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PAPER CHROMATOGRAPHIC IDENTIFICATION OF THE AMINO ACIDS OF GLUTATHIONE An Experiment in Biochemistry FABIAN LLONETTI Boston University, Ebston, Massachusetts
C a I t o n n ~ ~ o o ~ procedures n P ~ ~ c afi research tools for the separation and identification of heretofore difficultly distinguishable substances have advanced considerably in recent years. The facility and simplicity of many of these suggested their incorporation into modern student teaching.' Whereas such student experiments involve identification of synthetic mixtures, it is possible a t the expense of some additional effort to obtain more rewarding results with natural products and convey to the student simultaneously the satisfaction of duplicating in part the original researches. A technique involving the extraction of the tripeptide glutathione from baker's yeast and paper chromatographic identification of the constituent amino acids has been convincingly demonstrated in this laboratory by medical students working on a project basis for ten laboratory hours. The apparatus required is reasonably simple and the procedure straightforward. Essentially, the procedure is conveniently divided into three parts: A concentrated extract of glutathione is made by precipitation of the copper salt2 and decomposition with H,S; the extract is hydrolyzed by heating with dilute HC1; and chromatographic identification of the three amino acids in the mixture is effected. Several simplifying modifications have been made in the original method for the isolation of glutathione with consistentlv reliable results.
to allow the filtration to proceed overnight. The combined acid filtrates are adjusted to 0.5 N by first neutralizing to Congo red paper with 10 per cent NaOH and then adding the required amount of concentrated H2S04. (This is accomplished by rough titration uith the base, using a piece of Congo red paper swirled inside the flask to a color change from blue to red. The volume of concentrated H2SOI required to make the solution 0.5 N is found from the formula: z = 0.0135y, where x is the amount of acid to be added; and y is the volume of the "near" neutral filtrate.) A suspension of freshly prepared cuprous oxide (about 1 gm./lO ml. of H20)is stirred carefully in 2-ml. portions into the extract a t 50° on a water bath, care being taken to avoid an excess. The color of the oxide fades as it is added to the solution as in a volumetric titration; hence, an excess may he regarded as the first permanent pink coloration acquired by the solution. (Cuprous oxide is freshly prepared for this purpose by the slow addition of glucose to Fehling's solution followed with washing of the precipitate by decantation.) Gray cuprous glutathionate flocculates and settles out (15 to 20 minutes). A small excess of the oxide has not been found to cause re-solution of the precipitate and often stratifies above it. The amorphous precipitate is collected by centrifugation, washed with 0.5 N HzSOl to remove excess CuzO, and washed with distilled water until free from sulfate (about five washings EXPERIMENTAL PROCEDURE are necessary). The precipitate is suspended in five Twenty ml. of concentrated HzSOnis slowly added times its bulk of distilled water (about 5 ml.) and deto 100 ml. of 95 per cent ethanol with cooling under composed with HPS which has been washed xvith a rerunning tap water. To the cooled solution 80 ml. of versed wash bottle. The precipitated CuzSis removed ethyl ether is added, and this solution is ~ o u r e dover by centrifugation or filtration, and the supernate con1000 gm. (or 2 lb.) of baker's yeast in a 2-liter beaker. taining the glutathione freed from residual H2S by After a minute of stirring the cold solution is filtered warming the tube, or better by the passage of a slow through coarse filter paper on a battery of three grav- stream of nitrogen for about five minutes. In order to identify the amino acids composing the filters arranged in the refrigerator with the funnel stems passing through the grated openings in the shelves into tripeptide, the glutathione in solution is first hydrolyzed Erlenmeyer flasks sitting on the shelf below. (As by refluxing gently for about an hour with 10 Per cent (1 ml. Per 5 ml. filtrate). This is simply aceomalternatives, the solution may be centrifuged, or filtered plished by using a test tube with a 10% piece (18in.) of with aspiration-the latter being the most tedious and difficult.) In this laboratory it was found convenient glass tubing as an air condenser (under the hood). Considerable HCI may be lost and the hydrolysis re' GAGE,T. C., C. D. DOEGLAS, AND S. H, WENDBE, J. GEM. tarded if the solution is allowed to boil briskly. HCI EDUC., 27, 159 (1950). has been found to be the most effective acid for hyHAWK,P. B., B. L. O s m , AND W. H. S-MER~ON, "Practical Physiological Chemistry," 12th ed., The Blakikiston Company, drolysis because the resultant amino acids are hest Philadelphia, 1948, pp. 283-4. identified chromatographically as the hydrochlorides. 152
