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The isolation of a homologue of glutathione from bean seedlings
364
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Number of fractions
FIG. 2. Chromatography of 400 mg. of rat liver freeze-dried extract on a 2.2 X 35 cm. column of CM-cellulose with a potassium phosphate gradient. The eluting buffer was produced with a Varigrad mixing device (Technicon Chromatography Corp.). Six chambers of the Varigrad were charged, in order, with 100 ml. of potassium phosphate buffer of the following molarity and pH (1) 0.01 M, pH 6.5; (2) 0.01 M, pH 7.3; (3) 0.01 M pH 8.5; (4) 0.03 M, pH 8.5; (5) 0.05 M, pH 8.5; (6) 0.05 M, pH 8.5. Activity for cystathionase (unbroken line) and cysteine desulfhydrase (dotted line) is expressed as described in Fig. 1. the cleavage of cystathionine and for the desulfhydration of cysteine is very remote.
6. SMYTttE, C. V., Methods in Enzymology, (S. Colowiek and N. O. Kaplan eds.) Vol. II, p.
315. Academic Press, New York, 1955. B. MONDOVi h. SCIOSCIA-SANTORO 7. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Soc. 78,751 (1956). D. CAVALLINI 8. PORATH J., Biochim. Biophys. Aeta 22, 151 Institute of Biological Chemistry University of Rome (1956). Rome, Italy and Institute of Biological Chemistry University of Modena The Isolation of a Homologue of Glutathione Modena, Italy from Bean Seedlings Received February 14, I963
REFERENCES 1. BINKLEY, F., J. Biol. Chem. 186, 287 (1950). 2. MATSUO, Y., AND GREENBERG, D. M., J. Biol. Chem. 234,507,516 (1959). 3. CAVALLINI,D., MONDOVi, B., DE MARCO, C., ANn SCIOSCIA-SANTORO,A., Enzymologia 24, 253 (1962). 4. CHATAGNER,F., LABOUSSE, J., TRAUTMAN,O., ANn JOLLES-BERGERET,B., Compt. Rend. 253, 742 (1961).
5. BR?)GGEMAN, J., AND WALDSCttMIDT, M., Biochem. Z., 335,408 (1962).
During an investigation into acidic peptides from 4-day-old etiolated seedlings of Phaseolus aureus, I observed that one of the main peptides contained a disulfide group. The expressed juice (4 ml.) (1) was applied to a column (25 cm. X 1 cm.) of diethylaminoethyl-Sephadex A25 in the formate form, and the nonacidic compounds were washed off with water. Gradient elution with increasing concentrations of formic acid was used to elute the acidic amino acids and a complex mixture of acidic peptides. The disulfide peptide emerged in the tail of the glutamic acid peak. In later fractions there was a complex mixture of
L E T T E R S TO T H E E D I T O R S peptides containing glutamic acid, aspartic acid, cystine, f~-alanine, glycine, serine, and alanine as the main amino acids. The major peptide was ~,-glutamyl-S-methylcysteine (2, 3) and there were also traces of glutathione. The new disulfide peptide has now been isolated from two large-scale fractionations, in which a three-stage chromatographic process on anionexchange resin, Sephadex G25, and diethylaminoethyl-Sephadex A50 was used. The peptide is readily crystallized from water as fine colorless needles with melting point 194-197 ~ C. decomp. 120.5~ -- 58.9~ (uncorrected); specific rotation rLaiD (c 0.49 in water). It has been characterized by the usual procedures of peptide analysis as biS-L-Tglut amyl-L-cystinyl-bis -f~-alanine. Elemental analysis (C~H36OI~N6S:)
Calculated Found 1
C 41.24 41.61 41.54
H 5.66 5.78 6.13
O 29.97 30.48
N 13.12 12.53 13.22
S 10.01 9.87
This peptide, its SIt form, SO3I.I derivative, and its N-ethylmaleimide adduct all have slightly greater Ry values in butan-l-ol-acetie acid-water (4:1:5) than the corresponding derivatives of glutathione. This is in agreement with its formulation as a higher homologue (4). The name "homoglutathione" is suggested for this peptide. HomoGSH is an activator of glyoxalase. HomoGSSG will oxidize NADPI-I when a glutathione reductase preparation (prepared from an acetone powder of the seeds of Phaseolus aureus (5)), is added to the solution. Chromatography of the juice, after treating it with N-ethylmaleimide showed that 1Analysis performed by Pascher & Pascher Bonn, Western Germany.
