A STUDY OF GLUTATHIONE.

I. ITS PREPARATION IN CRYSTALLINE FORM AND ITS IDENTIFICATION.

BY EDWARD C. KENDALL, BERNARD F. MCKENZIE, AND HAROLD L. MASON.

(From the Section on Biochemistry, The Mayo Foundation, Rochester, Minnesota.)

(Received for publication, August 30, 1929.)

Knowledge of the chemical nature of those agents which modify and control physiologic processes is slowly gathered by extended investigations over years of time and in many laboratories. This has been true of epinephrine, thyroxine, insulin, and glutathione. The essential contributions in each case furnished definite chemical evidence concerning the respective compounds; additions and modifications have then been made. This paper is an addition to our knowledge of glutathione; it in no way alters the original fundamental work of Hopkins.

Theoretic Considerations.

An investigation concerning the chemical and physiologic properties of glutathione was begun in our laboratory in 1924. At that time, several gm. of glutathione were prepared according to the method of Hopkins (5). In order to continue this investi- gation, it became necessary to isolate more of the material and the first steps toward this end were made in the early part of 1928. After the first description of a method for the separation of gluta- thione by Hopkins (5), papers were published by Hunter and Eagles (6) and by Johnson and Voegtlin (7). Hunter and Eagles slightly modified the method of Hopkins, but the yield of gluta- thione was not quite so high and the final product contained a higher percentage of nitrogen and less sulfur than glutamyl cysteine. Johnson and Voegtlin separated glutathione in much lower yield,

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658 Study of Glutathione. I

TABLE I.

Yield Of Glutathione.* -

Lot NO. Neight.

:1uta- :1uta-

Date. Lot hione,

NO. kight. Date. not hione,

;;;Tl- ‘F,t,-

1 .

1028 kg. 13.29 w. om.

1 Aug. 22 25 45 Jan. 31 0.0 2 “ 28 26 45 Feb. 5 26.4 3 Sept. 4 27 45 CL 12 42.0 4 “ 18 28 45 “ 19 15.6 5 “ 25 29 45 “ 26 15.6 6 act. 2 30 45 Mar. 5 15.5 7 I‘ 9 31 45 “ 13 16.5 8 “ 16 32 45 [‘ 20 16.8 9 “ 23 33 45 “ 26 24.9

10 “ 30 34 45 Apr. 2 14.1 11 Nov. 6 35 45 “ 9 19.6 12 “ 13 36 45 “ 16 21.5 13 I‘ 15 37 45 “ 23 14 “ 20 38 45 ‘( 30

24.7

15 “ 27 39 45 May 7 22.0 6.6 16 Dec. 4 40 45 cl 14 5.1 17 “ 11 41 45 ‘l 22 17.0 7.1

18 LL 18 42 45 ‘I 29 14.9 19 “ 26 43 45 June 4 11.1

1929 44 45 “ 11 12.8 20 Jan. 2 45 45 I( 18

90.0 5.4 21 ‘I 9 46 45 “ 25 10.5 22 “ 15 47 45 July 2 5.0

23 ” 23 24 “ 29

- * The loss of glutathione in Lot 3 was due to the addition of large amounts

of uranium acetate. Lot 9 was heated at 100” in a steam kettle with a large amount of barium acetate; glutathione could not be separated after this treatment. Glutathione was lost in Lots 18 and 19 through adsorption on aluminum hydroxide cream. Lots 21, 22, 23, and 24 were heated by passing through a block tin pipe. No glutathione was isolated from Lot 25 because insufficient toluene was added.

kg.

22 22 22 45 45 45 45 45 45 45 45 45 45 45 45 45 45

45 45

45 45 45 45 45

Q%.

2.0 1.0 0.0 8.0

11.3 11.0 6.1

12.5 0.0

23.2 7.8 3.2

11.9 13.6 37.0 16.3 19.3

0.0 0.0

2.5

12.2

k 14.6

but their analysis agreed better with the theoretic values for glu- tamyl cysteine. It was decided, therefore, to follow the method of Johnson and Voegtlin, to use a large centrifuge recommended by them. and to treat 45 kilo lots of bakers’ yeast at a time.

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In addition to the separation of glutathione, the total amount of the thiol compound present in the yeast and in each of the solutions obtained during the isolation was determined. The quantitative study of each step of the method of isolation with each lot of yeast was of great help. The results are summarized in Table I, which is made clearer by the following paragraphs.

