A NEW DECOMPOSITION PRODUCT OF KERATIN WHICH GIVES MILLON’S REACTION. PRELIMINARY NOTE. BY ROSS AIKEN GORTNER. (From the Biochemical Laboratory of the Station for Experiment& Evolution, the Carnegie Institution of Washington.) Downloaded from www.jbc.org by guest, on September 21, 2009 (Received for publication, April 14, 1911.) The red coloration produced by the interaction of an aromatic phenol and Millon’s reagent (mercuricnitrate indilutenitrous acid) is one of the most characteristic of the color reactions of the pro- teins. Among the nineteen known compounds which form the protein molecule only one, p-oxy-a-amino phenyl propionic acid (tyros’n) is of such a nature as to give a coloration with Millon’s reagent and therefore a positive reaction is taken to prove the presence of tyrosin. In a recent article’ I have shown that the coloration of the in- teguments of the meal worm (Tenebris molitor) is due to the presence of a tyrosinase, acting upon a ch. omogen. In many respects this chromogen resembles tyrosin, in that it is not precipitated by phosphotungstic acid or by basic lead acetate and in that it gives a strong Millon’s reaction. It differs from tyrosin, however, by being extremely soluble so that when evaporation was carried al- most to dryness and a few drops of water were added no tyrosin- like crystals could be obtained, neither did anyotherof the tyrosin tests, excepting the coloration with tyrosinase, produce positive results. While this was a solitary instance no special attention was paid to it, the supposition being that the chromogen was present in exceedingly small amounts and was probably tyrosin. 1 This Journal, vii, p. 365, 1910. 355 356 A New Decomposition Product of Keratin In a later study of the pigment of black wool’ a melanin was isolated by the action of 0.2 per cent sodium hydroxide which was soluble in alkalies, dilute mineral acids and strong acetic and formic acids. Further study of this body has shown that it is probably a melano-protein, i.e., an acid albumin in which a portion of the pro- tein molecule is so modi$ed as to possess the properties usually as- cribed to melanins; that is, the pigmented portion of the molecule is readily soluble in dilute acid while combined in the protein molecule, but, when hydrolyzed by strong mineral acid or acted on by strong alkali, a pigment is obtained which is insoluble in mineral acids. Downloaded from www.jbc.org by guest, on September 21, 2009 In my earlier paper, I define melanin as “those dark pigments which occur normally or pathologically in the animal body, skin, hair or feathers,” and I still adhere to this nomenclature and call the “melano-protein” a melanin, since it is doubtless in this form that the pigment occurs in the wool.2 Approximately 20 grams of this melanin were hydrolyzed by boiling for 30 minutes with fuming hydrochloric acid. The solution was evaporated to dryness on a water-bath, distilled water added and the black melano-humin filtered off. The remainder of the hydrochloric acid was then precipitated by addition of silver sul- phate and the sulphuric acid quantitatively removed by baryta. The clear filtrate was concentrated almost to dryness and allowed to crystallize. Then a small amount of cold water was added and the solution filtered from the crj stals. Thesewere well washedwith cold water and dried in vacua over concentrated sulphuric acid. The dry crystals weighed about 0.3 gram, were of the character- istic tyrosin shape and gave all of the usual tyrosin tests (Millon’s, Pyria’s, DenigW and Mijrner’s and the coloration with tyrosinase). To my surprise, however, the mother liquor still gave Millon’s re- action with undiminished intensity. Thinking that perhaps hydroly- sis was incomplete and tyrosin polypeptides might be present, the solution was diluted to 150 cc. and sulphuric acid added to 25 1 Gortner: This Journal, viii, p. 341, 1910. 2 Although I havetested many varieties of keratin, including four samples of human hair, horn, black and white feathers, albino hair and feathers, negro hair, horse hair, black and white wool, etc., “melano-proteins” have been obtained only from black wool, red and brown horse hair and auburn human hair. The detailed report on these pigments, alkali albumins, etc., will shortly be ready for publication. Ross Aiken Gortner 357 per cent by weight, and the mixture boiled for 22 hours. The acid was then neutralized with baryta and tyrosin, determined in the usual manner. No tyrosin was present, but the Millon’s reaction was still very intense. A difference was, however, observed in the reaction, for if a large excess of the reagent were added or if the heating had been too vigorous the coloration did not appear or else was rapidly decolorized. The solution containing this material was evaporated to dryness on a water bath and insufficient water was added to dissolve the residue. On filtering the most soluble portion was found to contain Downloaded from www.jbc.org by guest, on September 21, 2009 the Millon reacting material showing beyond doubt that the substance could not be tyrosin. Abandoning hope of obtaining the pure compound from this small quantity of material I hydrolyzed 300 grams of air-dried black wool with two liters of 25 per cent sulphuric acid, boiling the mixture for 24 hours. After separating tyrosin (6.1 grams) in the usual manner, I found the Millon’s reaction of apparently un- diminished intensity and, although I was able to concentrate the reacting material in the most soluble fraction (after 20 grams of leucin (?) had been separated which gave no coloration with the reagent and a third fraction was removed which only gave a faint color). I was, however, unable to obtain the substance in any semblance of purity by this method. It is very evident, however, that some aromatic phenolic body is present in this keratinmolecule which is not identical with any of the known products of protein hydrolysis. I expect to take up the study anew in a short time and with the aid of a Geryk vacuum pump it may be possible to separate this component.