2.1 Chemicals and biochemicals

Catalase (bovine liver), digitonin, and GSSG reductase (Baker's yeast) were from Fluka (Buchs, Switzerland). Glucose oxidase and NADPH were from Boehringer (Mannheim, Germany). DTPA, GSH, H₂O₂, NaN₃, and Triton X-100 were from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.

2.2 Cell culture

Jurkat cells obtained from ATCC (clone E6-1) were cultured in complete medium (RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine, and antibiotics from Life Technologies (Rockville, MD, USA). Cells were incubated at 37°C in humidified air with 5% CO₂, and kept in logarithmic phase by routine passage every 2 days. Before use, cells were spun down, resuspended in fresh medium at 1×10⁶ cells/ml, and incubated for at least 1 h. Cell viability was determined by propidium iodide uptake.

2.3 Biochemical measurements

H₂O₂ was measured with an oxygen electrode following the addition of catalase, which caused a rapid conversion of H₂O₂ to H₂O and O₂, the release of the latter being monitored by the oxygen electrode. With a clean and stable electrode, H₂O₂ concentrations as low as 5 μM could be measured. A calibration curve was made with H₂O₂ standards before each experiment. Catalase activity was measured as previously described [9] in 0.5 M potassium phosphate buffer, pH 7.0, containing 0.01% digitonin in the presence of 1×10⁶ cells. H₂O₂ (10 mM initial concentration) consumption was followed at 240 nm at room temperature for 2 min (ϵ ₂₄₀=43.4 M⁻¹ cm⁻¹). Alternatively, catalase activity was measured with a cell lysate. H₂O₂ concentrations were plotted on a semi-logarithmic graph against time and the first-order rate constant (catalase activity) was calculated. Titration of catalase activity with digitonin was carried out in a similar manner, but digitonin was added using dimethyl sulfoxide (DMSO) as a vehicle (corrections accounting for the slight inhibition of catalase activity by DMSO at the level added (1% v/v) were considered). Glutathione peroxidase (GPx) activity was measured by studying the kinetics of the enzyme in the whole cell applying a well-established method [10]. The assay mixture contained (final concentrations): 10×10⁶ cells/ml, 0.05 M potassium phosphate buffer, pH 7.0, 1 mM DTPA, 50 μM NaN₃, 1.1 U/ml glutathione reductase, 0.1 mM NADPH, 35 μM H₂O₂, and 1% (v/v) Triton X-100; the concentration of GSH varied between 0.335 and 3.35 mM. Alternatively, glutathione peroxidase activity was measured on a cell lysate without the addition of Triton X-100 to the assay mixture. All reactants, with the exception of H₂O₂, were pre-incubated at 37°C for 10 min. NADPH consumption was followed at 340 nm (ϵ=6.2×10³ M⁻¹ cm⁻¹) at 37°C until all the hydroperoxide was used, recording the absorbance every 0.1 s. For the kinetic analysis, the part of the curve corresponding to 1.6–16 μM H₂O₂ was used as suggested [10]. Glucose oxidase activity was measured by following O₂ consumption with an oxygen electrode.

3.1 Consumption of H₂O₂ by intact Jurkat T-cells

The consumption of H₂O₂ by intact Jurkat T-cells was examined by two different experimental approaches: (a) exposure of cells to a bolus addition of H₂O₂ and (b) exposure of cells to a continuous flow of H₂O₂. Neither approach altered cell viability during the experiment(s).

In the former instances, the decay of H₂O₂ concentration after supplementing Jurkat T-cells with 100 μM H₂O₂ followed first-order kinetics (Fig. 2) with a k _cell value of 1.0±0.1×10⁻³ s⁻¹ per 10⁶ cells (n=16). The growth medium (used within a few hours after resuspending cells) did not exhibit significant H₂O₂ consumption (not shown).

In the latter instances, cells were incubated with a low steady state of H₂O₂ (9 μM), by simultaneously exposing them to H₂O₂ and glucose oxidase, an enzyme that reduces O₂ to H₂O₂ during glucose oxidation. Cells were able to maintain the steady state of H₂O₂ up to 2 h (Fig. 2); hence, their capacity to consume H₂O₂ did not decrease with time. The k _cell obtained with this steady-state incubation approach was similar, within experimental error, to that obtained with a bolus addition of H₂O₂ and independent of cell density.

