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An assay for glutathione in acid solution
ANALYTICAL
BIOCHEZMISTBY
18,
49%i98
(1!367)
An Assay for Glutathione
in Acid Solution
P. C. JOCELYN Department
of Biochemistry,
New Buildings,
Teviot
Place, Edinburgh,
Scotland
Received October 21, 1966 The few known specific methods for assaying GSH, whether by enzymes (1, 2) or by purely chemical methods (3, 4)) all require a pH of 7-3. At this pH, the thiol is subject to oxidation. It also undergoes thiol-disulfide exchange (5) and so cannot be assayed accurately by existing methods in the presence of other disulfides. Since these reactions do not occur at low pH, a method of assay in acid solution would be useful. Calvin (6) found that, when GSH was treated with concentrated hydrochloric acid, the solution developed an ultraviolet absorption spectrum with a peak at 265 rnp. This was ascribed to the formation of a thiazoline ring by dehydration (6, 12) though it is uncertain (7, 8) whether this can be the correct explanation. The appearance of this absorption has now been made the basis of a method for assaying GSH in acid solution. MATERIALS
AND
METHODS
GSH, cystine, cysteine, and iodoacetic acid were obtained from British Drug Houses Ltd., GSSG from Boehringer Ltd., iodoacetamide from Kodak Ltd., Liverpool, and DTNB’ from Aldrich Chemical Co., Milwaukee, Wisconsin. Cysteinylglycine was prepared according to Gutcho and Laufer (9) . Erythrocytes obtained from fresh oxalated blood were washed once with saline and hemolyzed with 12.5 vol water, and the proteins were precipitated by addition of 1.5 vol 10% metaphosphoric acid. NPSH of filtrates was determined with DTNB (10). GSH solutions were also standardized by this method. ASSQ~ Procedure. Duplicate aliquots (1.5 ml) of the test solution containing up to 0.6 eole GSH in either neutral or acid (1% metaphosphoric acid) medium were frozen in w’ X 1” test tubes. Concentrated sulfuric acid (AR, 2.0 ml) was then added all at once and the ’ Abbreviations protein thiol.
used:
DTNB,
5,5’-dithiobis@nitrobenzoic 493
acid) ; NPSH,
non-
494
P.
C.
JOCELYN
tubes shaken in ice water until melting and mixing were complete (2-4 min). Not more than 10 min later one of the tubes was immersed in boiling water for 30 min, then cooled, while the other tube was kept at 0-20°C. Absorbances were determined at 265 rnp against a blank of 1.5 ml water (or lo/O metaphosphoric acid) and 2 ml sulfuric acid. The absorbance difference between the heated and unheated tubes was converted into GSH concentration by comparison with standards. Absorbantes are stable for at least 30 min. Values are given as the means of duplicate assays. RESULTS
Calvin (6) found that GSH gave an absorption peak at 265 rr+ in concentrated HCl. In quantities of up to 2 pmoles/ml, however, this peak develops only if the acid solution is first heated at 100°; even when heated the absorption increase is very small if more dilute acid is used (Table 1). No increase is obtained on replacing HCl with phosTABLE 1 Effect on Absorbance at 265 mp of Varying Conditions sampk.
state
Acid
Time between acidifying and incubating, min
Incubation time nt lOO”, min
HCl (12 N) HCl (6 N) HaPOd (9 M) HzSOc (10.5 M)
Nil Nil Nil Nil Nil 5 5 15
5 5 5 4 2 10 30 30
Liquid
Frozen
of Incubation Absorbance
0.66 .021 Nil 0.56 0.86 0.88 0.87 0.91
phoric acid but sulfuric acid (10-15 M) gives a large increase at 100’ and this has been used as the basis for an assay method. A suitable strength of acid (10.5 M) is obtained by adding 1 vol concentrated H&O, to 0.75 vol test solution. Maximum absorbance is then obtained by heating at 100”. Best results have been obtained after first dissipating the heat of solution by adding the acid to the frozen sample with ice cooling. No absorbance at 265 rnp develops if the solutions obtained in this way are kept at O-20” but there is a consistent increase if they are heated at 100’ for 10-30 min (Table 1). The unheated solution thus serves as a convenient blank. The absorption spectrum of the heated solution is not significantly different from that obtained by Calvin (6). If the acid concentration
GSH
ASSAY
IN
ACID
495
0*70.6 ;L
FIQ. 1. Variation of absorbance with aSO, concentration. Concentrated H80, w&9 added to the frozen solutions containing GSH (0.375 pmole) to give the required strength and the mixtures were then incubated at 100” for 30 min.