MARCH, 1951
Excellent details and clear drawings have been given for student participation in paper-partition chromatography of simple mixtures of amino acids by Gage, Douglas, and W e ~ ~ d e rThe . ~ essence of their procedure is the same as that used in the present report, and either may be used. The method used is that of Consden, Gordon, and Martiq4 as modified by Williams and . . Kirby." The correct dilution of the hydrolyzate for chromatography is found by placing on filter paper a few spots of different dilutions (1 : 10, 1 : 1000, and 1 : 1,000,000), quick drying in the oven a t SO", and spraying with ninhydrin, followed by another quick drying. From these, the spots of maximum intensity and hence the optimum dilution for the chromatograms is ohtained. A penciled line is ruled on a sheet of filter paper (Whatman No. 1, 8'/z X 11 in.) one inch from the edge and parallel to the long dimension. With fine-tipped medicine droppers solutions are spotted (about 1 cm. diameter) every 2 in. along the line. Solutions of 1 per cent glycine, glutamic acid, and an equi-volume mixture of the two are used as knowns (very few ml. of these are needed), as well as the solution of the glutathione hydrolyzate in the predetermined dilution. The paper is then rolled into a cylinder and stapled a t each end. This is allowed to stand in a glass cylinder (9 in. tall and approximately 6 in. in diameter) the bottom of which contains about 50 ml. of phenol saturated with water. I n order to insure saturation of the atmosphere, a small beaker containing water saturated with phenol is placed inside the paper cylinder. A large watch glass is used to cover the apparatus. The liquid front is allowed to climb for about 8 to 10 hours or sufficient time for a climb of approximately 8 in. The cylinder of paper is then opened and carefully dried at 80" for just sufficient time to effect drying without charring (about 10 minutes). Best results are achieved when this step is carefully supervised and the paper removed from the oven when it is just dry. A 0.1 per cent solution of ninhydrin (triketohydrindene sulfate in nbutyl. alcohol) is then sprayed from an atomizer over the surface of the paper with care t o saturate the paper evenly hut to avoid excesses of liquid which could run on the surface and cause errors in interpreting the re-
= Op cit. CONSDEN, R., A. H. GORDON, AND A. J. P. MARTIN, Biochem. J., 38, 224-32 (1944). ' \$'ILLIAMS,R. J., AND H. KIRBY,Science, 107, 481 (1948).
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sultant spots. The moist paper is given a final drying a t 80' for an additional 10 minutes or until the colored spots appear on the paper. The Rf values for the spots are found by measuring the distance from the starting line to the points of maximum density for the spots, and finding their fractions of the total distance which the solvent front has climbed, i. e.: Rf (rateof flow)
=
Cm. traveled by the amino acid Cm. traveled by the solvent
The us! of two known solutions as standards, which are run simultaneously with the unknown solution, gives a convenient identification without taking the precautions usually necessary to duplicate the conditions required to get Rf values which agree with the literature. The spots derived from the unknown are compared with those of the known solutions (assuming that no other a-amino acids are present in the hydrolyzate). It has been possible t o achieve consistently satisfactory results with values of the usual order and correct magnitude for the three amino acids. Amino acid Glyoine Glutamie acid Cystine
The observed values are the average of three determinations which agreed within 4 per cent of one another, and for which a single student conducted the isolation and hydrolysis of the glutathione in two periods of four hours each. (A value for cystine instead of cysteine is presented on the assumption that the former is produced under the conditions of the experiment, as is frequently observed with hydrolyzates of peptides and polypeptides.) Of eleven groups which have conducted the experiment, eight have been able to make positive identification of the amino acids present in the glutathione hydrolyzate; whereas one group ohtained faint spots which did not correlate well with their standards and the remaining two groups were able to obtain chromatograms only with the synthetic mixture. Sources of trouble or failure were excessive drying of the chromatograms in the oven, the failure to use freshly prepared CulO in the isolation step, and excessive handling of the chromatograms with resultant spots derived from impurities. However, it has been demonstrated that little beyond ordinary care will lead to fruitful and s a b isfying results.