Temperature and Duration of the Initial Heating of the Yeast Suspension.-Lots of yeast, from Lots 1 to 24 inclusive, were heated. 45 kilo lots of yeast were suspended in 70 liters of tap water and were heated for various lengths of time. The results showed that prolonged heating at or near the boiling point carried a large amount of material into solution, and although the ap- parent thiol content was high, it was invariably lost by adsorption during the precipitation of the impurities. This was particularly true of Lot 3 in which all glutathione was removed from solution before the final precipitation with absolute alcohol.

In order to reduce the time of heating to a minimum, the suspen- sion of 45 kilos of yeast in 130 liters of water was rapidly passed through a coil of block tin pipe, 10 mm. in diameter and 9 meters long. The coil was placed in a container holding 30 liters of water which was kept boiling with a steam coil. The suspension of yeast cells was poured from the end of the coil directly on ice which was placed in 75 liter crocks. The temperature of the solution as it left the coil was about 80” and the time required for the solution to pass from the inlet to the outlet of the coil was 25 seconds. By this method of heating, the temperature of the solution was raised from 20” to 80” and back to 15” within less than 60 seconds. The thiol content of the solution was the same as that of suspensions which were heated longer. The solutions were much more easily treated for the separation of glutathione.

The work of Raymond (9) on the coenzyme of yeast showed that toluene liberated the coenzyme from yeast cells. If toluene at room temperature could liberate glutathione from the yeast cell, then the amount of impurities carried into solution would be reduced to the minimum, Toluene was tried and found to liberate all the glutathione from the yeast cell. Titration of the thiol group, either with iodine or with potassium ferricyanide, indicated the presence of even more glutathione than that in similar solutions which were heated. The action of toluene will

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be more fully described in another report, but it is nom evident that in the isolation of crystalline glutathione from yeast, the use of a hydrocarbon to liberate the thiol compound is one of the most important modifications of Hopkins’ method. Lots 25 and 26 were treated with toluene. All lots from Lots 27 to 47 inclusive were treated with benzene, which was shown to have the same effect as toluene.

Volume of the Suspension of Yeast.-Hopkins originally recom- mended a suspension of 45 kilos of yeast in 10 liters of water. This was repeated three times. Johnson and Voegtlin used 45 kilos of yeast in 100 liters of water and reextracted with 50 liters of water. In Lots 4 to 10, 45 kilos of yeast were suspended in 70 liters of water, but after it was shown that the glutathione lost in the extracted cells was inversely proportional to the total volume, 45 kilos of yeast were suspended in 210 liters of water; the extracted cells were not reextracted; not more than 15 per cent of the total glutathione was lost in the extracted cells.

Precipitation of Glutathione as the Lead Salt.-The first separa- tion of glutathione by all investigators has been precipitation as the lead salt. Five factors have been varied: (1) the volume of the solution, (2) the pH of the solution, (3) the amount of lead salt added, (4) the time which the solution stands before separation of the lead precipitate from the solution, and (5) the amount of extractives from the yeast cell.

The lead salt is so insoluble that the volume of the solution is immaterial. Lots of yeast from Lots 10 to 47 were extracted with a total volume of 210 liters to which the lead acetate was added.

If the pH of the solution is increased to 9.5 or 10, a water-white filtrate may be obtained and all reducing substances are precipi- tated. If the suspension of yeast is heated to liberate the gluta- thione, a heavy precipitate is produced by lead acetate. A much smaller precipitate is formed when lead acetate is added to the extract of yeast made with cold water and benzene; the pH of such a solution is about 5.5. At this pH, about 85 to 90 per cent of the glutathione in solution is precipitated. Any increase in the pH carries down impurities which cause subsequent losses of glutathione.

The amount of lead salt necessary to give maximal precipitation of glutathione depends on the amount of impurities. For Lots 10

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to 47, we have used 2 kilos of lead acetate for the extract of 45 kilos of yeast.

Hopkins allowed the solution to settle before separation of the lead salt, probably not more than an hour. Johnson and Voegtlin removed the lead salt after the solution had stood overnight. We have found that the precipitation of the glutathione is complete within less than 5 minutes. The lead precipitate was therefore removed by centrifugation of the solution without delay after the addition of the lead acetate, After precipitation in this way, the solution contains appreciable amounts of thiol compounds but the precipitate contains a much smaller percentage of com- pounds which have to be removed by subsequent steps.

The amount of total extractives in the solution has an important bearing on the completeness of the precipitation of glutathione with lead. If the suspension of yeast is heated, a large amount of material goes into solution and precipitation with lead acetate is less complete.

