ISOLATION OF A NEW SULFUR-CONTAINING AMINO ACID (LANTHIONINE) FROM SODIUM CARBONATE-TREATED WOOL

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1 ISOLATION OF A NEW SULFUR-CONTAINING AMINO ACID (LANTHIONINE) FROM SODIUM CARBONATE-TREATED WOOL BY MILLARD J. HORN, D. BREESE JONES, AND S. J. RINGEL (Prom the Protein and Nutrition Research Division, Bureau of Agricultural Chemistry and Engineering, United States Department of Agriculture, Washington) (Received for publication, November 7, 1940) In a recent publication (1) the authors reported the isolation of a crystalline organic selenium compound from plant material grown on seleniferous soil. This compound agreed in its properties and composition with an isomorphous mixture of a sulfur and selenium amino acid having the formula, HOOC-CH(NHz)-CH2--Se-CH2-CH-CH(NH,)-COOH (S) -4s far as we are aware, the only instance recorded of the isolation of a compound of this type is that of the thio ether amino acid which Kiister and Irion (2) claimed to have obtained from the products of reaction of sodium sulfide solution on wool at room temperature, and to which they ascribed the formula, HOOC-CH(NHz)-CHZ-CHyS-CH,CH(NH,)-COOH Subsequent attempts, however, on their part to isolate the compound were unsuccessful. As the quantity of our selenium-sulfur compound was small, and the supply of the source material was exhausted and could not be replenished for several weeks, it was decided that in the meantime it would be worth while to repeat the work of Kiister and Irion with the hope of isolating the thio ether from wool. Familiarity gained with its properties should be of value later when our work on the analogous selenium-sulfur compound is resumed. Several attempts to isolate the thio ether by carefully following 141

2 142 New Amino Acid from Wool Ktister and Irion s procedure proved unsuccessful. Some departures from their technique were then followed in which was included the use of pyridine to precipitate the free amino acids from the acid hydrolysates. Five attempts were made with this procedure with 100 gm. of wool each time. In every case active cystine in the form of hexagonal plates was isolated, and also some crystals which were identified as inactive cystine. In one instance only was a small quantity of a crystalline substance isolated which showed the properties of a thio ether. Tests for disulfide and for sulfhydryl groups in this substance were negative. The crystals were similar in appearance to those of the symmetrical thio ether amino acid which is herein described. The quantity obtained, however, was too small to permit closer examination. The fact that Kuster and Irion were unsuccessful in repeating the isolation of their thio ether suggests that its formation may be limited to a very narrow range of conditions, which, if not rigidly controlled, would yield negative results. Slight changes in ph, temperature, duration of the reaction, and other factors may have a pronounced effect upon the nature of the endproducts. Hoffman (3) found that when hair was heated with 1 per cent Na2C03 solution for a short time, it lost about 24 per cent of its sulfur content. The hair thus treated was identical in appearance to that which had not been treated, and although it retained about three-fourths of its original sulfur content, no cystine could be isolated from it. Consideration of Hoffman s results raised the question as to the type of linkage of the sulfur in the alkalitreated wool and suggested that the disulfide bonds may have been split in some manner such as occurs when wool is treated with sodium sulfide with the possible formation of thio ether amino acids. Evidence has now been obtained showing that such is the case. When wool was boiled for a short time with dilute Na&03 solution, and then hydrolyzed with HCl, there was isolated from the hydrolysate a crystalline thio ether amino acid. This is not, however, the thio ether that Kiister and Irion claimed to have isolated, but the symmetrical compound, /%amino+carboxyethyl sulfide (I). Two other compounds having the properties

