aqueous solution of its chloride gives a precipitate with a very little salt which is insoluble in stronger saline solutions.

The most important difference between these histons and edestan is that the former appear to be soluble in water when neutral, whereas neutral edestan is very insoluble in water, the reactions, which I have described, being given by aqueous solutions of its chloride. This combined acid, however, is present in such small proportion that it can be detected only in concentrated solutions by using very delicate litmus paper, so that it might easily be overlooked were its presence unknown. Bang gives no evidence that his solutions did not contain a similar proportion of acid and it seems probable that some of them at least did contain it, since the substances were extracted by dilute hydrochloric acid and the solutions made "neutral" presumably to litmus, this indicator being the one commonly employed by physiologists for such purposes. That any near relation exists between edestan and the bodies enumerated by Bang as histon is not probable, except in the case of globin, which seems to be more nearly related to edestan than to the histons since globin is a true protein substance, obtained from hemoglobin by the action of acids under conditions similar to those leading to the formation of edestan. With thymus histon and with scombron, it would seem that edestan and globin have little in common, since the two former yield little or no proteoses on pepsin digestion, whereas edestin yields such abundantly, and globin doubtless does the same.

From the facts now at our command it is evident that we have two different classes of bodies which conform pretty closely to the reactions characteristic of the histons. It is important to recognize this fact, since otherwise confusion will result if these two classes are not distinguished from one another, and protein derivatives produced by the acid used in preparing these substances may be regarded as actual constituents of the tissues.

SUMMARY.

By the action of water or very dilute solutions of acids, the globulin edestin is converted into a substance insoluble in saline solutions of moderate concentration.

This derivative of edestin is formed by hydrolysis, the amount formed being proportional to time and the concentration of the solution in hydrogen ions. In pure water less is formed in a given time than in water containing carbonic acid. More is

formed by a given quantity of hydrochloric acid than by an equivalent quantity of phosphoric acid, and by either of these acids much more is formed than by an equivalent of acetic acid.

This substance is the same as that designated as "albuminate” by Weyl, which is formed in greater or less amount in preparations of crystallized edestin made in the usual manner, and is without doubt the first product of the hydrolytic changes leading to the formation of the so-called acid albumin.

It is probable that the products insoluble in saline solutions which are formed from other globulins, originate from the same cause, and that these form a distinct class of hydrolytic derivatives of the native protein molecules.

For this derivative of edestin the name edestan is proposed.

The ultimate composition of edestan is the same, so far as can be determined by analysis, as that of edestin from which it originates.

Edestan forms salts with hydrochloric acid which react acid toward phenolphthalein to the full extent of the combined acid. One salt, having an acidity equivalent to 20 cc. of a centinormal solution per gram, is very sparingly soluble in water. It is this salt which forms the so-called "albuminate" found in edestin preparations. If edestan has a molecular weight near that of edestin, assumed to be about 14,500' this acidity would correspond to that of a trichloride, being just three times that of edestin monochloride and one and one-half times that of the bichloride.

Edestan is insoluble in water, far less soluble in solutions of potassium hydroxide than is edestin and insoluble in ammonia water, unless the solution of the latter is relatively very strong.

The aqueous solution of edestan chloride, when concentrated, reacts acid with litmus. It is precipitated by neutralization, the precipitate being soluble in strong ammonia, yielding a solution which is precipitated by ammonium chloride but not by sodium chloride.

The aqueous solution of edestan chloride gives a precipitate with nitric acid which dissolves on warming and reappears on cooling; a precipitate with ovalbumin solutions with the alkaloidal reagents and with sufficient mercuric chloride if its solution is concentrated. These reactions agree closely with those given 1 See Osborne: This Journal, 21, 486 (1899); also the paper following.

by Kossel as characteristic of histons, but with the true histons edestan has little in common.

RESEARCH LABORATORY OF THE CONNECTICUT
AGRICULTURAL EXPERIMENT STATION.

THE BASIC CHARACTER OF THE PROTEIN MOLECULE AND THE REACTIONS OF EDESTIN WITH DEFINITE QUANTITIES OF ACIDS AND ALKALIES.

THA

BY THOMAS B. OSBORNE.
Received September 28, 1901.

I. INTRODUCTION.

The Basic Character of the Protein Molecule.

HAT the proteins are ionized and highly reactive bodies is indicated by the rapidity with which they unite with both bases and acids, by the readiness with which, in many cases, they respond to changes in the ionization of their solutions, and also by the fact that they are, chemically, the most active constituents of protoplasm.

That they are neutral bodies, like the carbohydrates, is not in harmony with what is known of them.

Nevertheless, it appears to be generally assumed that a solution containing protein matter, which shows neither acid nor alkaline. reaction with litmus, is chemically neutral.

Observations are on record which show that some protein solutions, when neutral to litmus, are acid to phenolphthalein and alkaline to lacmoid. It is also well known that a notable quantity of acid can be added to a protein solution before an acid reaction with tropaeolin, alizarine, or phloroglucin and vanillin appears.