Adsorption of Glutathione on Precipitates Formed with Aluminum Hydroxide Cream, Uranium Acetate, Barium Phosphotungstate, and Barium Suljate.---In order to remove protein material as much as possible before the addition of lead acetate, aluminum hydroxide cream, 1.0 per cent, was added to the extract of the yeast. Alumi- num hydroxide cream at pH 5 removes protein material from the solution and carries down almost no glutathione, but at pH 7.4 this reagent may remove glutathione almost completely from solution. The loss of all the glutathione in Lots 18 and 19 showed this reagent to be useless and it was not added to any of the other preparations.

Hopkins proposed the use of uranium acetate to remove impuri- ties from the solution prepared from the lead precipitate with sulfuric acid. If the yeast suspension is heated and a large amount of material is extracted from the yeast, the addition of uranium acetate to the solution will precipitate a large amount of impuri- ties, but this separation is attended with great loss of glutathione. In a slightly acid solution, uranium acetate will not precipitate glutathione, but as the pH is increased, the amount of glutathione carried down increases: it is possible to precipitate all glutathione at this step. In Lot 3 all glutathione was lost because of the addi- tion of too much uranium acetate to the solution. The qualitative

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test for uranium with potassium ferrocyanide indicates the pre- sence of uranium long before the maximal precipitation of impuri- ties with uranium acetate is reached. It was subsequently shown that uranium acetate does not remove impurities which phospho- tungstic acid does not remove; uranium acetate was not used in Lots 4 to 47.

Quantitative determination of glutathione after treatment with phosphotungstic acid showed that under proper conditions, no glutathione is precipitated, but that barium phosphotungstate at pH 7.4 to pH 8.0 carries down large amounts of glutathione. This loss is unavoidable and is best controlled by reducing to a minimum the amount of impurities which are removed with phosphotungstic acid.

Large amounts of glutathione may be adsorbed on barium sulfate and removed from solution. This is particularly true if the pH is 7.4 or above. The best yields of glutathione are obtained if both sulfuric acid and barium salts are used in minimal amounts.

Decomposition of Lead Precipitate with Sulfuric Acid.-Hopkins decomposed the lead salt of glutathione with 0.5 N sulfuric acid by grinding in a mortar. Johnson and Voegtlin carried out this step in the same way. This method is so time-consuming that we have modified it. The lead precipitate is suspended in 5 liters of water containing the sulfuric acid and is vigorously stirred with a nickel stirrer. The maximal decomposition of the lead salt is obtained in the minimum of time.

Precipitation of Impurities with Phosphotungstic Acid.-For the precipitation of impurities not removed with uranium acetate, Hopkins used phosphotungstic acid. Hunter and Eagles and Johnson and Voegtlin also have used phosphotungstic acid at this step. We have used both tungstic acid and phosphotungstic acid. Tungstic acid is objectionable since it does not precipitate as many impurities as does phosphotungstic acid, and its use introduces sodium sulfate into the solution.

Two important observations were made concerning the use of phosphotungstic acid : (1) a large amount of material is precipitated by phosphotungstic acid at 0" which is soluble at 20”; not only is it necessary to cool the solution to 0” during this step but the solution must be filtered at 0" to prevent resolution of the impuri- ties, and (2) the limited solubility of the phosphotungstic acid salt

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of glutathione at 0” requires this step to be carried out in a large volume (from 8 to 10 liters).

It was not found necessary to have the solution 0.5 N with respect to sulfuric acid during the precipitation with phosphotungstic acid. This reagent does not precipitate glutathione in a solution 0.1 N with respect to sulfuric acid, and the impurities are as completely removkd. Reducing the sulfuric acid content to about 4 times the equivalent of glutathione minimizes the loss of glutathione with the barium sulfate which results from the addi- tion of barium hydroxide used in the removal of the excess phos- photungstic acid.

Precipitation with Mercury.-No essential change was found necessary to give the maximal precipitation. The mercury sulfate solution was changed slightly to reduce as far as possible the amount of sulfuric acid present. The mercury precipitate was filtered immediately, as it was found that the precipitate that slowly separates from the solution contains more impurities than it does glutathione. The mercury precipitate was suspended in distilled water and thoroughly agitated with a mechanical glass stirrer to disintegrate it completely before treatment with hydro- gen sulfide. The precipitation of glutathione with mercury sulfate is remarkably complete; only traces of the thiol compound are left in the filtrate.

The steps during which sulfuric acid was removed, the solution concentrated to small volume, and the glutathione precipitated with absolute alcohol and ether were not changed.