3 Horn, Jones, and Ringel of thio ether amino acids were also isolated, which were found to differ from (I) with respect to crystalline form and solubility. The fact that both had about the same nitrogen content as (I) suggests that the three may be optical isomers. They will be the subject of further study and the results will be published later. The isolation of these compounds from wool according to the same procedure has been repeated seventeen times. EXPERIMENTAL 100 gm. of wool, freed mechanically from some extraneous material, were washed with cold water and then boiled for 1 hour with 1.5 liters of 2 per cent Na&Os solution. The NazCOstreated wool was then separated on cheese-cloth and washed with water. It is of interest to note that when the alkaline filtrate from the treated wool was acidified a strong odor of H&3 was observed. The treated wool was hydrolyzed by boiling with 200 cc. of concentrated HCl for 15 hours. After decolorization with norit the hydrolysate was then concentrated to a thick sirup in VUCUO. In order to remove most of the free HCI the residue was taken up in about 100 cc. of 95 per cent alcohol and the solution was again concentrated as before. The thick, sirupy residue was dissolved in 600 cc. of absolute alcohol. A small quantity of NaCl which separated was removed by filtration, and the volume of the filtrate was made up to 1 liter with absolute alcohol. Pyridine was then cautiously added in small quantities with shaking until an additional drop caused no further precipitation. After the mixture had stood for about an hour the precipitate was separated by centrifugation and washed with absolute alcohol. It was then heated to boiling with 100 cc. of water until the greater part had dissolved. After cooling, the mixture was allowed to stand in a refrigerator overnight. The material which had separated was removed by filtration and redissolved by heating with 100 cc. of water to which 5 cc. of concentrated ammonia had been added. After standing for about half an hour the small quantity of a reddish, flocculent substance which had settled was removed by filtration. The clear filtrate was then concentrated by distillation in vucuo. As the ammonia was being removed, the thioamino acid separated. It was removed by filtration and recrystallized from dilute am-

4 144 New Amino Acid from Wool monk by allowing the solution to stand exposed to the air until most of the ammonia was expelled. It separated in the form of thin plates (Fig. 1). About 0.5 gm. of this product was isolated. The other two compounds previously referred to, which are much more soluble, were obtained from the filtrate of (I). The joint yields obtained of these two compounds were about 0.5 gm. Compound (I) had the following percentage composition. CsHInSNtOa. Calculated. C 34.59, H 5.81, N 13.46, S Found , 5.99, 13.48, Tests for sulfhydryl and disulfide groups were negative. A strong positive test was obtained with ninhydrin reagent, indi- FIG. 1. Lanthionine from wool. (Photomicrograph by Mr. G. L. Keenan.) eating the presence of amino groups in the CY position to the carboxyl. All the nitrogen was found to be in the amino form. Titration with NaOH showed the presence of two carboxyl groups, and corresponded to an equivalent weight of Iodine Absorption Number-One of the characteristic properties of thio ethers is the capacity to absorb 2 atoms of halogen under certain conditions. It was, therefore, considered of interest to determine the iodine absorption value of the new amino acid. This determination was carried out according to the Hanus method (4). For comparison, a similar determination was made on methionine. The results are given in Table I. The new com-

5 Horn, Jones, and Ringel 145 pound was found to absorb the theoretical 2 atoms of iodine in 10 minutes, while mcthionine, on the other hand, required 40 minutes. The absorption values for both compounds remained constant thereafter for several hours. Assuming that the constant absorption value found for the new compound represented 2 atoms of iodine, a calculated molecular weight of 209 was derived. This is in fairly close agreement with that corresponding to the empirical formula. Optical Rotation-A preliminary test on 5 mg. of the compound showed that it was optically inactive. A determination was then Time min hrs TABLE I Iodine Absorption Values for Thio Ether Amino Acid (I), and for Methionine Thio ether (I) ( pm.) Iodine absorbed - ml _- - per cent 100 loo Metbionine (020824gm.) 1 [odine absorbed - I- mg. per cent made at 20 on mg. of the substance dissolved in 10 cc. of normal HCl. No optical rotation was observed. Dibenzoyl Derivative-O.1 gm. of the compound was dissolved in 5 cc. of N NaOH in 10 cc. of water. 1 cc. of benzoyl chloride was added in small portions with vigorous shaking. The mixture was maintained at a slightly alkaline reaction by addition of NaOH. After the odor of benzoyl chloride had disappeared, 20 cc. of water were added and the solution was acidified with HCl. The voluminous precipitate was filtered off and extracted with benzene in order to remove benzoic acid. The residue was then recrystallized from 50 per cent alcohol. The dibenzoyl derivative l loo

