ELECTROLYTIC REDUCTION AND DETERMINATION OF OXIDIZED GLUTATHIONE BY JANETTA SCHOOPU‘OVER DOHAN AND GLADYS E. WOODWARD (From The Biochemical Research Foundation of the Franklin Institute, Philadelphia) (Received for publication, May 13, 1939) Oxidized glutathione (GSSG) usually is det,ermined in its sulf- hydryl form (GSH) after reduction. The accuracy of its de- termination therefore depends upon (1) the completeness of re- duction, and (2) the specificity of the method used for estimation of GSH. A specific method for determining GSH based upon the activat- ing effect of the latter on the enzyme glyoxalase was described previously by one of us (1). At that t,ime, the procedure could not be applied to oxidized glutathione since no means was known for reducing the oxidized to the reduced form without introducing materials which were toxic to the enzyme. This difficulty now has been overcome. Electrolytic reduction in an acid medium with a mercury cathode has been found to change GSSG to GSH rapidly and completely; the resulting solution has been found to be suitable for GSH estimation by the specific glyoxalase method (1, 2) as well as by the more common but less specific iodometric pro- cedures. The electrolytic reduction of GSSG is complete even in the presence of protein-free filtrates of biological material; this is not the case with t,he Zn reduction used by many investigators. Quensel and Wachholder (3) after adding GSSG to blood or tissues were able to recover only a small part of it after Zn reduction. They assumed that GSSG was bound to the protein and did not pass into the filtrate. Electrolytic reduction, however, shows that all of the added GSSG was in the filtrate. There appears to be some condition of the filtrate which leads to inhibition of the Zn reduction. The observation of Oberst (4) that GSH added to 393 by guest on April 10, 2018 http://www.jbc.org/ Downloaded from
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ELECTROLYTIC REDUCTION AND DETERMINATION OF OXIDIZED GLUTATHIONE
BY JANETTA SCHOOPU‘OVER DOHAN AND GLADYS E. WOODWARD
(From The Biochemical Research Foundation of the Franklin Institute, Philadelphia)
(Received for publication, May 13, 1939)
Oxidized glutathione (GSSG) usually is det,ermined in its sulf- hydryl form (GSH) after reduction. The accuracy of its de- termination therefore depends upon (1) the completeness of re- duction, and (2) the specificity of the method used for estimation of GSH.
A specific method for determining GSH based upon the activat- ing effect of the latter on the enzyme glyoxalase was described previously by one of us (1). At that t,ime, the procedure could not be applied to oxidized glutathione since no means was known for reducing the oxidized to the reduced form without introducing materials which were toxic to the enzyme. This difficulty now has been overcome. Electrolytic reduction in an acid medium with a mercury cathode has been found to change GSSG to GSH rapidly and completely; the resulting solution has been found to be suitable for GSH estimation by the specific glyoxalase method (1, 2) as well as by the more common but less specific iodometric pro- cedures.
The electrolytic reduction of GSSG is complete even in the presence of protein-free filtrates of biological material; this is not the case with t,he Zn reduction used by many investigators. Quensel and Wachholder (3) after adding GSSG to blood or tissues were able to recover only a small part of it after Zn reduction. They assumed that GSSG was bound to the protein and did not pass into the filtrate. Electrolytic reduction, however, shows that all of the added GSSG was in the filtrate. There appears to be some condition of the filtrate which leads to inhibition of the Zn reduction. The observation of Oberst (4) that GSH added to
plasma, serum, or hemolyzed blood disappeared and could not be recovered even after Zn reduction may be explained on the same basis. GSH added to plasma or serum rapidly becomes oxidized, and in the oxidized form would not be recovered by this method. It is entirely recovered after electrolytic reduction.
By the combined use of electrolytic reduction and the glyoxalase method of estimation, no oxidized glutathione has been found in sulfosalicylic acid extracts of blood or tissues.
Electrolytic Reduction Procedure
Fig. 1 shows a diagram of the reduction system. Direct current is supplied from any convenient source (E).
The voltage is regulated by a slide-wire potentiometer (P) and the current passing through the electrolytic circuit is measured by the milliammeter (M). Connection is made with the cathode, a layer of mercury in the bottom of a small beaker or test-tube (C), by means of a platinum wire electrode dipping below the surface of the mercury. Over the mercury is placed the solution to be reduced. The mercury or mercury-solution interface is stirred mechanically by means of a small glass paddle stirrer. A KCl-agar salt bridge (B) is used to make connection with the anode compartment (A) which contains 4 per cent sulfosalicylic acid. Into the acid dips the anode itself, another platinum wire electrode. Connection with the potentiometer then completes the circuit.