is reduced the absorbance developed is smaller and is insignificant below 5 M. An increase in the acid concentration from 10.5-13 M makes little difference to the absorbance but at higher concentrations it declines (Fig. 1). With 10.5M acid the absorbance is linear with GSH concentration (Fig. 2). The effect of other substances on the reaction is shown in Table 2. GSSG, cysteine, and oysteinylglycine develop no absorbance on heating themselves and do not affect that due to GSH. Glutamic acid gives a slightly decreased absorbance when heated and so does GSH pretreated with iodoacetic acid or iodoacetamide. The absorbance developed by GSH is also abolished by first adding DTNB to the neutral GSH solution. This reagent probably oxidizes GSH to GSSG (11). Cystine also considerably reduces the absorbance when added to GSH in neutral solution. This is due to thiol-disulfide exchange because, if cystine is added after acidifying the GSH solution, it then has no effect on the absorbance. The method can be used to estimate GSH in erythrocytes after hemolysis and precipitation of the proteins (Table 3). The difference between the absorbance of the heated and unheated protein-free solution after adding sulfuric acid is most probably due to the GSH content
496
P. C. JOCELYN
II.9-
0.2
O-3 0-L Ii SH AODEO, p moles
O-5
04
FIG. 2. Absorbance of GSH solutions in 3.5 ml 10.5 M eulfuric acid after incubation at the stated temperatures for 30 min. The technique is described in “Methods.”
because no difference is found when a neutralised solution is treated with DTNB prior to adding the acid. Values obtained for GSH by this method are not significantly different from those found for nonprotein thiol. The method (like the alloxan method) cannot be used to assay GSH present in rat liver tissues because of the high initial absorption of the extracts at 265 mp. DISCUSSION
The method described is not as sensitive as the alloxan method (3) and much less so than the recently described fluorometric method (4). These are the only specific purely chemical methods available. Its chief virtue is that its functions in acid solution and is specific for GSHcysteine, cysteinylglycine, GSSG, or S-alkyl derivatives of GSH do not give the reaction or interfere. The method can thus be used to assay GSH
491
QSH ASSAY IN ACID
TABLE 2 Relative Absorbance at 265 rnp of Some Substances Incubated in 10.5 A4 H&G, with or without GSH The difference in absorbance between the solution heated at 100” for 30 min or kept at 20” is given as a percentage of the difference found with a solution containing only GSH. % Change on heat& Substance
Nil GSSG Cyst&e Cystine Cystine Cysteinylglycine DTNB Glutamic acid Iodoacetamide Iodoacetic acid
I!&dbm~
%=”
1.2 2.4 0.4 0.4 0.5 0.25 0.5 1.2 1.2
Without
Water
Nil +1
HzSOd (0.1 M) Buffer* HzSO, (0.1 M) Water Buffer Water c c
-3 1-5 +5
With GSH (0.4 pmole)
GSH
+100 +102 +99 +49 +1os
+2 +2
+2
-8 -6 -7
-10 -12
0 Solutions in 1.5 ml were froeen and 2 ml concentrated H&Q added ae described in “Methods.” * 0.1 M phosphate, pH 7.4. c Iodoacetamide or iodoacetic acid and GSH in 0.05 M phosphate buf?er, pH 3.2 (0.5 ml), were kept at 37” until no sulfhydryi remained (15 min); then 1 M H&Q (l.Omi) was added.
TABLE 3 Assay of GSH and NPSH in Filtrates from Eythrocyte Hemolyzates (GSH and NPSH were determined as described in “Methods”)
Solution
GSH standard Fiitratec Filtrate Filtrate + DTNB” Filtrate f DTNB
‘%n? tempersture, ‘C
100 20 100 20 100
Found,* pmolea Ab%E-r
0.77 0.64 1.00 0.55 0.57
a
(0.01) (0.01) (0.015) (0.01) (0.01)
NPt3H 0.48 (0.005) 0.25 (0.005)
GSH 0.48 (0.005) 0.22 (0.02)
Nil Nil
8 All results are given as the mean and, in brackets, RD. from 4 readings. * 0.2 ml of DTNB (1 mole/ml) in 0.5 M phosphate, pH 7.4, was added to 1.3 & filtrate bsfore freezing. Absorbance was measured against an acid blank containing DTNB. c Contained in 1.5 ml of I:15 filtrate. Hence concentration per 106 ml of eryt,hrocytes; GSH, 220 (20) ; NPSH, 250 (5).