Yield of Glutathione.-When yeast is treated as outlined, about 23 gm. of glutathione may be separated from 45 kilos of yeast. This is the average of Lots 39 to 47 inclusive. Determination of the total thiol compounds present indicates that the total amount of glutathione in the form as separated is approximately 45 gm. in 45 kilos of the yeast which was used.

Chemical Properties of Glutathione.-Hopkins described gluta- thione as a compound containing carbon, hydrogen, oxygen, nitro- gen, and sulfur, easily soluble in water and insoluble in ethyl al- cohol and other organic solvents. Its nitrogen and sulfur content were 11.70 and 12.31 per cent, respectively. After hydrolysis, he separated glutamic acid in 88 per cent yield as the hydrochloride and cystine in 60 per cent yield. He suggested the structure to be glutamyl cysteine.

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The product obtained by Hunter and Eagles contained too much nitrogen and too little sulfur to be glutamyl cysteine and they therefore suggested a more complex structure for glutathione but were unable to secure satisfactory chemical evidence for their hypothesis. Johnson and Voegtlin separated glutathione, the composition of which agreed more nearly with that prepared by Hopkins; its composition was much closer to that of glutamyl cysteine. Benedict and Newton (2) recently found that gluta- thione prepared from blood contains too much nitrogen and too little sulfur to be glutamyl cysteine.

Analysis of the glutathione which we have prepared gave about 13 per cent of nitrogen and about 11 per cent of sulfur. Titration of the SH group in alcohol with iodine or with potassium ferricya- nide electrometrically showed that the material contained about 75 per cent of the theoretic amount of the SH group required for glutamyl cysteine.

Fractional precipitation with lead acetate at varying pH, or fractional removal of phosphotungstic acid precipitates in aconcen- trated solution, or fractional precipitation with increasing amounts of copper hydroxide or mercury sulfate did not bring about any significant change in either the nitrogen or sulfur content. The material appeared to be homogeneous.

By the time we had treated thirty-nine lots of yeast, of 45 kilos each, the method as outlined had been developed to a point at which so little extraneous material was present that the thiol compound could assert its physical properties. In short, the solution of Lot 40 which was prepared for the precipitation of the glutathione with absolute alcohol set to a mass of crystals just before the alcohol was added. The crystals were washed with glacial acetic acid, and then with absolute alcohol in a centrifuge tube, and were filtered on a Buchner funnel. 5 gm. were obtained. Lots 39 and 41 were then carried through in the same way but the solution was seeded with some of the crystals. 13 gm. of crystals were separated. Six more lots of yeast of 45 kilos each were treated and the crystals were separated from each lot; a total of 78 gm. of crystalline glutathione has been isolated. (See Fig. 1.)

Identi$cation of Crystalline Glutathione.-It was soon shown that the crystals contained a thiol group which was equivalent to 81 per cent of the theoretical amount in glutamyl cysteine: this

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indicated a molecular weight of 307. It was next shown that the percentage composition indicated the presence of 3 atoms of nitrogen for each atom of sulfur. These results pointed to the probability that the crystals were a tripeptide and the difference in molecular weight (57) between glutamyl cysteine and the tripeptide indicated the probable presence of glycine. Hydrolysis with hydrochloric acid confirmed this.

The hydrochloric acid was removed in a vacuum, the cysteine was precipitated with mercury sulfate. The excess mercury was

FIG. 1. Crystalline glutathione. On the left are crystals of the tri- peptide as it separates from the original solution. On the right are the recrystallized tripeptide crystals. The crystals are coarser and of much greater cross-section. X about 16.

removed as sulfide and the solution was concentrated and benzoy- lated. Hippuric acid was separated in 75 per cent of the theoretic amount. Another portion of crystals was hydrolyzed and gluta- mic acid was separated as its hydrochloride in a yield of 66 per cent of the theoretic amount.

The mercury precipitate of cysteine was decomposed with hydro- gen sulfide, and the solution was concentrated to small volume. 95 per cent alcohol was added and the thiol group present was titrated with iodine. 94 per cent of the theoretic amount was

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required. The alcohol was removed and cystine was separated in pure crystalline form by the addition of sodium acetate. The yield of crystals was 76 per cent of the theoretic amount. Nitro- gen (Kjeldahl method) and sulfur (Parr bomb) determination showed 13.68 and 10.42 per cent respectively. These results leave no doubt that the crystals are a tripeptide of glutamic acid, glycine, and cysteine. They do not show the manner in which the three amino acids are combined.

