An Improved Method for the Determination of Lanthionine*

An Improved Method for the Determination of Lanthionine* By M. X. SULLIVAN and CHESTER F. MIJALt to the discovery of cystine by S Wollaston in 1810 in a urinary calculus, a great deal of work was done as to its nature and UBSEQUENT

(1)

occurrence. Baudrimont and Malaguti (2) found that cystine contained sulfur, which previous workers had missed. Then Morner (3) isolated cystine from the hydrochloric acid hydrolyzate of horn. Subsequently, cystine was found widespread in proteins by a host of investigators. Dependent on the method of purification of the particular protein, the procedure of isolation of cystine, and its determination, great variation was found in the cystine content of individual proteins. Hoffman (4), in 1925, reported that hair treated with hot sodium carbonate (1 to 4 per cent) for a short time lost about 24 per cent of its sulfur. The treated hair was identical in appearance with that which had not been treated. However, although the ,treated hair retained about i 5 per cent of its original sulfur, no cystine could be isolated from an acid hydrolyzate. About the same time Sullivan ( 5 ) , employing his highly specific test for cystine, found that grain-curd casein, never in contact with alkali, had a higher cystine content then Hammarsten casein because the latter had gone through a sodium carbonate treatment. Later, Sullivan and Hess (6), using the Sullivan cystine test, found that treating various proteins with dilute alkali lowered the cystine content, and they ( i )also found that treatment of insulin with pyridine lowered the cystine content of the hydrolyzed insulin. The pyridine treatment apparently did not affect the physiological unitage. All our work pointed to the conclusion that treatment with weak alkali made changes in the nature of the protein, especially as to the amount of cystine found in an acid hydrolyzate. In the meantime, Kiister and Irion (8) reported the isolation of a thioether from the acid hydrolyzate of wool heated with sodium sulfide. Horn, Jones, and Ringel (9) were unable to repeat this isolation, but did succeed in isolating a thioether by boiling wool in a 2 per cent

* Received September 18, 1958, from Georgetown University, Washington, D. C . Taken in part from a thesis submitted by Chester F . Mijal in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Georgetown University, Washinaton. D. C. t Present address: Army Quartermaster Research Center, Natick. Mass.

sodium carbonate so!ution, and hydrolyzing the treated wool with 20 per cent hydrochloric acid solution. On concentrating the hydrolyzate, dissolving the concentrate in absolute ethyl alcohol, and adding pyridine to the alcohol extract, they obtained a certain amount of a thioether which they named “lanthionine” and found to be structurally (COOH-CHNH2CH2S-CH2-CHNH2-COOH)-a symmetrical thioether. The structure was confirmed by du Vigneaud and Brown (10) by synthesis of meso-lanthionine from 1-cysteine and methyl d,Z-a-aminorfl-chloropropionate hydrochloride in strongly alkaline solution. Horn and Jones (1 1) then isolated lanthionine from human hair, chicken feathers, and lactalbumin, after treatment with sodium carbonate, and du Vigneaud and co-workers (12) found that amorphous insulin, similarly treated, yielded lanthionine. Others who have worked on the formation of lanthionine in a detailed, comprehensive way are Cuthbertson and Phillips (13) and Lindley and Phillips (14). Lanthionine resembles methionine in that it is a thioether. Baernstein (15) had shown that hydriodic acid containing KH2POz (potassium hypophosphite) converts methionine into the thiolactone of homocysteine, CHXSCH2CH2CHNH2COOH

+ HI 7

S

=

T

l

CHzCH?CHhHzC

=

0

Later, Hess and Sullivan (16) considered that one mole of lanthionine heated with hydriodic acid should yield 1 mole of cysteine, and developed a method for the quantitative determination of lanthionine. Lanthionine plus a 57 per cent solution of hydriodic acid, reinforced with potassium hypophosphite (KHzP02) to prevent liberation of iodine, was hydrolyzed a t 135-140’ for four hours under a steady stream of nitrogen. A quantitative splitting resulted, with rupture of the carbon-sulfur bond of lanthionine and with the formation of cysteine. The Sullivan (15) procedure for the estimation of cysteine was then applied. Lanthionine, as such, does not react in the Sullivan cysteine or cystine reaction; hence, if either of the latter compounds is present, it could be determined first, after hydrochloric acid hydrolysis, and then