365
in the freshly expressed juice homoglutathione was present as the thiol form. In the future, when studying glutathione in tissues it will be necessary to check for the presence of homoglutathione. Fortunately there is a simple and rapid means of distinguishing between homoGSSG and GSSG by electrophoresis at pH 4 (6). HomoGSSG had a mobility (105 ~, cm. 2 volts -1 see. -1) of 3.0 whereas GSSG had 6.3, and for comparison glutamic acid had 4.6, on Whatman 3MM paper at 4 ~ C. Price (7) observed a new thiol in several species of Phaseolus but did not characterize the compound. He suggested the name "phaseothione" which is rather inappropriate since his own results showed that it was also present in Glycine max and Trifolium repens. A full account of the isolation and characterization of homogluthathione is to be published later. I thank S. Kanason for technical assistance. PATRICK R. CARNEGIE Department of Biochemistry Faculty of Medicine University of Singapore Singapore, Malaya Received March 3, 1963 REFERENCES 1. CARNEGIE, P. R., Biochem. J. 78, 697 (1961). 2. RINDERKNECHT, n . , THOMAS, D., AND ASLIN, S., Helv. CAirn. Acta 41, 1 (1958). 3. ZACHARIUS,R. M., MORRIS, C. J., AND THOMPSON, J. F., Arch. Biochem. Biophys. 80, 199 (1959). 4. HOWE, J. R., J. Chromatog. 3, 389 (1960). 5. MAPSON, L. W., AND GODDARD, D. R., Biochem. J. 49, 592 (1951). 6. WALEY, S. G., Biochem. J. 68, 189 (1958). 7. PRICE, C. A., Nature 180, 148 (1957).
LETTERS TO T H E EDITORS
-~ 0,80,5-
44-
~--~o
~o
~o
go
~o
Jo
70
Number of fractions
FIG. 2. Chromatography of 400 mg. of rat liver freeze-dried extract on a 2.2 X 35 cm. column of CM-cellulose with a potassium phosphate gradient. The eluting buffer was produced with a Varigrad mixing device (Technicon Chromatography Corp.). Six chambers of the Varigrad were charged, in order, with 100 ml. of potassium phosphate buffer of the following molarity and pH (1) 0.01 M, pH 6.5; (2) 0.01 M, pH 7.3; (3) 0.01 M pH 8.5; (4) 0.03 M, pH 8.5; (5) 0.05 M, pH 8.5; (6) 0.05 M, pH 8.5. Activity for cystathionase (unbroken line) and cysteine desulfhydrase (dotted line) is expressed as described in Fig. 1. the cleavage of cystathionine and for the desulfhydration of cysteine is very remote.
6. SMYTttE, C. V., Methods in Enzymology, (S. Colowiek and N. O. Kaplan eds.) Vol. II, p.
315. Academic Press, New York, 1955. B. MONDOVi h. SCIOSCIA-SANTORO 7. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Soc. 78,751 (1956). D. CAVALLINI 8. PORATH J., Biochim. Biophys. Aeta 22, 151 Institute of Biological Chemistry University of Rome (1956). Rome, Italy and Institute of Biological Chemistry University of Modena The Isolation of a Homologue of Glutathione Modena, Italy from Bean Seedlings Received February 14, I963
REFERENCES 1. BINKLEY, F., J. Biol. Chem. 186, 287 (1950). 2. MATSUO, Y., AND GREENBERG, D. M., J. Biol. Chem. 234,507,516 (1959). 3. CAVALLINI,D., MONDOVi, B., DE MARCO, C., ANn SCIOSCIA-SANTORO,A., Enzymologia 24, 253 (1962). 4. CHATAGNER,F., LABOUSSE, J., TRAUTMAN,O., ANn JOLLES-BERGERET,B., Compt. Rend. 253, 742 (1961).