3.07 gm. of the crystals were treated with nitrous acid and the solution was then refluxed for 18 hours after the addition of con- centrated hydrochloric acid. Glycine was separated as hippuric acid in the same yield as before but no glutamic acid could be isolated. This is evidence that the glycine is attached through its amino group and not through its carboxyl group, also that the amino group of the glutamic acid is not substituted. The reaction toward nitrous acid does not show whether the glycine is attached to the carboxyl group of the glutamic acid or to the carboxyl group of the cysteine.

HH 0

I I II H-S-C-C-C-O-H

I I H N-H

I c=o

H--C-H

I H-C-H

’ /H H-C-N

I ‘H c=o

I N-H

I H-C-H

I c=o I O-H

The structural formula tentatively suggested for the tripeptide, gluta- thione, glutamyl-glycine-cysteine.

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Evidence for this was secured by the action of hydrogen peroxide in the presence of ammonia. Carbon dioxide was given off and after hydrolysis neither glutamic acid nor glycine could be isolated, but succinic acid was separated. This shows that probably the amino group of the glycine is attached to the carboxyl group of the glutamic acid which is next to the amino group in the glu- tamic acid. The crystals are therefore glutamyl-glycine-cysteine.

After it has been shown that 15 gm. of the tripeptide can be separated in crystalline form from 45 kilos of yeast the question immediately arises as to whether cysteine is present in any other form. Further work on this question is necessary but analysis of the material precipitated from the mother liquor of the crystals with absolute alcohol shows it to contain nitrogen and sulfur in amounts which are almost theoretic for the tripeptide. Glycine can also be separated in the same yield as from the crystalline tripeptide after hydrolysis with hydrochloric acid. These results make it highly probable that practically all of the cysteine is in the form of the tripeptide.

The results obtained by Hopkins on the identification of gluta- thione leave no doubt that the substance which he isolated was glutamyl cysteine. The formation of this dipeptide from the tripeptide is an obvious explanation of the source of the dipeptide. Preliminary experiments indicate that the amount of the tripeptide which can be isolated greatly diminishes if the yeast extract is allowed to stand before the addition of lead acetate. Further work on the stability of the tripeptide is in progress. It has been clearly shown that the crystalline tripeptide can be heated to 100” in water or in 0.1 N acid for several hours without loss of glycine but after the crystals were heated in water at this temperature, subsequent crystallization of the material was greatly retarded and the weight of crystalline material recovered was much less than that from a similar solution which was heated only to 60”.

EXPERIMENTAL.

Method for Isolation of Crystalline Glutathione from Yeast.- 45 kilos of bakers’ yeast, free from starch, are broken into small lumps and evenly distributed between four 75 liter crocks, each of which contains 50 lit.ers of distilled water. The suspension of yeast is thoroughly stirred with a mechanical stirrer. 2400 cc.

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of benzene are added to each jar and the suspension is again vigor- ously stirred. 15 liters of distilled water are added to each jar and the solution is allowed to stand about 2 hours. 210 cc. of concentrated sulfuric acid dissolved in 4 liters of distilled water are added to each jar and the solution is again stirred. 1200 gm. of crystalline barium hydroxide containing 8 molecules of water dissolved in 3 liters of hot water are added to each crock. The solution is stirred and centrifuged immediately in a Sharples supercentrifuge. The precipitation of barium sulfate greatly facilitates the removal of the extracted yeast cells. The extract of the yeast is about 210 liters at this time; it is slightly turbid. It is again divided evenly among four crocks. 500 gm. of neutral lead acetate dissolved in 2 liters of distilled water are added to each crock. The pH of the solution is approximately 5.5. After thorough mixing the solution in the four crocks is centrifuged. The lead precipitate is suspended in 4 liters of water to which are added 900 cc. of 5 N sulfuric acid. This step is carried out in a 6 liter glass museum jar. The suspension of the lead salt is vigorously stirred with a nickel stirrer for 2 hours until it is re- duced to a smooth homogeneous creamy consistency without lumps. This step is not time-consuming, as the agitation is entirely mechanical. The solution is filtered and made to a volume of 7.5 liters. Barium hydroxide is added until the pH of the solution is 4. The amount of barium hydroxide required varies between 600 and 700 gm. The barium precipitates a large amount of material not glutathione. This is removed by filtration. Sulfuric acid is then added until the barium hydroxide is just neutralized and then 150 cc. of 5 N sulfuric acid are added in excess, This acidity is about equivalent to four times the glutathione present. The solution is filtered in order to remove the barium sulfate which interferes with the rapid filtration which is required at the next step. The solution is cooled to 0” in an ice and salt pack and phosphotungstic acid is added until no further precipitation is produced. Between 45 and 50 gm. are required. The solution is filtered while the temperature is main- tained as close to 0” as possible. Barium hydroxide is added to the filtrate until the pH is 7.0. The solution is again filtered. This removes the excess phosphotungstic acid. The barium is removed with sulfuric acid. Approximately 200 cc. of 5 N sulfuric

