ON THE PREPARATION AND PROPERTIES OF PURE GLUTATHIONE (GLUTAMINYL-CYSTEINE).

BY J. M. JOHNSON AND CARL VOEGTLIN.

(From the Division of Pharmacology, Hygienic Laboratory, United States Public Health Service, Washington.)

(Received for publication, October 6, 1927.)

Glutathione was first. isolated in 1921 from yeast, mammalian liver, and muscle by Hopkins (5), who also presented evidence indicating that the substance is a dipeptide of cysteine and glutamic acid. In 1925 Stewart and Tunnicliffe (8) published their work on the synthesis of glutathione. Their second synthetic method yielded a product, which possessed all of the properties of the substance found in nature. Since then, a number of other workers have prepared the natural product, but so far no analytic findings have been published which would support the data given by the English investigators. Brand and Sandberg (3), for instance, obtained a product from ox blood with a sulfur tiontent of 8.6 per cent instead of the theoretical figure of 12.87 per cent. They therefore considered their sub- stance impure. The other workers, with the exception of Hunter and Eagles (7), do not give any analytical findings at all. About a year ago there appeared a paper by Hunter and Eagles (7) which led these workers to the conclusion that glutathione prepared from yeast, blood, and liver in reality is not a simple dipeptide composed of cysteine and glutamic acid, but more likely a tripeptide of Iunknown composition. They base this conclusion largely on the fact that their products had a much lower sulfur and higher total nitrogen content than the products of the English workers. Hunter and Eagles’ report was immediately followed by a short note by Hopkins (6) in which he suggests that the low sulfur content of the products obtained by Hunter and Eagles was probably due to the splitting off of sulfur from the substance by the alkali used in the course of isolation. Bergmann and Stather (2) had previously called attention to the great ease with which alkali splits off sulfur from dialanyl-Z-cystine dianhydride. Re- ferring to this work Hopkins in his note reported some experiments which indicate that exposure of pure glutathione to alkali results in the splitting off of sulfur. He concludes with the following remarks: “In any case, although I have myself no doubt as to the nature of glutathione, the ap- pearance of Hunter and Eagles’ papers makes it desirable that I should if possible give greater precision to the account of its isolation. This I hope to do in the near future.”

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During the last 5 years we have had a large amount of experience in the isolation of glutathione, which material we used in work dealing with the physiological and pharmacological action of the substance. Suffice it to say here that we worked up twenty-three batches of pressed yeast, totaling over 1000 kilos. The purpose of the present communication is to report briefly our findings in view of the fact that a report on the subject by independent investiga- tors is needed to clear up the discrepancy between the published claims. We may state at once that we have never had any reason to doubt the reliability of’the statements of the English workers as to the structure of glutathione. On the other hand we may state that in our earlier experience we sometimes obtained products which analyzed low in sulfur and high in total nitrogen. These products were obtained by following Hopkins’ method in every essential detail to the best of our ability. Hence it was obvious that the method described by Hopkins may or may not yield a pure product and our efforts were therefore turned towards modi- fying the method in such a way as to avoid the production of im- pure material. In the experimental part we shall presently de- scribe this method as accurately as possible so that other workers may have no difficulty in repeating the work. It is well here to emphasize the fact that the isolation can only be satisfactorily accomplished with patience and careful observation of every required detail. It will be seen that our description of the method deviates only slightly from that given by Hopkins, but we are convinced that it will overcome the difficulties inherent in his method. In addition we report the preparation and analysis of some compounds of glutathione with certain heavy metal salts. S-S glutathione always indicates the oxidized form and SH glutathione the reduced form.

EXPERIMENTAL.

Preparation of Glutathione from Yeast.-Fresh, pressed bakers’ yeast (45 kilos), free from starch,’ is dropped in small pieces into 100 liters of water previously heated to about 80°C. The mixture is brought to the boiling point under constant stirring, cooled, and

1 Ordinary commercial bakers’ yeast contains a considerable amount of starch, which in our experience renders the first extraction very difficult.