The current necessary is dependent upon the area of the mercury surface. Current densities of about 2 to 4 milliamperes per sq. cm. (calculated from the cross-section of the cathode vessel) have been found most suitable. Since current density = current + area, less current will be needed for a small cathode vessel than for a larger one. In the system described, a current of greater than 40 milliamperes never is required. The resistance of the circuit is such that this current is produced by about 35 volts. The current may be supplied from radio B batteries, but direct current from a supply line, or alternating current which has passed through a rect’ifier, is preferable. A potentiometer having a resistance of about 1800 ohms with a capacity of 0.5 ampere will be required to regulate the current as described.
The cathode vessel should be chosen so that the depth of solu-
tion to be reduced is about 0.5 to 0.7 cm. This requirement is met by using approximately 10 cc. in a 100 cc. beaker (4.6 cm. diameter), 5 cc. in a 30 cc. beaker (3.2 cm. diameter), or 2.5 cc. in a 22 mm. test-tube. With more liquid the reduction is very slow. With less liquid it is difficult to immerse the end of the salt bridge completely without making direct contact with the mercury. The amount of mercury used as the cathode must be sufficient to cover all of the exposed wire of the platinum electrode.
@Eq h\\\\w
FIG. 1. Apparatus for electrolytic reduction of GSSG
Stirring is an essential factor. Since reduction occurs only at the mercury-solution interface, it will be more efficient the more rapidly t,he layer of solution at the mercury surface is changed. The stirrer is placed just below the surface of the mercury, in- stead of in the solution, in order to avoid striking the platinum electrode or the salt bridge. Stirring of the mercury agitates the solution sufficiently. The speed of stirring should be as rapid as possible without breaking up the surface of the mercury into small globules. When t,his occurs, reduction is seriously impaired. Any stirring unavoidably increases the surface area of the mercury, but this effect is disregarded, as is customary, in the calculation of current densities.
Salt bridges are made as usual from 1 per cent agar in saturated KCl. The bore of the tubing used should be 3 to 4.5 mm. For 40 milliamperes a bridge of at least 4.5 mm. bore is desirable. This will become slightly warm during passage of current, while with smaller bore the higher resistance causes enough heat to melt the agar. However, a bridge of 3 mm. bore suffers no heating when the current is 16 milliamperes or less, and is more convenient to use when the cathode vessel is the 22 mm. tube. Eventually, the cathode end of the salt bridge may collect a harmless white deposit. Also the agar disappears slowly from this end, but the empty portion need be cut off only when the space is sufficient for a bubble of hydrogen to collect and decrease the current.
The purpose of the 4 per cent sulfosalicylic acid in the anode compartment is merely to make connection with the anode. With continued use this acid becomes yellow and finally brown, after which it should be replaced.
In actual practice, all of the apparatus remains assembled according to the diagram (Fig. 1) except the cathode vessel and salt bridge, which are chosen each time according to the volume of solution to be reduced (Table II). For each reduction, the proper amount of mercury and the solution to be treated are placed in the cathode vessel; when the latter and the salt bridge are placed in position, as shown in Fig. 1, the circuit becomes complete. Stirring is started and adjusted to the proper speed. The current is turned on, regulated by the potentiometer, and allowed to flow through the circuit, for a suitable time. After current and stirring have been stopped, the solution is pipetted off and analysis for GSH is made directly on the solution without further treatment. The mercury and salt bridge are washed with distilled water and dried with filter paper between reductions.
EXPERIMENTAL
E$ect of Acidity and GSSG Concentration on Degree of Reduction -Sulfosalicylic acid had been shown previously to be an ideal medium in which estimations of reduced glutathione could be made. The acid protected against autoxidation of GSH (5), and such acid extracts of tissues, when neutralized, were not toxic to glyoxalase (1). Furthermore, no toxicity has been found to develop in acid submitted to the electrolytic reduction process.
The degree of reduction was found to be equally satisfactory in all of the acid concentrations used, 1.8 to 3.4 per cent. A slightly
1 The oxidized glutathione was prepared from a pure sample of reduced glutathione by the method of Pirie (6). The purity of the oxidized sample was established by iodometric (5) estimation after Zn reduction in 2 per cent sulfosalicylic acid solution.
higher acid concentration than the usual 2 per cent (about 2.3 per cent) is preferable because the reduction causes a decrease in the acidity of the medium. Otherwise, when the reduction is stopped, reoxidation of the GSH is likely to occur.
Concentrations of GSSG up to 50 mg. per cent were quantita- tively reduced in t.he blood and tissue filtrates as well as in the plain acid. Therefore, the method should enable the determina- tion of any amount of GSSG which might occur naturally in the usual extracts of blood or tissues.
TIME (MIN.)
FIG. 2. Reduction of 50 mg. per cent GSSG in 1:2 serum filtrate with different current densities. 0 represents current density of 4.2 milli- amperes per sq. cm.; X, 2.1 milliamperes per sq. cm.; 0, 1.05 milliamperes per sq. cm.