6 146 New Amino Acid from Wool separated in the form of long prisms, melting at (uncorrected). Analysis- c2oh2006n2s. Calculated. N mg. substance. Found ( I I 6.60 Properties of the Compound-The compound is difficultly soluble in water and insoluble in alcohol, ether, chloroform, and acetone. It is readily soluble in dilute mineral acids and alkalies. It is optically inactive. When heated, it softens at 270, and decomposes at about 304. It is stable toward alkalies. When heated with 40 per cent NaOH in the presence of lead, there is no lead darkening. When dissolved in dilute ammonia and the solution allowed to evaporate slowly at room temperature, the thio ether separates in the form of six-sided plates having a triangle-like appearance (Fig. 1). We are indebted to Mr. G. L. Keenan for the following characterization of the crystals. When examined in ordinary light, it consists of six-sided plates, more or less wedge-shaped in habit. In parallel polarized light (crossed nicols) t,he plates extinguish sharply and no interference figures were observed in convergent polarized light (crossed nicols). On account of the absence of interference figures, a variation of the statistical method was used in determining the refractive indices, selecting plates showing maximum double refraction in determining the lowest and highest indices of refraction. The refractive indices as determined by the Becke line method are na = 1.605, n, = 1.647, both f The composition, chemical behavior, and properties of the new amino acid pointed very definitely to a thio ether amino acid having the structure, HOOC-CH-CHt-S-CH2--CH-COOH I I NHe NH2 I. &Amino+carboxyethyl sulfide That the above represents the correct structure has now been definitely established by synthesis by du Vigneaud and Brown (5), an account of which is described in the following article. A microscopical examination by Mr. Keenan of the synthetic compound, kindly furnished us by Dr. du Vigneaud, showed

7 Horn, Jones, and Ringel 147 that the crystal habit and the optical-crystallographic data were identical with those of the compound isolated from wool. Because this amino acid has been first isolated from wool, and contains sulfur, it has been named Zanthionine. DISCUSSION Until recently the number of sulfur-containing amino acids which have been isolated from naturally occurring material included only the two well known hydrolytic products of proteins; namely, cystine and methionine. Recently, the isolation of two other amino acids containing sulfur has been reported, (a) the thio ether of Ktister and Irion (2), COOH-CH(NHz)- CH2--CH2-S-CH2-CH(NH2)-COOH, which was obtained by the action of sodium sulfide on wool, and (b) djenkolic acid, COOH-CH(NH2)-CHZ-S-CHs-S-CHZ-CH(NHz)- COOH, isolated from the djenkol bean by van Veen and Hyman (6) in 1933, and synthesized by du Vigneaud and Patterson (7) in Goddard and Michaelis (8) have shown that keratins, such as wool and feathers, are dissolved in a few hours at room temperature at alkaline reaction with a solution of Na& KCN, or thioglycolic acid. Products prepared from the solutions behave as true proteins, and not as products of hydrolysis. They are soluble in dilute acids and alkalies, and are digestible by pepsin or trypsin. The effects are produced chiefly by the splitting of disulfide bonds. The action of dilute Na2C03 solution on wool, however, is quite different from that produced by the reagents used by Goddard and Michaelis, although both involve a splitting of disulfide bonds. When wool is heated for 1 hour with dilute Na2C03, very little is apparently dissolved and the wool retains its fibrous appearance. Nevertheless, marked changes occur. The Na&03- treated wool is no longer soluble in sodium sulfide, and unlike the product obtained by treating wool with sodium sulfide, it is not digestible by pepsin or trypsin. Although containing about threefourths of the sulfur of the untreated wool, it yields no cystine when hydrolyzed with HCl, whereas both optically active and inactive cystines were isolated from the sodium sulfide product. Evidently preliminary treatment of a protein with reagents