498
P. C. JOCELYN
directly in the presence of closely related thiols or disulfides, as in thiol-disulfide exchange reactions with cystine, IGSH synthesis from cysteine, or GSHJNPSH ratios in erythrocytes. SUMMARY
A method is described for assaying GSH by heating solutions in 10.5 M sulfuric acid at 100” and determining the absorbance developed at 265 ~JJ. Substances closely related to GSH do not interfere. The method can be used to assay GSH in erythrocytes. REFERENCES 1. WOODWARD, G. E., J. Bill, Chem. 109, 1 (1935). 2. LACK, L., AND SMITH, M., Anal. Biochem. 8, 217 (1954). 3. PATITU~SON, J. W., LAZAROW, A., AND LETY, S., J. Biol. Chem. 177, 187 (1949). 4. C%HN, V. H., AND LYLE, J., Anal. Biochem. 14, 434 (1966). 5. PIHL, A., ELDJARN, L., AND BREMER, J., J. Biol. Chem. 227, 339 (1957). 6. CALVIN, M., in “Gluthathione Symposium,” p. 3. Academic Press, New York, 1954. 7. PREUJX, G., AND LONTIE, R., B&hem. J. 66, 2&P (1957). 8. GARFINKEL, D., J. Amer. Chem. Sot. 80, 4837 (1958). 9. GVTCHO, M., AND LAUFEB, L., in “Gluthathione Symposium,” p. 79. Academic Press, New York, 1954. 10. BE-, E., DURON, O., AND KELLEY, B. M., J. Lab. Clin. Med. 61, 882 (1963). 11. OWN, C. W. I., AND BEL~HER, R. V., B&hem. J. 94, 705 (1965). 12. GOODMAN, I., AND SALcq L., Biochim. Biophys. Acta 109, 283 (1965).
BIOCHEZMISTBY
18,
49%i98
(1!367)
An Assay for Glutathione
in Acid Solution
P. C. JOCELYN Department
of Biochemistry,
New Buildings,
Teviot
Place, Edinburgh,
Scotland
Received October 21, 1966 The few known specific methods for assaying GSH, whether by enzymes (1, 2) or by purely chemical methods (3, 4)) all require a pH of 7-3. At this pH, the thiol is subject to oxidation. It also undergoes thiol-disulfide exchange (5) and so cannot be assayed accurately by existing methods in the presence of other disulfides. Since these reactions do not occur at low pH, a method of assay in acid solution would be useful. Calvin (6) found that, when GSH was treated with concentrated hydrochloric acid, the solution developed an ultraviolet absorption spectrum with a peak at 265 rnp. This was ascribed to the formation of a thiazoline ring by dehydration (6, 12) though it is uncertain (7, 8) whether this can be the correct explanation. The appearance of this absorption has now been made the basis of a method for assaying GSH in acid solution. MATERIALS
AND
METHODS
GSH, cystine, cysteine, and iodoacetic acid were obtained from British Drug Houses Ltd., GSSG from Boehringer Ltd., iodoacetamide from Kodak Ltd., Liverpool, and DTNB’ from Aldrich Chemical Co., Milwaukee, Wisconsin. Cysteinylglycine was prepared according to Gutcho and Laufer (9) . Erythrocytes obtained from fresh oxalated blood were washed once with saline and hemolyzed with 12.5 vol water, and the proteins were precipitated by addition of 1.5 vol 10% metaphosphoric acid. NPSH of filtrates was determined with DTNB (10). GSH solutions were also standardized by this method. ASSQ~ Procedure. Duplicate aliquots (1.5 ml) of the test solution containing up to 0.6 eole GSH in either neutral or acid (1% metaphosphoric acid) medium were frozen in w’ X 1” test tubes. Concentrated sulfuric acid (AR, 2.0 ml) was then added all at once and the ’ Abbreviations protein thiol.
used:
DTNB,
5,5’-dithiobis@nitrobenzoic 493
acid) ; NPSH,
non-
494
P.