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the extract separated by means of a Sharples centrifuge. The solid residue is again extracted with 50 liters of boiling water. The combined extracts are cooled to about 15°C. and a cold satu- rated solution of neutral lead acetate is added to complete precipi- tation. Concentrated NH,OH is added until the mixture is only slightly acid to litmus. After standing overnight at ordinary tem- perature, the lead precipitate is separated by means of the Sharples centrifuge and washed with a small volume of cold water. The precipitate is ground up in mortars with the addition of pure quartz sand and 0.5 N HzS04 until the filtrate contains a slight excess of free HzS04. The PbSOe is filtered off on Buchner filters. Further extraction of the residue with H&304 does not increase the yield in glutathione, though such extract still gives a nitroprusside test and material precipitable with HgS04. The acid extract, amount- ing to about 10 to 12 liters, is treated in 2 liter portions with a saturated solution of uranium acetate until a little of the filtered fluid gives a strong color with potassium ferrocyanide. The mix- ture after thorough cooling with an ice-salt mixture is treated with a hot saturated solution of Ba(OH)2 and filtered without delay and as rapidly as possible on several Buchner filters. The filtrate is immediately acidified with a slight excess of H&SO4 and filtered. The filtrate is now precipitated with the Hopkins-Cole HgS04 reagent, avoiding an excess of the reagent. After standing for several hours the precipitate is collected on a Buchner filter, well washed with cold water, suspended in water, and decomposed with H&S. The filtrate (1200 cc.) from the HgS is aerated to remove H&4, made roughly 0.5 N with H&SO, and precipitated with an excess of a saturated solution of phosphotungstic acid in 0.5 N

H&301. A small amount of gummy precipitate is filtered off and the phosphotungstic acid removed from the cooled filtrate by the addition of a cooled, saturated solution of Ba(OH)2. The mixture is filtered as rapidly as possible on a Buchner funnel and the filtrate acidified at once with H,S04 and again precipitated with HgS04 in the manner previously described. After standing for several hours the mercury precipitate is collected on a Buchner filter, washed several times with HzO, and finally suspended in about 100 cc. of H,O. After complete decomposition with H&S and removal of HgS by filtration and washing of the latter with water, the combined filtrates are aerated with purified (NH3-free) air in

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order to remove H2S. The solution is then carefully adjusted so as to be free from both HzS04 and barium and is concentrated under reduced pressure (below 50°C.) to a thin syrup. This syrup and watery washings from the distilling flask, amounting to IOcc., are poured into a cooled mixture of 100 cc. of absolute alcohol and 100 cc. of dry ether. After standing for about 12 hours in the ice box the supernatant alcohol-ether mixture is poured off and the somewhat gummy precipitate treated with 100 cc. of absolute alco- hol. After standing for another 12 hours the precipitate is friable and is removed to a vacuum desiccator and dried over Ps05 and paraffin to constant weight. Yield of SH glutathione, 4 gm.

Conversion of SH Glutathione into S-S Glutathione.-500 mg. of the above SH glutathione are dissolved in 15 cc. of Hz0 and carefully made very slightly alkaline to litmus by the addition of cold saturated Ba(OH)2 solution. A rapid stream of pure oxygen is run through this solution until the nitroprusside test is just negative (in this case after 2 hours). Without delay the solution is freed from barium with H,S04, an excess of the latter being avoided. The use of the centrifuge for testing the freedom of the solution from both barium and H.SOd is time-saving. After concentration to a thin syrup (3 cc.) under reduced pressure (below 50°C.) the material is precipitated by being poured into 40 cc. of absolute alcohol. The precipitat.e becomes friable after stand- ing overnight. It is dried in a vacuum desiccator over PZOs and paraffin and finally brought to constant weight at 100°C. in vacua over Pz06. Yield of S-S glutathione, 450 mg.

Analytical Methods.-Total nitrogen was estimated by the Kjeldahl method; the total sulfur by the method described by Hoffman and Gortner (4), use being made of an electric furnace for the ashing. This method was first tested as to its reliability by applying it to the analysis of C.P. cystine and cysteine hydro- chloride with satisfactory results. The specific rotation was de- termined by means of a Schmidt and Haensch polarimeter, either mercury light or an electrical bulb (Mazda) of 500 watts being used.

The gold was estimated as the metal after ashing of the com- pound in the electric furnace at 500°C. The copper was estimated as CuO after fusion in an electric furnace at 500°C. with a mixture of NaN03 and NH4N03, followed by precipitation with KOH.

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The lead was determined as PbS04, by treating the compound with dilute HzS04, concentration on the water bath, and heating over the free flame until fumes of HzS04 came off. After cooling and dilution with water the precipitate was collected on a Gooch filter.