5. BR?)GGEMAN, J., AND WALDSCttMIDT, M., Biochem. Z., 335,408 (1962).
During an investigation into acidic peptides from 4-day-old etiolated seedlings of Phaseolus aureus, I observed that one of the main peptides contained a disulfide group. The expressed juice (4 ml.) (1) was applied to a column (25 cm. X 1 cm.) of diethylaminoethyl-Sephadex A25 in the formate form, and the nonacidic compounds were washed off with water. Gradient elution with increasing concentrations of formic acid was used to elute the acidic amino acids and a complex mixture of acidic peptides. The disulfide peptide emerged in the tail of the glutamic acid peak. In later fractions there was a complex mixture of
L E T T E R S TO T H E E D I T O R S peptides containing glutamic acid, aspartic acid, cystine, f~-alanine, glycine, serine, and alanine as the main amino acids. The major peptide was ~,-glutamyl-S-methylcysteine (2, 3) and there were also traces of glutathione. The new disulfide peptide has now been isolated from two large-scale fractionations, in which a three-stage chromatographic process on anionexchange resin, Sephadex G25, and diethylaminoethyl-Sephadex A50 was used. The peptide is readily crystallized from water as fine colorless needles with melting point 194-197 ~ C. decomp. 120.5~ -- 58.9~ (uncorrected); specific rotation rLaiD (c 0.49 in water). It has been characterized by the usual procedures of peptide analysis as biS-L-Tglut amyl-L-cystinyl-bis -f~-alanine. Elemental analysis (C~H36OI~N6S:)
Calculated Found 1
C 41.24 41.61 41.54
H 5.66 5.78 6.13
O 29.97 30.48
N 13.12 12.53 13.22
S 10.01 9.87
This peptide, its SIt form, SO3I.I derivative, and its N-ethylmaleimide adduct all have slightly greater Ry values in butan-l-ol-acetie acid-water (4:1:5) than the corresponding derivatives of glutathione. This is in agreement with its formulation as a higher homologue (4). The name "homoglutathione" is suggested for this peptide. HomoGSH is an activator of glyoxalase. HomoGSSG will oxidize NADPI-I when a glutathione reductase preparation (prepared from an acetone powder of the seeds of Phaseolus aureus (5)), is added to the solution. Chromatography of the juice, after treating it with N-ethylmaleimide showed that 1Analysis performed by Pascher & Pascher Bonn, Western Germany.
365
in the freshly expressed juice homoglutathione was present as the thiol form. In the future, when studying glutathione in tissues it will be necessary to check for the presence of homoglutathione. Fortunately there is a simple and rapid means of distinguishing between homoGSSG and GSSG by electrophoresis at pH 4 (6). HomoGSSG had a mobility (105 ~, cm. 2 volts -1 see. -1) of 3.0 whereas GSSG had 6.3, and for comparison glutamic acid had 4.6, on Whatman 3MM paper at 4 ~ C. Price (7) observed a new thiol in several species of Phaseolus but did not characterize the compound. He suggested the name "phaseothione" which is rather inappropriate since his own results showed that it was also present in Glycine max and Trifolium repens. A full account of the isolation and characterization of homogluthathione is to be published later. I thank S. Kanason for technical assistance. PATRICK R. CARNEGIE Department of Biochemistry Faculty of Medicine University of Singapore Singapore, Malaya Received March 3, 1963 REFERENCES 1. CARNEGIE, P. R., Biochem. J. 78, 697 (1961). 2. RINDERKNECHT, n . , THOMAS, D., AND ASLIN, S., Helv. CAirn. Acta 41, 1 (1958). 3. ZACHARIUS,R. M., MORRIS, C. J., AND THOMPSON, J. F., Arch. Biochem. Biophys. 80, 199 (1959). 4. HOWE, J. R., J. Chromatog. 3, 389 (1960). 5. MAPSON, L. W., AND GODDARD, D. R., Biochem. J. 49, 592 (1951). 6. WALEY, S. G., Biochem. J. 68, 189 (1958). 7. PRICE, C. A., Nature 180, 148 (1957).