324

SCIENTIFIC EDITION

June 1959

again, following hydrolysis with hydriodic acid, which yields only cysteine. The amount of lanthionine present would be measured by the increase in cysteine found after t h e hydriodic acid hydrolysis (Le., hydriodic acid hydrolysis minus hydrochloric acid hydrolysis). Multiplying the figure for the excess cysteine b y 1.72 gives the amount of lanthionine. T h e procedure of Hess a n d Sullivan for determining lanthionine is rather long, involves two separate hydrolyzates, a n d requires a special hydriodic acid. Accordingly, an investigation was made as t o the possibility of splitting the lanthionine b y cyanogen bromide, since t h e latter had been used by Cahours (1'7) t o split such thioethers as dimethyl sulfide (CHaSCH3) b y heating t h e thioether with solid cyanogen bromide in a sealed tube. Cahours considered that the reaction products were methyl thiocyanate (CH3SCN) and a bromide of trimethyl sulfine [(CH3)$Br]. Later, von Braun and Engelbertz (18) reported that t h e reaction is: (CH&S

+ BrCN = CHBSCN+ CH3Br

and that, as a n intermediary product, [(CH3)2CNSBr] may occur. I n the light of t h e work b y Cahours a n d especially b y von Braun a n d Engelbertz, Sullivan and Folk (19) studied the action of cyanogen bromide on lanthionine isolated from hair b y the procedure of Horn, Jones, a n d Ringel (9). Sullivan a n d Folk found t h a t lanthionine, heated with excess sodium cyanide a n d cyanogen bromide, would react in the Sullivan cysteine reaction as cysteine does. Lanthionine treated with sodium cyanide or cyanogen bromide (separately) does not give the Sullivan cysteine reaction. Also, when cysteine or cystine is similarly heated with sodium cyanide and cyanogen bromide, no evidence of cysteine could be obtained. T h e free amino acids are either destroyed b y the procedure or are changed t o some compound not reacting as cysteine does. T h e Sullivan a n d Folk (19) procedure was offered as a test for lanthionine. However, the procedure of alternately heating the lanthionine-sodium cyanide-cyanogen bromide mixture in a n open tube is somewhat tedious a n d requires care in manipulation. Accordingly, we sought a n easier method. Such a method was devised and is herein given.

EXPERIMENTAL Procedure -To 5 ml. of a 0.1 N hydrochloric acid solution containing 0.05-0.5 mg. of lanthionine in a 20 x 150 mm. test tube, add 1 ml. of 5 % sodium cyanide solution. Shake gently and add 1 ml. of

325

cyanogen bromide.' Stopper the test tube and heat for five minutes in a water bath held a t 70'. Cool in cold water. T o the cooled solution add 1 ml. of freshly prepared 0.5% aqueous solution of sodium 1,2-naphthoquinone-4-sulfonate. Shake the test tube for forty seconds and add 5 ml. of a n alkaline sodium sulfite solution (10% of sodium suEte in 0.5 N sodium hydroxide), stopper the tube tightly, and place it in the 70' water bath for fifteen minutes. Then cool in cold water and add 1 ml. of freshly prepared 2% sodium hydrosulfite (Na2S2Od) in 0.5 N sodium hydroxide. Read the density of the color in the Klett-Summerson photoelectric colorimeter with the aid of the green filter (filter 54). Preparation of a Standard Curve -The precision and reproducibility of the quantitative lanthionine procedure as outlined above were studied. Ten duplicate samples of lanthionine solution of each concentration shown in Table I were run. The standard deviation and percentage standard deviation were calculated. The standard curve is shown in Fig. 1. The reddish color produced fades quite rapidly, as may be seen in Fig. 2. Accordingly, after the addition of the sodium hydrosulfite, the interval of elapsed time before reading the absorbance in the Klett-Summerson colorimeter should be the same for all tubes. TABLE I. Av. mg.