Copper, Gold, and Lead Derivatives of SH Glutathione.-To 500 mg. of SH glutathione, dissolved in 20 cc. of HzO, there are added 5 cc. of a solution of CuCL containing 169.3 mg. of CuC& + 2Hz0, i.e. 4 mols of glutathione to 2 mols of CuCL The grad- ual addition of 6.5 cc. of 0.25 N NaOH produces a grayish green precipitate which is separated by centrifugation and is washed free from chlorides by water. The addition of more 0.25 N NaOH to the mother liquor does not cause any further precipitation. The precipitate is dried to constant weight over PZOs and represents an amorphous grayish green powder. Yield, 82 mg.

Found. Cu 33.20 per cent. Calculated for CsHtzOsNpSCua 33.88 per cent. “ s 8.07 “ “ “ “ ‘I 8.55 i‘ “

To 500 mg. of SH glutathione, dissolved in 7.5 cc. of H,O, there is gradually added a solution of AuCL, previously neutralized with NaOH, until no further precipitation occurs. This requires Q of an atom of Au to 1 mol of glutathione. A very small amount of precipitate was centrifuged off and discarded. The pale brown supernatant fluid is precipitated by the addition of 10 cc. of abso- lute alcohol. The precipitate is separated by centrifugation, washed once with absolute alcohol, and dried to constant weight over P205 and paraffin. Yield, 258 mg. of a white, amorphous powder, which is easily soluble in water.

Found. Au43.75 per cent. Calculated for CsHlsOsNzSAu. 44.20 per cent. “ S 6.52 “ “ “ “ “ 7.21 “ “

From the mother liquor of the above gold compound there are recovered (after removal of the gold) by mercury precipitation 220 mg. of glutathione.

To 200 mg. of SH glutathione, dissolved in 5 cc. of H20, there is added gradually a solution of basic lead acetate (Goulard’s extract) until complete precipitation has occurred, which requires 1.25 cc. The precipitate is separated by centrifugation, washed

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repeatedly with water, and dried to constant weight in a vacuum desiccator over Pz05. Yield, 276 mg. of a white amorphous powder.

Found. Pb 44.92 per cent. Calculated for CsH1205N&Pb. 45.50 per cent. I‘ N 6.96 “ “ ‘I “ ‘I 6.15 “ “

DISCUSSION.

Preparation.-In our experience fresh, pressed, and starch- free bakers’ yeast is the most suitable raw material for the prepara- tion of pure glutathione, though we have found brewers’ yeast (bottom) quite satisfactory. Yeast is superior to ox liver, as the preliminary extraction is rather cumbersome with ox liver. We found a single extraction with water quite sufficient in remov- ing all but a small amount of the substance. The use of a Sharples centrifuge in the preliminary extraction and for the separation of the lead precipitate is very convenient and time-saving and it is quite feasible to obtain the lead precipitate from 45 kilos of yeast ready for acid decomposition within 12 to 36 hours. Particular care is required to speed up the steps in the process when the material is in contact with alkali, and the temperature should belowered during these stages by proper cooling. Contrary to Hunter and Eagles, and confirming Hopkins, we consider the precipitation with phos- photungstic acid essential for the removal of impurities. We have eliminated the precipitation with copper or lead following the second mercury precipitation, as in our experience these reagents do not yield a pure product if at this stage the impurities have not been removed. We have repeatedly attempted to purify products low in sulfur by precipitation with copper, mercuric sulfate, or lead acetate without success, as analysis of the final products showed. Once formed, these impurities cannot be removed by the present methods. We also found that it is easier to obtain SH glutathione in pure form than S-S glutathione. We are inclined to attribute this to the possibility of sulfur being split off during the final oxidation. In fact we have shown that a given sample of pure SH glutathione will yield S-S glutathione low in sulfur if the solution is not worked up as soon as the nitroprusside test has disappeared. A few hours standing in even a slightly alkaline medium will lead to deterioration. We also recommend using

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relatively small batches (0.5 to 1 gm.) of SH glutathione for carry- ing out the oxidation.

