Glutathione (GSH) is the major low molecular weight thiol in most eukaryotic and many prokaryotic cells and is regarded as one of the cell’s first lines of defense against several oxidative insults [1
]. In healthy non-stressed cells, usually nearly all of the GSH in the cytosolic glutathione pool is in the reduced form, reflecting the highly reducing environment of that cell compartment. Indeed, recent estimates using reduction-potential-dependent fluorescent protein probes suggest a cytosolic EGSH
of <−320 mV in yeast. Assuming a cytosolic [GSH] of 10 mM would mean a ratio of GSH:GSSG of 50,000:1 [3
The dedicated biosynthetic pathway to GSH consists of two steps. The enzyme γ-glutamylcysteine synthetase (GSH1) catalyzes the coupling of glutamate with cysteine and the γ-glutamylcysteine product is coupled with glycine by glutathione synthetase (GSH2) to make the final product. Cells deleted for the GSH1
gene must be supplemented with GSH for survival and growth. Upon oxidative stress, caused for example by reactive oxygen species, GSH is oxidized to its dimeric form glutathione disulfide GSSG (Scheme 1
). The reaction with H2
is rather slow and in vivo is catalysed by enzymes called peroxiredoxins [4
]. Furthermore, under oxidative stress conditions, cysteine thiols in proteins may be reversibly glutathiolated to protect them from over-oxidation to sulfenic and sulfinic acid residues [6
]. The GSH pool within the cell is restored by the reduction of GSSG to GSH by the NADPH-dependent enzyme glutathione reductase (GR, E.C. 1.8.17) (Scheme 2
] whereas glutathiolated proteins are usually reduced by glutaredoxins [7
A GR/NADPH-dependent cycling assay was developed to determine [GSH] and [GSSG], whereby GSH is oxidized to GSSG by 5,5′-dithiobis-2-nitrobenzoate (DTNB) in the presence of excess enzyme (Scheme 3
]. The 2-nitro-5-thiobenzoate (TNB) formed can be measured spectrophotometrically (λmax
= 412 nm) and its rate of production depends upon the rate of GSH formation due to GR activity with GSSG as substrate. In contrast, under conditions of substrate excess, the rate of TNB production is limited by the amount of GR activity present and the assay can be used to measure GR activity.
Allicin (diallylthiosulfinate) is produced in garlic when cells are damaged and the enzyme alliinase mixes with its substrate alliin which is separately compartmentalized in the cell. A single clove of garlic can produce up to 5 mg of allicin [9
], which is the first and major volatile sulfur compound produced and gives fresh garlic its characteristic odor. Allicin was identified as the major antimicrobial substance produced by garlic [10
] and it was shown to oxidize and deplete the cellular GSH pool, reacting with GSH to yield S
-allylmercaptoglutathione (GSSA) (Scheme 4
). The thiosulfinate group in allicin reacts readily with thiols, particularly in the thiolate ion form, without the need for enzymic catalysis [12
]. Furthermore, allicin can react with accessible cysteine thiols in proteins by S
GSH and GR have been shown to play an important role in the resistance of cells against allicin [16
]. GR replenishes the GSH pool from GSSG and this protective effect would be strengthened if GSSA were also a substrate for GR. In this case one mol GSSA would be reduced to one mol each of GSH and allylmercaptan (ASH) (Scheme 5
). The goal of this investigation was to clarify whether GSSA can serve as a substrate for GR and commercially available GR from yeast was chosen for this purpose. However, the DTNB assay is not suitable to assay for GR activity with GSSA as a substrate because any GSH formed by GR action on GSSA would be oxidized to GSSG by the DTNB and this would enter into the cycling reaction and lead to complicated mixed-substrate kinetics. Therefore, in order to test whether GSSA is a substrate for GR it is necessary to leave out the DTNB reagent and measure the disappearance of NADPH + H+
, which can be observed as a reduction in A340
over time. In the work reported here we show that indeed GSSA is a substrate for GR from yeast, but has a higher Km
and therefore a lower substrate affinity with the enzyme than GSSG.
2. Materials & Methods
2.1. Allicin Synthesis
Allicin was synthesized as previously described by the formic acid-catalyzed oxidation of redistilled diallyl disulfide by hydrogen peroxide [18
2.2. Synthesis and LC-MS of S-allylmercaptoglutathione
S-allylmercaptoglutathione was synthesized after a method modified from Miron et al. [12
]. GSH (400 mg dissolved in 5 mL distilled H2
O) were added dropwise with stirring to 130 mg allicin dissolved in 2 mL 50% (v
) methanol and stirred for a further 2 h at room temperature. The white precipitate (yield 450 mg) was washed repeatedly with dichloromethane and the product gave a single peak which eluted from HPLC at 5.8 min (conditions as in [18
The identity of the product was confirmed by electrospray ionization mass spectrometry (ESI-MS). Measurements were carried out on a Thermo Fisher Scientific Orbitrap XL (in high resolution mode using methanol/water (50%/50%) with 0.1 mM acetic acid.
2.3. Glutathione Reductase Assay
Yeast glutathione reductase was purchased from Sigma-Aldrich GmbH, Steinheim, Germany. The GR enzyme solution (20 U mL−1) was prepared in 143 mM sodium phosphate buffer (pH 7.5) containing 6.3 mM Na2EDTA. DTNB (5,5’-dithiobis-(2-nitrobenzoic acid), 6 mM) and NADPH (0.3 mM) were prepared in phosphate buffer. The assay mix for GSSG with DTNB present was as follows: 12.5 µL substrate solution (1.0, 0.5, 0.25, 0.1 mM), 5 µL GR solution, 50 µL DTNB solution, 350 µL NADPH solution and 332.5 µL H2O. The assay mix for GSSA without DTNB present was as follows: 12.5 µL substrate solution (7.5, 5.0, 4.5, 3.0 mM), 5 µL GR solution, 350 µL NADPH solution and 382.5 µL H2O. Substrate concentration ranges giving linear kinetics were established at enzyme excess ensuring the rate of substrate conversion was proportional to the substrate concentration.
The haploid Saccharomyces cerevisiae
yeast strain BY4742 (Matα; his3∆1; leu2∆0, lys2∆0, ura3∆0) was used. The BY4742 mutant ∆gsh1
(Y17097) used in this study lacks gene for γ-glutamylcysteine synthetase (YJL101C) which catalyzes the first step in glutathione biosynthesis. The mutant was obtained from the EUROSCARF Collection, University of Frankfurt (Main), Germany (http://www.euroscarf.de/
Yeast was grown in complete synthetic mixture (CSM) medium (0.79 g L−1 CSM Drop-Out: Complete [ForMedium, Norwich, United Kingdom]; 6.9 g L−1 Yeast Nitrogen Base [ForMedium, Norwich, United Kingdom]; 40 g L−1 D-Glucose [Carl Roth, Karlsruhe, Germany], 15 g L−1 agar for solid medium.