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acid are required. Barium sulfate is filtered out and the gluta- thione is precipitated with mercury sulfate. From 200 to 350 cc. of the modified Hopkins’ solution of mercury sulfate’ are required. This reagent is added until no more precipitate forms. The mercury precipitate is filtered, is broken up with a glass mechanical stirrer, and is then decomposed with hydrogen sulfide. The solu- tion is filtered from mercury sulfide and after being cooled barium hydroxide is added until the pH is 7.5. The solution is treated with hydrogen sulfide to precipitate heavy metals. It is filtered and the barium is exactly removed with sulfuric acid. The barium sulfate is removed by filtration. The solution is evaporated to about 30 or 40 cc., is placed in a wide, deep container, and is allowed to stand after the addition of a small amount of the tripeptide previously isolated. The solution sets to a firm crystalline mass during the next few hours, The crystals are suspended in glacial acetic acid, then transferred to 50 cc. centrifuge cups, and sepa- rated from the solution by short centrifugation. They are then washed with glacial acetic acid and again centrifuged. They are washed out of the centrifuge cup with absolute alcohol and are filtered on a Buchner funnel.

The compound is purified by recrystallization from water. Crystallization of the Tripeptide.-11 gm. of the crude material

were suspended in 25 cc. of water. This was not sufficient to dissolve the crystals. The solution was heated to 60” over a free flame and was stirred with a thermometer. It was filtered through paper on a small Buchner funnel, placed in a small beaker, and put in the ice box after the addition of a few crystals of the tripep- tide. After 2 days the crystalline mass was broken up and the solution was again placed in the ice box for 24 hours. The crystals were then filtered off, washed with glacial acetic acid, and finally with absolute alcohol. After drying in the desiccator, the weight was 6.4 gm.

Hydrolysis of the Tripeptide, Separation of Glutamic Acid- 2 gm. of Lot 40 were dissolved in 100 cc. of 25 per cent hydrochloric acid and the solution was refluxed for 8 hours. It was then evapo- rated in a vacuum to 10 cc. ; the solution was saturated with hydrochloric acid gas at 0”. On the following day, 620 mg. of

1200 gm. of mercury sulfate, 50 cc. of 10 N sulfuric acid, 40 cc. of con-

centrated sulfuric acid, and 600 cc. of water are mixed in the order given.

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crystals were filtered from the solution. The filtrate was again concentrated and saturated with hydrochloric acid gas at 0”. 170 mg. more of the crystals were separated. Finally a crop of 20 mg. were separated. The total weight was 710 mg. After recrys- tallization, the melting point was found to be 201”. When mixed with glutamic acid hydrochloride, the melting point was unchanged.

0.1524 gm. substance: 8.35 cc. 0.1 N NHI. Calculated. N 7.63.

Found. 7.66. 0.1534gm. substance: 8.42~~. 0.1 NNH3. Found. N 7.67.

This is a yield of glutamic acid hydrochloride equivalent to 66 per cent of the theoretic amount.

Separation of the Benzoyl Derivative of Glycine.-2 gm. of the recrystallized tripeptide were refluxed in 100 cc. of constant boiling hydrochloric acid for 15 hours. The solution was evaporated to dryness in a vacuum in order to remove the hydrochloric acid. The residue was dissolved in 150 cc. of water and mercury sulfate was added until precipitation was complete. The precipitate was filtered out. The mercury was removed from the filtrate with hydrogen sulfide; the solution was concentrated to 50 cc. 50 cc. of 5 N sodium hydroxide were added and 5 cc. of benzoyl chloride. This solution was shaken until the odor of benzoyl chloride was very slight. Hydrochloric acid was added and the solution was extracted four times with 20 cc. portions of ethyl acetate. The ethyl acetate was removed in a vacuum and the residue was dis- solved in chloroform. Hippuric acid (1) separated almost imme- diately. After 24 hours, 880 mg. were filtered off. The melting point was 188”.

