicates that, as 4 rule, the 1 Moss, W. L., Johns Hopkins Hospital Bulletin, 1910, xxi., 62. sera agglutinate the blood corpuscles of individuals of the other groups, but not those of individuals belonging to the same group. The phenomenon that a serum agglutinates no corpuscles (Group i), or that the corpuscles are agglutinated by no serum (Group 4), are the exceptions. It is obvious that, as far as our problem is concerned, only Groups 2 and 3 are to be considered. There is no Mendelian character which refers only to one half of the individuals except sex. Since nothing is said about a relation of Groups 2 and 3 to sex such a relation probably does not exist.

8. The facts thus far reported impty the suggestion that the heredity of the genus is determined by proteins of a definite constitution differing from the proteins of other genera. This constitution of the proteins would therefore be responsible for the genus heredity. The different species of a genus have all the same genus proteins, but the proteins of each species of the same genus are apparently different again in chemical constitution and hence may give rise to the specific biological or immunity reactions.

We may consider it as established, by the work of McClung, Sutton, E. B.Wilson, Miss Stevens, Morgan, and many others, that the chromosomes are the carriers of the Mendelian characters. These chromosomes occur in the nucleus of the egg and in the head of the sperm. Now the latter consists, in certain fish, of lipoids and a combination of nucleinic acid and protamine or histone, the latter a non-coagulable protein, more resembling a split product of one of the larger coagulable proteins.