Yield.-The yield of pure substance we found to be about the same as that given by Hopkins (100 to 150 mg. per kilo). This represents only a fraction of the substance present in pressed yeast (containing about 25 per cent solids) as indicated by iodine titra- tion of a trichloroacetic acid extract. The low yield is partly explained by the incomplete precipitation of the substance by lead acetate, copper salts, and mercuric sulfate, which is evident from a consideration of the experiments described by us, where every precaution was taken to obtain complete precipitation. In other preparations of these metal derivatives somewhat different yields were obtained, but in every case the loss of substance was considerable. We believe that this loss in the copper precipitation is partly due to the conversion of part of the SII glutathione into S-S glutathione by the copper salt, and S-S glutathione is not precipitated by copper. A similar, though perhaps smaller loss occurs in the use of lead and mercury, due undoubtedly to the solubility of these metal derivatives. At any rate we have often recovered from the filtrates of the heavy metal precipitates, after removal of the metal with H&S, considerable amounts of gluta- thione. The alcohol-ether mother liquors in the final precipita- tion also yield some additional substance after evaporation of the solvent, but as a rule this is only a small amount and of uncertain purity.

Purity.-The results in Table I are given as an illustration of the agreement in the analytical findings of two of our products with those of Hopkins, and Stewart and Tunnicliffe. As stated by Hop- kins, the behavior of the substance in the melting point deter- mination is of minor significance as glutathione has no sharp melting point. Making allowance for this fact it will be seen that our figures agree with those given by Hopkins for S-S glutathione.

The figures for total nitrogen and sulfur are in perfect agreement with those of Hopkins and those required by theory. Our sulfur values have always been slightly lower than the theoretical, but this is to be expected.

The figures for the optical rotation of S-S glutathione are some- what lower than those given by Stewart and Tunnicliffe for the

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natural and synthetic product; i.e., -93.9” in aqueous solution and -84.7” in 10 per cent HCI instead of -98.3” and -89.2” respectively. The difference of the rotation in water and in acid is in both cases the same, 9.1 to 9.2. We used for these deter- minations the same concentrations and the same light as the

TABLE I.

Physical Properties and Analytical Findings from Yeast Glutathione.

Melting point. Softening at.. . Swelling at.

Total nitrogen, per cent ....... Sulfur, per cent .................

Specific rotation.

3.46 Der cent in Hz0 at 28.5”C. 1.73 -I( ‘( “ lo-per cent HC

at26.6”C . . . . . . . . . . . . . Electric light.

3.46 per cent in Hz0 at 28.5%. 1.73 “ (‘ “ 10 per cent HC

at 26.6”C.. . . . . . . . . . . . .

MHgi 3.46 per cent in Hz0 at 15°C.. 1.73 “ “ “ 10 per cent HCl

at 15”C... . . . . . . . . . . . . . 10 per cent in Hz0 at 27°C.. .

5 “ ‘< “ 10 per cent HCl at27”C.....................

S-S glutnthione.

Hopkins.

165-167% 182-185 “

11.70 12.31

Stewart and Tunnicliffe.

-98.3”

-89.2”

Johnson and Voegtlin.

162-163°C 170-195 ((

11.24 12.06

Johnson and Voegtlin.

-93.9”

-84.7”

-83.8”

-78.3”

-

.-

.

-

1’ SH glutatbione. Calcu-

lated. Johnson and

Voegtlin.

111°C. 185-190°C.

(melts).

11.14 11.24 12.33 12.85

lohnson and Voegtlin.

-10.2”

-1.6”

English workers, but a different temperature (28.5 and 26.6”C. respectively instead of 15’C.). The room temperature was always too high to permit readings at 15°C. and we had no rotation tubes available which would permit the keeping of a constant lower tem- perature. We found, however, that in the determination of the

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rotation of glutathione the temperature coetilcient has a consider- able influence on the result. This was shown by cooling the 3.46 per cent solution ([a]npj -93.9” at 28.5”C.) to about 5°C. in a refrigeration room. When this solution was brought to the polarimeter room the solution with the same light, as soon as it could be read, gave a value of - 100.9” and then returned to -93.9” after standing for some time at 28.5%. The range of rotation between a temperature of 5-28.5”C. is therefore from -100.9” to - 93.9”. A similar temperature coefficient was found for cystine solutions by Andrews (1). The figures obtained with electric light are given for the convenience of workers who are not provided with a mercury light. These figures are considerably lower, as could be predicted.