8 148 New Amino Acid from Wool such as NazS and dilute Na&03, under favorable conditions, can bring about structural changes in the molecule so that it will give on subsequent hydrolysis products that are very different from those yielded by direct hydrolysis of the original protein. This consideration suggests the interesting possibility that other mild treatments, chemical or physical, might determine to some extent the kind of amino acids a protein will yield on hydrolysis. The new thio ether here described is of interest from a number of different angles. It is the first instance of the isolation of a symmetrical thio ether amino acid obtained from the hydrolytic products of a protein. The question whether other proteins than wool, or the fibrous keratins, will yield thio ethers under similar treatment remains an interesting possibility. In this connection it is suggestive that van Veen and Hyman isolated djenkolic acid by prolonged treatment of the beans with barium hydroxide at 30. The mechanism of the reaction between wool and dilute sodium carbonate, whereby the disulfide bonds of cystine are split with the formation of a grouping which on hydrolysis with acid yields thio ether amino acids, opens up a field of investigation which may be of great value in elucidating problems relating to protein structure. The structural relationship of the thio ether amino acid to cystine and methionine suggests interesting questions in connection with sulfur metabolism. The thio ether may also be of particular interest to the organic chemist as a basic material for use in the synthesis of new organic compounds. Feeding experiments are in progress in order to find out whether the thio ether amino acid which we have isolated from wool can replace cystine or methionine in the diet with respect to their growth-promoting properties. A study of hair and feathers is in progress in order to see whether these keratins will also yield thio ethers. We wish to express our thanks to Mr. G. L. Keenan of the Microanalytical Division of the Food and Drug Administration, Federal Security Agency, for kindly supplying us with the opticalcrystallographic data recorded in this paper, and for the photomicrograph of the crystals of the thio ether amino acid.

9 Horn, Jones, and Ringel 149 SUMMARY A new thio ether diamino acid, HOOC-CH(NHz)-CHX-S- CH2-CH(NHJ-COOH, P-amino-p-carboxyethyl sulfide, has been isolated by acid hydrolysis of wool that had been boiled for 1 hour with 2 per cent sodium carbonate solution. It crystallizes in plates, which are difficultly soluble in water and readily soluble in dilute acids and alkalies. It is optically inactive, and decomposes at about 304. Because it was first isolated from wool, and contains sulfur, the name Zanthionine has been given to this amino acid. BIBLIOGRAPHY 1. Horn, M. J., and Jones, D. B., J. Am. Chem. Sot., 62, 234 (1940). 2. Ktister, W., and Irion, W., 2. physiol. Chem., 184, 225 (1929). 3. Hoffman, W. F., J. Biol. Chem., 66, 251 (1925). 4. Official and tentative methods of analysis of the Association of Official Agricultural Chemists, 4th edition, 410 (1935). 5. du Vigneaud, V., and Brown, G. B., J. Biol. Chem., 138, 151 (1941). 6. van Veen, A. G., and Hyman, A. J., Geneesk. Tijdschr. Nederl.-IndiE, 73, 991 (1933). 7. du Vigneaud, V., and Patterson, W. I., J. Biol. Chem., 114, 533 (1936). 8. Goddard, D. It., and Michaelis, L., J. Biol. Chem., 106, 605 (1934).

10 ISOLATION OF A NEW SULFUR-CONTAINING AMINO ACID (LANTHIONINE) FROM SODIUM CARBONATE-TREATED WOOL Millard J. Horn, D. Breese Jones and S. J. Ringel J. Biol. Chem. 1941, 138: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1