C.
JOCELYN
tubes shaken in ice water until melting and mixing were complete (2-4 min). Not more than 10 min later one of the tubes was immersed in boiling water for 30 min, then cooled, while the other tube was kept at 0-20°C. Absorbances were determined at 265 rnp against a blank of 1.5 ml water (or lo/O metaphosphoric acid) and 2 ml sulfuric acid. The absorbance difference between the heated and unheated tubes was converted into GSH concentration by comparison with standards. Absorbantes are stable for at least 30 min. Values are given as the means of duplicate assays. RESULTS
Calvin (6) found that GSH gave an absorption peak at 265 rr+ in concentrated HCl. In quantities of up to 2 pmoles/ml, however, this peak develops only if the acid solution is first heated at 100°; even when heated the absorption increase is very small if more dilute acid is used (Table 1). No increase is obtained on replacing HCl with phosTABLE 1 Effect on Absorbance at 265 mp of Varying Conditions sampk.
state
Acid
Time between acidifying and incubating, min
Incubation time nt lOO”, min
HCl (12 N) HCl (6 N) HaPOd (9 M) HzSOc (10.5 M)
Nil Nil Nil Nil Nil 5 5 15
5 5 5 4 2 10 30 30
Liquid
Frozen
of Incubation Absorbance
0.66 .021 Nil 0.56 0.86 0.88 0.87 0.91
phoric acid but sulfuric acid (10-15 M) gives a large increase at 100’ and this has been used as the basis for an assay method. A suitable strength of acid (10.5 M) is obtained by adding 1 vol concentrated H&O, to 0.75 vol test solution. Maximum absorbance is then obtained by heating at 100”. Best results have been obtained after first dissipating the heat of solution by adding the acid to the frozen sample with ice cooling. No absorbance at 265 rnp develops if the solutions obtained in this way are kept at O-20” but there is a consistent increase if they are heated at 100’ for 10-30 min (Table 1). The unheated solution thus serves as a convenient blank. The absorption spectrum of the heated solution is not significantly different from that obtained by Calvin (6). If the acid concentration
GSH
ASSAY
IN
ACID
495
0*70.6 ;L
FIQ. 1. Variation of absorbance with aSO, concentration. Concentrated H80, w&9 added to the frozen solutions containing GSH (0.375 pmole) to give the required strength and the mixtures were then incubated at 100” for 30 min.
is reduced the absorbance developed is smaller and is insignificant below 5 M. An increase in the acid concentration from 10.5-13 M makes little difference to the absorbance but at higher concentrations it declines (Fig. 1). With 10.5M acid the absorbance is linear with GSH concentration (Fig. 2). The effect of other substances on the reaction is shown in Table 2. GSSG, cysteine, and oysteinylglycine develop no absorbance on heating themselves and do not affect that due to GSH. Glutamic acid gives a slightly decreased absorbance when heated and so does GSH pretreated with iodoacetic acid or iodoacetamide. The absorbance developed by GSH is also abolished by first adding DTNB to the neutral GSH solution. This reagent probably oxidizes GSH to GSSG (11). Cystine also considerably reduces the absorbance when added to GSH in neutral solution. This is due to thiol-disulfide exchange because, if cystine is added after acidifying the GSH solution, it then has no effect on the absorbance. The method can be used to estimate GSH in erythrocytes after hemolysis and precipitation of the proteins (Table 3). The difference between the absorbance of the heated and unheated protein-free solution after adding sulfuric acid is most probably due to the GSH content
496
P. C. JOCELYN
II.9-
0.2
O-3 0-L Ii SH AODEO, p moles
O-5
04
FIG. 2. Absorbance of GSH solutions in 3.5 ml 10.5 M eulfuric acid after incubation at the stated temperatures for 30 min. The technique is described in “Methods.”