Separation of Cystine.-The mercury precipitate which was removed before the isolation of benzoyl glycine was decomposed with hydrogen sulfide and the mercury sulfide was removed by filtration. The solution was evaporated to dryness in a vacuum, and was dissolved in 50 cc. of 95 per cent alcohol and oxidized with iodine dissolved in alcohol. 780 mg. were required. This is 94 per cent of the theoretic amount. The alcohol was removed by evaporation in a vacuum and the residue was dissolved in 50 cc. of water. 5 gm. of sodium acetate in 25 CC. of water were added. After 24 hours, 596 mg. of cystine were filtered from the solution. This is a yield of 75 per cent. The cystine was identified by its

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quantitative determination by a modification of the Folin-Looney method. 50 mg. in 50 cc. of 0.1 N sulfuric acid gave a colori- metric reading of 20:20 with a solution of an equal amount of pure cystine.

Ultimate Analyses of the Tripeptide.

0.149 gm. substance: 0.2104 gm. CO* and 0.0764 gm. HzO. CIOHITOBNBS. Calculated. C 39.06. Found. 38.47.

H 5.58. “ 5.63. 0.1503 gm. substance: 0.1151 gm. BaSOa. Calculated. S 10.42. Found.

10.46. 0.1635 gm. substance: 0.1239 gm. BaS04. Found. S 10.35.

0.1565 “ “ : 15.29 cc. 0.1 N NH,. Calculated. N 13.68.

Found. 13.67. 0.1535 gm. substance: 15.36 cc. 0.1 N NH,. Found. N 14.01.

Melting Point of the Tripeptide.-The melting point of the tripeptide is affected by the rate of heating. Two samples of the crystals melted at 190-192”. There was evolution of gas but no charring.

Constitution of the Tripeptide, Treatment with Nitrous Acid.-

3.07 gm. of the recrystallized tripeptide were dissolved in 50 cc. of water. 2.1 gm. (3 equivalents) of sodium nitrite were added. The solution turned bright red. 6 cc. of 5 N sulfuric acid, which was equivalent to the nitrite, were added slowly with cooling. A large volume of gas was given off. The solution was placed in a vacuum to remove the last trace of nitrous acid. Concen- trated hydrochloric acid was added so that the resulting volume of 100 cc. contained approximately 25 per cent of hydrochloric acid. The solution was refluxed overnight. The red color rapidly dis- appeared when hydrochloric acid was added; the solution was light yellow. After removal of the hydrochloric acid by evapora- tion to dryness in a vacuum, 100 cc. of water were added and cystine was precipitated with mercury sulfate. Mercury was removed from the filtrate, the sulfuric acid was removed with a measured weight of barium hydroxide. The barium sulfate was removed by filtration and 30 cc. of N sulfuric acid, equivalent to the barium were again added. The solution was concentrated to 25 cc. and 150 cc. of alcohol were added. Sodium sulfate crystallized out and was removed. The solution was filtered, the alcohol removed, and concentrated to a small volume which

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672 Study of Glutathione. I

was saturated with hydrochloric acid gas. No crystals of glutamic hydrochloride formed. A small amount of sodium chloride was present. The solution was again placed under a vacuum and, after removal of the hydrochloric acid, benzoyl chloride and sodium hydroxide were added as already described. From this, 1.05 gm. of hippuric acid were separated. The melting point was 188”.

Oxidation ojthe Tripeptide (8).-l gm. of the recrystallized gluta- thione was dissolved in 10 cc. of water to which were added 4 cc. of concentrated ammonia, 5 mg. of ferrous sulfate, and 50 cc. of 5 per cent hydrogen peroxide (3, 4). The solution, was warmed to 70”. It was then just acidified with hydrochloric acid and oxygen was passed through the solution to force out the carbon dioxide which was passed through a standardized solution of barium hydroxide. 98 cc. of 0.1 N carbon dioxide were given off. This indicates the liberation of 1.5 equivalents of carbon dioxide. The solution was evaporated to dryness in a vacuum, redissolved in a small volume of water, and was extracted with ether. No organic acid was soluble in the ether. Hydrochloric acid was then added and the solution was refluxed for 20 hours. It was evaporated to a small volume and again extracted with ether. The organic acid in the ether weighed 235 mg. By carboxyl titration and formation of the silver salt this was identified as succinic acid. The sohrtion was benzoylated but benzoyl glycine could not be separated. These results show that both the glutamic acid and the glycine were destroyed by oxidation. Oxalic acid was not present.