Relation of Current Density and Time to Amount of Reduction- Successive 5 cc. samples of 50 mg. per cent GSSG in a 1: 2 serum filtrate, made with sulfosalicylic acid in such a way that the acid concentration of the filtrate was 2.75 per cent, were reduced in a 30 cc. beaker as the cathode vessel, with a 4 mm. salt bridge. The extent of reduction was determined by iodate titration, since it had been shown previously that such titrations give reliable figures for GSH in blood filtrates, agreeing with those obtained by the specific glyoxalase method (1). The results obtained by variation of the time of reduction and the current density are plotted in Fig. 2.
Fig. 2 shows that the reduction rate is nearly proportional to the
current density. Treatment continued after the reduction is complete does not cause, in general, any loss of GSH within 60 minutes, even with the highest current density. The GSH value also remains constant with GSH and GSSG in plain 2.75 per cent sulfosalicylic acid and with GSSG in a 1: 5 liver filtrate containing 1.8 per cent sulfosalicylic acid. Determinations for GSH in t,he liver filtrate were made by the manometric glyoxalase method because of the inaccuracy of iodometric procedures in tissue extracts.
In contrast to serum or tissue filtrates, plasma filtrates contain- ing GSSG may not be submitted safely to the electrolytic treat- ment much beyond the minimum time required for complete re- duction, unless the GSH so obtained is measured manometrically.
TABLE II
Standard Conditions for Electrolytic Reduction of GSSG
cc. cm.
2.5 2.2 5 3.2
10 4.6
__-
mm.
3 4 4.5
s
r:
Cathode urface area :calculated ~omca;;od~
diameter)
sq. cm.
3.8 8.04
16.6
Current
mnilliamperes
16 34 40
Time of reduction
I----
milliamperas min per sp. em.
4.2 10 4.2 10 2.4 20
When the reduction proceeds longer, the titration value begins to decrease. This phenomenon is attributable to the interference of the oxalate used as an anticoagulant. Oxalate added to pure sulfosalicylic acid solutions of GSSG has the same effect. It is conceivable that, in time, a carboxyl group on the oxalic acid is reduced to an aldehyde (7) which combines with the sulfhydryl group of the reduced glutathione (8). The titration value is lowered thereby, but for some reason the glyoxalase reaction is not affected.
In view of these results, the foregoing sets of conditions have been adopted as standard procedure (Table II).
GSSG Content of Blood and Tissues-Blood and tissue extracts were made with sulfosalicylic acid as previously reported (1). For the reduction of the blood filtrate, 5 cc. were used in a 30 cc.
beaker with 34 milliamperes; for the tissue filtrates, 1.5 to 2.5 cc. in a 22 mm. tube with 16 milliamperes. A reduction time of 10 minutes was used. Estimations of GSH were made by the mano- metric glyoxalase method.
Table III shows that approximately the same GSH content of the filtrates was found after reduction as before, indicating that none of these tissues or blood contains any oxidized glutathione. This is in distinct disagreement with the work which Fujita and Numata (9) recently reported. Their method, consisting of re- duction by H&S and removal of ascorbic acid by ascorbic acid
oxidase, followed by iodometric titration, indicated that up to 63 per cent of the total glutathione in blood was in the oxidized form. Considerable amounts were also found in most tissues. There is some likelihood that the increases in titration which these authors found after reduction, and which they attributed to GSSG, are due to incomplete removal of H&S. The presence of traces of H8 in filtrates reduced by their method was suggested when these filtrates were submitted to analysis for GSH by the glyoxalase method. The GSH value in such filtrates was always lower than before reduction, and filtrates with added GSH showed incomplete recovery of the GSH corresponding to the toxic effect of about
0.00005 N HZS on glyoxalase. This amount of H,S in the dilute (1:20) filtrates can consume enough iodine to produce a tre- mendous error when the data are calculated as mg. of GSSG per 100 gm. of tissue.
E$ect of Electrolytic Reduction on Cystine and Dehydroascorbic Acid-2 cc. of 20 mg. per cent cystine or dehydroascorbic acid in 3 per cent sulfosalicylic acid were submitted to reduction in the 22 mm. cathode tube with a current of 8 milliamperes (current density 2.1). In 10 minutes the cystine was reduced quantita- tively, while with dehydroascorbic acid no reduction had taken place in 20 minutes.