The fact that acids combine with protein bodies is, therefore, well known, and, in making preparations of these substances, the necessity of removing such acids has long been recognized. This has been supposedly accomplished by adding potassium or sodium hydroxide or carbonate until the reaction with litmus becomes neutral. I am not aware that any one has offered any evidence, however, that by this procedure this object is fully accomplished. It is of importance, therefore, to know whether litmus can be used to determine the point when all combined acid has been converted into neutral salts of potassium or sodium and all the protein substance has been set free, or whether, as we know is the

case when tropaeolin or lacmoid is used as an indicator, more acid still remains combined.

Solutions in water of preparations of crystallized ovalbumin, in sodium chloride brine of excelsin, amandin, vignin, conglutin, glycinin, corylin, phaseolin and legumin, and in 75 to 90 per cent. alcohol of zein, gliadin and hordein,' which were either neutral or acid when tested with a strip of sensitive, neutral litmus paper, capable of showing distinctly the presence of 0.25 cc. of centinormal hydrochloric acid in 10 cc. of water, when made neutral to litmus, were, in every case, still acid towards phenolphthalein. With the exception of ovalbumin, these preparations had been made by the methods usually employed and had come in contact with no acid except that contained in the seeds from which they were obtained. The ovalbumin preparations were made both by Hopkins's and by Hofmeister's methods, the acidity of all of them being the same.

To render gram portions of these several protein preparations neutral to litmus required in a few cases not any, in most cases from 0.1 cc. to 1.5 cc. of, decinormal alkali; while to make the same 1-gram portions neutral to phenolphthalein required the further addition of from 0.7 to 1 cc. of decinormal alkali, except for legumin, which required 2 cc.

This reaction with phenolphthalein is sharp and definite, like that with strong mineral acids, and is independent of dilution, the same result being obtained in a volume of 10 cc. or in one of

100 CC.

The question now arises, whether complete neutralization of the combined acid is indicated by phenolphthalein or by litmus.

Preparations of edestin, which are neutral or acid to litmus, when suspended in water and made neutral to phenolphthalein by adding potassium hydroxide are not in any perceptible degree dissolved, but yield to the solution potassium salts of simple acids, which may be obtained therefrom by evaporation. When thus freed from these acids, the edestin immediately begins to dissolve if more alkali is added.

Edestin made neutral to phenolphthalein and dissolved in sodium chloride solution reacts distinctly alkaline towards litmus. This alkaline reaction is caused by the edestin itself and not by

1 These protein bodies are described in Reports of the Connecticut Agricultural Experiment Station, 1890 to 1899; also Osborne: Am. Chem. J., 13, 14 and 15; This Journal, 16, 17, 18, 19, 20 and 21; also "Die Proteide," etc., Heidelberg, 1897.

organic salts of the alkali, since such preparations yield a very small amount of ash, less than 0.05 per cent., which is neutral to both litmus and phenolphthalein.

Crystalline preparations of excelsin, obtained from the Brazil nut, Bertholetia excelsa, are undissolved when suspended in water and made neutral to litmus, but are completely dissolved when enough alkali, either potassium or its equivalent of ammonium hydroxide, is added to render the solution neutral to phenolphthalein. With less alkali, solution is not complete, the amount of excelsin dissolved depending upon the quantity of alkali added. Likewise, preparations of legumin from the pea, horse bean, or vetch, when suspended in water, are completely dissolved only when enough potassium, or an equivalent of ammonium hydroxide is added to neutralize their acid reaction to phenolphthalein. It is highly improbable that excelsin and legumin can form soluble compounds with potassium which are neutral to phenolphthalein, and the fact that an exactly equivalent quantity of ammonium causes solution, is also evidence that this is not so, since a much larger proportion of ammonium hydroxide than of potassium hydroxide is required to dissolve edestin. Thus, 1 gram of a preparation of edestin, which was completely dissolved by 1 cc. of decinormal potassium hydroxide solution, was not entirely dissolved by 13 cc. of decinormal ammonium hydroxide. Furthermore, the proteins being very weak acids, it is scarcely possible that these form salts with potassium which are neutral to phenolphthalein. Certainly this is not the case with edestin, as the slightest excess of potassium hydroxide above that required to neutralize the combined acid at once turns phenolphthalein red.

Solutions of all the other protein bodies that I have examined, when similarly made neutral to phenolphthalein react decidedly alkaline with litmus.

From these facts it seems certain that the proteins are true bases, as I have previously pointed out,' and that they are not pseudoammonium bases, as Cohnheim and Krieger assume."

II. COMPOUNDS of edESTIN WITH ACIDS.

Since edestin, when neutral to phenolphthalein, is insoluble in 1 Osborne: This Journal, 21, 486; Report of Connecticut Agricultural Experiment Station for 1899; also This Journal, 22, 402.

2 Cohnheim and Krieger: Ztschr. Biol., n. f., 22, 95.