0.05 0.1 0.2 0.4

Reading

31 60.3 121.2 240.6

Sum Diff.2

29 50 161 264

S. D.

1.78 2.36 4.31 5.41

S. D. mg.

f0.002

zt0.004 f0.006 f0.009

Determination of Lanthionine Produced in Wool -As a check on the method, a sample of white wool of unknown previous treatment (but possibly bleached) was employed. Thus 1 Gm. of the wool in question was boiled for thirty minutes with 200 ml. of a 2% solution of sodium carbonate. The treated wool was filtered on a sintered-glass funnel and washed with 200 ml. of distilled water. After being air-dried, the wool was hydrolyzed with 50 ml. of 20y0 hydrochloric acid solution until it no longer gave a biuret test. An equal volume of distilled water was added, and the solution (which was slightly colored) was treated with an iron-free Norit, boiled for two minutes, and filtered on a small Biichner funnel. The Norit was washed with 20 ml. of 0.1 N hydrochloric acid, and the washings were added to the main filtrate. The pH of the clear. colorless solution was adjusted to 3.5 with 5 N sodium hydroxide added dropwise with stirring, and the solution was transferred quantitatively to a 200-nil. volumetric flask and brought to volume by addition of 0.1 N hydrochloric acid. The quantitative lanthionine determination was made by taking separately ( a ) a reagent blank (5 ml. of 0.1 N hydrochloric acid); ( b ) a hydrolyzate blank (1ml. of the wool hydrolyzate plus 4 ml. of 0.1 N hydrochloric acid); (c) a standard control (2 ml. of standard lanthionine solution containing 0.2 mg. of lanthionine plus 3 ml. of 0.1 N hydrochloric acid); 1The cyanogen bromide is prepared by adding a 5% solution of sodium cyanide dropwise to a saturated, aqueous solution of bromine until a colorless solution results.

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JOURNAL OF THE

AMERICAN PHARMACEUTICAL ASSOCIATION v01. XLVIII. NO. 6

YC OF S l Y P L E

Fig. 1.-Standard

curve, avcrag- of 10 duplicate szmplcs.

tion of lanthionine, since it is performed more easily and obeys Beer's law over a wide range. I t is somewhat different from that of Sullivan and Folk who, by strong heating, split the carbon-sulfur bond with the formation of a free sulfhydryl group (-SH), as shown by a positive nitroprusside test and by the cysteine estimated by the Sullivan cysteine reaction performed a t room temperature. In the new reaction, no sulfhydryl (-SH) group is brought into play since the nitroprusside reaction is negative. Also, whereas Sullivan and Folk developed a color reaction a t room temperature, the new procedure gives no color a t room temperature on addition of the sodium cyanide, naphthoquinone derivative, and alkaline sodium sulfite solution. In short, the color only develops, with these reagents, on heating a t 70" The specificity of the new lanthionine reaction is good. Related thioethers, such as thiodiglycolic acid ( COOHCH2SCH2COOH), methionine (CH,SCHZ-CHXHNH~COOH), and cystathionine (COOHCHNHzCH2CH2SCH2CHN\lTH?COOH),treated with sodium cyanide and cyanogen bromide, gave no color. Free cysteine and free cystine are oxidized a t once by cyanogen bromide and no longer give a nitropiusside reaction with or without sodium cyanide and became negative in the regular Sullivan reactions for cysteine or cystine. Based on the work of von Braun and Engelbertz (18), it is possible that, in the lanthionine treatment, there is first folmed a complex such as: COOH-CHNHzCH2

CN

\ YO

Fig. 2.-Fading

of U Y R L

COOHCHNH2CH2

of color with time.