because no difference is found when a neutralised solution is treated with DTNB prior to adding the acid. Values obtained for GSH by this method are not significantly different from those found for nonprotein thiol. The method (like the alloxan method) cannot be used to assay GSH present in rat liver tissues because of the high initial absorption of the extracts at 265 mp. DISCUSSION
The method described is not as sensitive as the alloxan method (3) and much less so than the recently described fluorometric method (4). These are the only specific purely chemical methods available. Its chief virtue is that its functions in acid solution and is specific for GSHcysteine, cysteinylglycine, GSSG, or S-alkyl derivatives of GSH do not give the reaction or interfere. The method can thus be used to assay GSH
491
QSH ASSAY IN ACID
TABLE 2 Relative Absorbance at 265 rnp of Some Substances Incubated in 10.5 A4 H&G, with or without GSH The difference in absorbance between the solution heated at 100” for 30 min or kept at 20” is given as a percentage of the difference found with a solution containing only GSH. % Change on heat& Substance
Nil GSSG Cyst&e Cystine Cystine Cysteinylglycine DTNB Glutamic acid Iodoacetamide Iodoacetic acid
I!&dbm~
%=”
1.2 2.4 0.4 0.4 0.5 0.25 0.5 1.2 1.2
Without
Water
Nil +1
HzSOd (0.1 M) Buffer* HzSO, (0.1 M) Water Buffer Water c c
-3 1-5 +5
With GSH (0.4 pmole)
GSH
+100 +102 +99 +49 +1os
+2 +2
+2
-8 -6 -7
-10 -12
0 Solutions in 1.5 ml were froeen and 2 ml concentrated H&Q added ae described in “Methods.” * 0.1 M phosphate, pH 7.4. c Iodoacetamide or iodoacetic acid and GSH in 0.05 M phosphate buf?er, pH 3.2 (0.5 ml), were kept at 37” until no sulfhydryi remained (15 min); then 1 M H&Q (l.Omi) was added.
TABLE 3 Assay of GSH and NPSH in Filtrates from Eythrocyte Hemolyzates (GSH and NPSH were determined as described in “Methods”)
Solution
GSH standard Fiitratec Filtrate Filtrate + DTNB” Filtrate f DTNB
‘%n? tempersture, ‘C
100 20 100 20 100
Found,* pmolea Ab%E-r
0.77 0.64 1.00 0.55 0.57
a
(0.01) (0.01) (0.015) (0.01) (0.01)
NPt3H 0.48 (0.005) 0.25 (0.005)
GSH 0.48 (0.005) 0.22 (0.02)
Nil Nil
8 All results are given as the mean and, in brackets, RD. from 4 readings. * 0.2 ml of DTNB (1 mole/ml) in 0.5 M phosphate, pH 7.4, was added to 1.3 & filtrate bsfore freezing. Absorbance was measured against an acid blank containing DTNB. c Contained in 1.5 ml of I:15 filtrate. Hence concentration per 106 ml of eryt,hrocytes; GSH, 220 (20) ; NPSH, 250 (5).
498
P. C. JOCELYN
directly in the presence of closely related thiols or disulfides, as in thiol-disulfide exchange reactions with cystine, IGSH synthesis from cysteine, or GSHJNPSH ratios in erythrocytes. SUMMARY
A method is described for assaying GSH by heating solutions in 10.5 M sulfuric acid at 100” and determining the absorbance developed at 265 ~JJ. Substances closely related to GSH do not interfere. The method can be used to assay GSH in erythrocytes. REFERENCES 1. WOODWARD, G. E., J. Bill, Chem. 109, 1 (1935). 2. LACK, L., AND SMITH, M., Anal. Biochem. 8, 217 (1954). 3. PATITU~SON, J. W., LAZAROW, A., AND LETY, S., J. Biol. Chem. 177, 187 (1949). 4. C%HN, V. H., AND LYLE, J., Anal. Biochem. 14, 434 (1966). 5. PIHL, A., ELDJARN, L., AND BREMER, J., J. Biol. Chem. 227, 339 (1957). 6. CALVIN, M., in “Gluthathione Symposium,” p. 3. Academic Press, New York, 1954. 7. PREUJX, G., AND LONTIE, R., B&hem. J. 66, 2&P (1957). 8. GARFINKEL, D., J. Amer. Chem. Sot. 80, 4837 (1958). 9. GVTCHO, M., AND LAUFEB, L., in “Gluthathione Symposium,” p. 79. Academic Press, New York, 1954. 10. BE-, E., DURON, O., AND KELLEY, B. M., J. Lab. Clin. Med. 61, 882 (1963). 11. OWN, C. W. I., AND BEL~HER, R. V., B&hem. J. 94, 705 (1965). 12. GOODMAN, I., AND SALcq L., Biochim. Biophys. Acta 109, 283 (1965).