As to the rotation of SH glutathione, it should be pointed out that Hunter and Eagles first called attention to the probability that this substance had ‘la very small negative rotation, or possibly a positive rotation.” These workers dealt with an impure product. The figures in Table I for the rotation of the sample of SH gluta- thione were obtained with a product made according to the direc- tions given in this paper, which should avoid partial oxidation. It is seen that this product has a small negative rotation in water (- 10.2’) and a very slight negative rotation (- 1.6’) in 10 per cent HCl. Other batches of SH glutathione showed a slightly different rotation, but of the same order of magnitude.

Absence of Cyst&e or Cysteine.-The samples of glutathione referred to in Table I were tested for the presence of free cystine or cysteine by the naphthoquinone test of Sullivan (9) and were found to be free from either of these substances., This test is of some value for testing the purity of samples of glutathione. The majority of our products gave a negative test, but a few of the early batches prepared by the original (Hopkins) method gave a curious purple coloration different from that produced by cystine and cysteine.

In view of the fact that Van Slyke’s amino acid nitrogen estima- tion yields unreliable results in the case of cystine-containing substances, as was also found for glutathione by Hopkins, we dispensed with this determination. We also thought it superfluous to carry out a hydrolytic analysis which is never quantitative, as stated by Hopkins.

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Finally, mention should be made of the heavy metal derivatives obtained from SH glutathione. Their exact constitution has not been determined. From the analysis of the copper compound, however, it is fairly certain that the copper substitutes the hydrogen of the SH group and that the copper is monovalent. This would indicate that part of the cupric salt used is reduced to cuprous salt with the simultaneous oxidation of part of the SH glutathione to S-S glutathione. We found that cuprous hydroxide also forms a compound with SH glutathione which appears to be of the same composition as that obtained with cupric salts. The second atom of Cu is probably attached to a carboxyl group. S-S glutathione does not give any precipitate with copper salts, as stated by Hopkins.

From the analysis and the color of the gold derivative it appears that the metal is in the aurous state and probably attached to sulfur. Auric salts are strong oxidizing agents and should easily oxidize part of the SH glutathione to S-S glutathione. This view would explain the small yield.

The analysis of the lead compound and the fact that S-S glu- tathione does not yield a precipitate when treated with lead ace- tate indicate that one valence of the lead is attached to sulfur and the other probably to a carboxyl group.

A few attempts to prepare a mercury compound of definite composition so far have failed. At all events the preparation of these heavy metal derivatives furnishes additional information for a proper understanding of the significance of glutathione in the chemical defense of the body against poisoning with certain heavy metals (Voegtlin, Johnson, and Dyer (10)).

SUMMARY.

1. The original method of Hopkins for the preparation of glu- tathione from yeast sometimes yields products which are impure.

2. A modified method is described in detail, which, if carefully followed, will yield a product having the same composition and properties as given by Hopkins for diglutaminyl-cystine. The previous claims of Hopkins as to the chemical nature of gluta- thione are therefore confirmed.

3. The optical rotation of pure SH glutathione was determined for the first time.

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J. 111. Johnson and C. Voegtlin 713

4. The copper, gold, and lead compounds of SH glutathione have been prepared.

BIBLIOGRAPHY.

1. Andrews, J. C., J. Biol. Chem., 1925, lxv, 147. 2. Bergmann, M., and Stather, F., 2. physiol. Chem., 1926, clii, 189. 3. Brand, E., and Sandberg, M., J. Biol. Chem., 1926, lxx, 381. 4. Hoffman, W. F., and Gortner, R. A., J. Am. Chem. Sot., 1923, XIV, 1033. 5. Hopkins, F. G., Biochem. J., 1921, xv, 286. 6. Hopkins, F. G., J. Biol. Chem., 1927, lxxii, 185. 7. Hunter, G., and Eagles, B. A., J. Biol. Chem., 1927, lxxii, 147. 8. Stewart, C. P., and Tunnicliffe, H. E., Biochem. J., 1925, xix, 207. 9. Sullivan, M. X., Pub. Health Rep., U. S. P. H. S., 1926, xli, 1030.

10. Voegtlin, C., Johnson, J. M., and Dyer, H. A., Proc. Nat. Acad. SC., 1925, xi, 344.

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J. M. Johnson and Carl Voegtlin(GLUTAMINYL-CYSTEINE)

GLUTATHIONEPROPERTIES OF PURE ON THE PREPARATION AND

1927, 75:703-713.J. Biol. Chem. 

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