and ( d ) the hydrolyzate ( 1 ml. of the wool hydrolyzate plus 4 ml. of 0.1 N hydrochloric acid). To each of the four test tubes was added 1 ml. of 5% aqueous sodium cyanide solution, followed by 1 ml. of the cyanogen bromide solution. The tubes were stoppered, kept in the water bath a t 70" for five minutes, and then cooled t o 15O in cold running water. Successively, to each tube (with the exception of the hydrolyzate blank where water was substituted) was added 1 ml. of sodium 1,2-naphthoquinone-4-sulfonate solution. The test tube was stoppered and shaken for forty seconds. Then, 5 ml. of sodium sulfite solution was added t o each tube and, after being restoppered, the tubes were placed in the water bath, kept a t T O o for fifteen minutes, and cooled to 15'. In succession, one ml. of the sodium hydrosulfite (Na2S204) solution was added and, after shaking, the absorbance was read immediately. The following readings were obtained: ( a ) reagent hlank 21, ( b ) hydrolyzate blank 10, ( 6 ) standard control 182, and ( d ) hydrolyzate 97. The calculations are: (a)standard control minus the reagent blank equals 161, and ( b ; the hydrolyzate minus the sum of the hydrolyzate blank and the reagent blank equals 66. These results indicate the presence of 0.082 mg. of lanthionine per ml. of the wool hydrolyzate, or 1.64'3&of lanthionine in this sample of alkali-trtated wool.

DISCUSSION The new procedure presented herein is a better colorimetric method for the quantitative determind-

S

/'

/ \

Br

which split into COOH-CHNH2CH2Brand COOKCHNH2CH?SCN. The RSCN complex, on heating to TO" with sodium cyanide, the naphthoquinone derivative, sodium sulfite, and alkali, wotild be converted t o a disulfide as suggested by the findings of Kaufmann (20) in the case of thiocyanates of fatty acids. However, the full explanation of the lanthionine reaction has not been ascertained.

REFERENCES (1) Wollaston, W. H., Phil. Trans. R o y . Soc. London., 1810, 223; A n n . Cham., 76, 21(1810). (2) Baudrimont. A , . and Malaeuti. Comal. rend.. 5 . 394( 1837). (3) Morner, K. A H . , Z. physiol. Chem. Hoppe-Seyler's, 2 A . 5Q5118QQ) (4) Hoffman, W. P . , J . B i d . Chem., 65, 251(1925). ( 5 ) Sullivan, M. X . . U. S. Public Healfh Seru. Suppl. 7 8 , 1929. (6) Sullivan, M. X . , and Hess, W. C . , Public Health Serv. Suppl. 86,1930. (7) Sullivan. M. X., and Hess, W. C . , J . B i d . Chem., 130 745(1939). Kiister, W., and Irion. W., Z . physiol. Chem. HoppeSeyler's, 184,225(1929). (9) Horn, M . J., Jones, D. B., and Ringel, S. J., J . Biol. Chem., 138,141(1941). (10) du Vigneaud, V.. and Brown, G. B., ibid., 138, 151 ( 1941). (11) Horn, M . J.. and Jones, D. B . . ibid. 139 473(1941). (12) du Vigneaud, V . . Brown, G. B . , anh B o k n e , R . W . , ibid.. 141,707(1941). (13) Cuthbertson. W. R., and Pbillips, H . , Biochem. J . , 39,7(1945). (14) Lindley, H., and Phillips, H., ibid.. 39, 37(1945). (15) Baernstein, H . D.. J . B i d . Chem., 106, 451(1934). (16) Hess, W. C.. and Sullivan, M . X . , ibid., 146, 15(1942). (17) Cahours, A,, A n n . chim. el phys., 10, 29(1877). (18) Von Braun, J . , and Engelhertz, P., Ber., 56, 1573 f 1923). (19) Sullivan, M. X., and Folk, J. E., Arch. Biochem. Biophys., 35,305(1952). (20) Kaufmann, H. P . , Gindsberg, E . , Rottig, W . , and Salchow, R . , Ber., 70, 2519(1937). ~