Xolubility and Stability of the Tripeptide.-The solubility of the tripeptide depends on the purity of the material. In the presence of the products precipitated with mercury and subsequently liberated with hydrogen sulfide, the tripeptide is exceedingly soluble. It seems highly probable that practically all of the cysteine present is in a form of the tripeptide. As shown in Table I, however, the amount of crystalline material which can be separated does not exceed about a half of the total weight. When the crude crystals are dissolved in water, only about a half of the weight of the material can be recrystallized. But as the crystal- lization is continued, the tripeptide becomes less and less soluble. When pure, it is non-hydroscopic, and is soluble to the extent of about 1 part in 10 parts of water at 0”. It is easily soluble in warm

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Kendall, McKenzie, and Mason 673

water. 3 gm. of the uncrystallized material which had been precipitated with absolute alcohol from the mother liquor of the crystals was boiled in 200 cc. of 0.1 N hydrochloric acid. The hydrochloric acid was removed by evaporation to dryness in a vacuum and the residue was redissolved in water and was precipi- tated with mercury sulfate. Mercury was removed from the filtrate which was then concentrated and treated with benzoyl chloride in the presence of sodium hydroxide. Benzoyl glycine could not be separated from the solution.

3 gm. of the same material which were hydrolyzed in concen- trated boiling hydrochloric acid for 18 hours gave a yield of 1.3 gm. of hippuric acid when treated in an entirely similar way. This is evidence that the glycine is not easily broken off from the tripep- tide in boiling 0.1 N hydrochloric acid. Preliminary experiments have indicated that the glycine may be broken off from the tripeptide by enzymic action.

Throughout this work, we have been greatly assisted in the analyses of the solutions and in some of the analytical work by Daisy Simonsen and Dr. A. E. Osterberg.

SUMMARY.

Hopkins’ method for the isolation of glutathione from yeast has been modified as follows:

1. The suspension of yeast is extracted with cold water in the presence of benzene. The cells are removed in a large centrifuge. The solution is precipitated with neutral lead acetate. The pH of the solution must be about 5.5. The lead precipitate is de- composed with sulfuric acid and some impurities are removed by raising the pH to 4.0 with barium hydroxide. The solution is made acid and is treated with phosphotungstic acid at 0”. The phosphotungstic acid is removed with barium and the glutathione is precipitated with mercury sulfate. The mercury precipitate is decomposed with hydrogen sulfide. Sulfuric acid is removed from the solution which is then concentrated to a small volume. On standing, the solution sets to a crystal mass and the crystals are washed with glacial acetic acid and absolute alcohol. They may be recrystallized from water.

2. This material is a tripeptide of glutamic acid, glycine, and cysteine. The glycine is attached to the carboxyl group of

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674 Study of Glutathione. I

glutamic acid, which is nearest to the amine group. Cysteine is attached to the other carboxyl group of the glutamic acid. The chemical reactions used in the determination of the structure of the tripeptide are given.

3. The material precipitated from the mother liquor of the crystals with absolute alcohol has very nearly the same percentage composition and glycine can be separated after hydrolysis from this material and from the crystalline tripeptide in about the same yield. It seems highly probable that practically all of the cysteine is present in the form of the tripeptide.

4. The formula suggested in this paper is based on three facts. First, succinic acid is obtained after hydrogen peroxide treat- ment only after hydrolysis. Second, glycine but no glutamic acid can be separated after treatment with nitrous acid. Third, neither glutamic acid nor glycine can be separated after oxida- tion with hydrogen peroxide. The fact last mentioned is nega- tive in character; therefore, until more evidence positive in nature is obtained, the structure of the tripeptide must remain in doubt.

BIBLIOGRAPHY.

1. Abderhalden, E., Biochemisches Handlexikon, Berlin, 4,397 (1911).

2. Benedict, S. R., and Newton, E. B., J. Biol. Chem., 83,361 (1929). 3. Dakin, H. D., J. Biol. Chem., 1,171 (190506).

4. Dakin, H. D., J. Biol. Chem., 6,409 (190809). 5. Hopkins, F. G., Biochem. J., 16,286 (1921). 6. Hunter, G., and Eagles, B. A., J. Biol. Chem., 72,147 (1927). 7. Johnson, J. M., andvoegtlin, C., J. BioZ. Chem., 76,703 (1927).

8. Quastel, J. H., Stewart, C. P., and Tunnicliffe, H. E., Biochem. J., 17, 586 (1923).

9. Raymond,A. L., J. BioZ. Chem., 79,637 (1928).

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Harold L. MasonEdward C. Kendall, Bernard F. McKenzie and

FORM AND ITS IDENTIFICATIONPREPARATION IN CRYSTALLINE

A STUDY OF GLUTATHIONE: I. ITS

1929, 84:657-674.J. Biol. Chem. 

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