Comparison of Zn and Electrolytic Reduction of GXSG-Reduc- tion by Zn in acid solution has been a common method for con- version of GSSG to GSH. Several investigators (3-5) haveshown that in plain sulfosalicylic acid solution the reduction was quanti- tative. However, Quensel and Wachholder (3) were able to re- cover as GSH after Zn reduction only a small part of GSSG added to blood or tissue. They believed that the GSSG was bound in some way by the proteins and did not pass into the filtrate, al- though added GSH was entirely filt.rable. Our experiments in- dicate that GSSG is also completely filtrable. As shown in Table I, GSSG could be added directly to blood plasma or serum and recovered quantitatively as GSH after electrolytic reduction of the filtrate. In substantial agreement with Quensel and Wach- holder, we were able to recover only about 35 per cent of the added GSSG by Zn reduction of the same filtrate. Therefore, it is shown that the added GSSG is not bound to t,he proteins, that it is com- pletely filtrable, but that Zn, in the presence of the plasma or serum filtrate, is not able to bring about complete reduction of the GSSG present.
Oberst (4) has observed a similar phenomenon which he called “the phenomenon of disappearance of glutathione (total) from blood.” On addition of GSH to plasma or serum, the GSH was rapidly lost, and he was able to recover only a small part of the material after Zn reduction of the protein-free filtrate. It ap- peared, therefore, that the loss was due to some cause other than oxidation, apparently associated with the plasma proteins. How- ever, by electrolytic reduction of such a filtrate, we have been able to recover all of the original GSH with incubation periods
up to 3 hours (Table IV), even when the GSH had disappeared completely from the incubation mixture. It was also observed (Experiment C, Table IV) that when GSH and serum or plasma were allowed to incubate in the absence of oxygen, no loss of GSH occurred in short periods of time. Furthermore, when the pH of the mixture was lowered by buffering (Experiment A, Table IV), the loss of the GSH in the presence of oxygen was considerably less than at a higher pH where, as is known, autoxidation of GSH readily occurs. The disappearance of glutathione, therefore, seems to be due entirely to oxidation, and the phenomenon with
TABLE IV
Disappearance and Recovery after Electrolytic Reduction of GSH
Added to Blood Plasma
The values represent the per cent found before and after reduction.
Aerobic incubation
I Time of
incubation (25”)
Experiment A, 100 mg. Experiment B. 50 mg. per cent GSH in 1:2 plasma per cent GSH in 1: 2 plasma
(buffered to pH 7) (unbuffered, pH 7.8 to 8.6)
Before After Before After ____
min.
30 93 101 60 84 101 39
120 61 97 9 240 2 99
Anaerobic incubation
Experiment C, l~y$gg
in 1: 2 serum (unbuffered,
PH 7.7)
Before
100
which Oberst was concerned was the inability of Zn to reduce GSSG completely in the presence of plasma or serum constituents which had also passed into the acid filtrates.
Zn t’reatment, however, will completely reduce GSSG in plasma or in red blood cell filtrates which are obtained by the use of tung- state in conjunction with sulfosalicylic acid for the protein pre- cipitation. Not, only is the reduction of GSSG added to filtrates or to the original material complete, but also the evolut;ion of hydrogen from the Zn is increased enormously, thus creating a desirable stirring effect. The proportions and amounts of tung- state and sulfosalicylic acid are different for plasma and red blood cells, and no compromise could be found that was successful in
the case of whole blood. As the Zn reacts with the filtrate, the tungstate is reduced, and a blue color is produced which turns brown in a few minutes. When the proper amount of tungstate has been used the color is not deep enough to interfere with the titration procedure, and usually disappears in an hour without any accompanying reoxidation of GSH. However, the color formation is objectionable, and, upon consideration of the facts that the glyoxalase method cannot be used after Zn treatment, and that the technique cannot be applied to whole blood, the procedure appears to be of no great practical value. It is of interest, how- ever, that a filtrate prepared by a method which is supposed to free it of polypeptides as well as of proteins is entirely susceptible to Zn reduction.
SUMMARY
1. ,4n electrolytic method for reduction of GSSG to GSH in acid solution at a mercury cathode is described. The GSH so formed may be estimated by the specific glyoxalase method, thus providing a specific method for the estimation of GSSG.
2. GSSG added to sulfosalicylic acid extracts of blood and tis- sues, or to the blood constituents directly, was completely re- covered as GSH in the protein-free filtrates after electrolytic reduction. Reduction of GSSG in these filtrates by Zn was only partial.
3. No GSSG was found to occur naturally in blood or in tissues.
BIBLIOGRAPHY
1. Woodward, G. E., J. Biol. Chem., 109, 1 (1935). 2. Schroeder, E. F., and Woodward, G. E., J. Biol. Chem., 129, 283 (1939). 3. Quensel, W., and Wachholder, K., 2. physiol. Chem., 231, 65 (1934). 4. Oberst, F. W., J. Biol. Chem., 111, 9 (1935). 5. Woodward, G. E., and Fry, E. G., J. Biol. Chem., 97, 465 (1932). 6. Pirie, N. W., Bioehem. J., 26, 614 (1931). 7. Nakata, H., Anniversary volume dedicated to Masumi Chikashige,