It may be surmised that a k _cell value of 1×10⁻³ s⁻¹ per 10⁶cells is a reliable measure of the capacity of Jurkat T-cells to consume H₂O₂, for low to moderate concentrations of H₂O₂. Furthermore, the similar k _cell values obtained with both experimental approaches, suggest that no substantial changes in GSH levels –sufficient to compromise H₂O₂ removal by glutathione peroxidase – occurred following a bolus addition of H₂O₂ to cells.

3.2 Consumption of H₂O₂ by disrupted Jurkat T-cells

To determine the consumption of H₂O₂ in disrupted Jurkat T-cells, the enzymatic activities that mainly remove H₂O₂, catalase and glutathione peroxidase were examined.

3.2.1 Catalase activity

In most tissues, but not all, this enzyme is present in peroxisomes. The subcellular location of catalase in Jurkat T-cells was examined by titration with digitonin, a drug that binds to cholesterol present in membranes forming pores; with this approach, membranes with higher levels of cholesterol are disrupted preferentially, thus allowing the study of enzyme compartmentation and latency [11]. Because only one latency threshold to digitonin was observed (Fig. 3), it may be surmised that in Jurkat T-cells all catalase activity is entrapped within one type of membrane, most likely the peroxisomal membrane.

Catalase activity in fully disrupted cells was 1.65±0.13×10⁻³ s⁻¹ per 10⁶ cells (n=5). As with the plasma membrane, the peroxisomal membrane constitutes a barrier to H₂O₂ diffusion and, thus, it is responsible for the latency of catalase activity. In intact cells, or in cells with only the plasma membrane disrupted, catalase activity represented 35% of the total catalase activity measured in fully disrupted cells (Fig. 3). Accordingly, the overall contribution of catalase to the removal of external H₂O₂ in intact cells was 35% of the value found in disrupted cells: 0.58±0.05×10⁻³ s⁻¹ per 10⁶ cells.

3.2.2 Glutathione peroxidase activity

Evaluation of the contribution of glutathione peroxidase to cellular H₂O₂ removal is more complex, because its reaction mechanism involves an oxidation–reduction cycle of the Se-cysteine moiety at the active center using GSH as the reducing agent [7]:

((2))

((3))

((4))

Glutathione peroxidase activity is usually measured by following the oxidation of GSH under relatively high concentrations of H₂O₂; under these conditions, catalysis is mainly limited by the reduction step ((3), (4), with their respective constants, k ₃ and k ₄). Conversely, under conditions entailing relatively low H₂O₂ concentrations (e.g. most in vivo conditions or addition of H₂O₂ to intact cells), catalysis is mainly limited by the oxidation step (Eq. 2, with a rate constant k ₂) [7]. The integrated approach in Eq. 5 [10] considers both the reductive and oxidative steps, though the latter (comprising Eq. 2 and applying to low [H₂O₂]) is of interest for this study.

((5))

where t is time, [GPx]_total refers to total concentration of glutathione peroxidase, φ ₁=1/k ₂ and φ ₂=1/k ₃+1/k ₄. The kinetics of glutathione peroxidase in the whole cell homogenate fitted Eq. 5 well (Fig. 4A,B); the following parameters were obtained from Fig. 4A:

and from Fig. 4B:

Because the concentration of glutathione peroxidase in Jurkat T-cells is not known, the kinetic parameters φ ₁ and φ ₂ could not be determined 1 . However, the ratio φ ₂/φ ₁ (reflecting the redox properties of glutathione peroxidase) obtained in Jurkat T-cells (4.7×10²) compares well with those obtained for the rat liver (5.3×10²) [10] and the bovine erythrocyte enzyme (2.1×10²) [7]. At low H₂O₂ concentrations, the rate of H₂O₂ consumption by glutathione peroxidase is given by:

((6))

and, hence, the term k ₂×[GPx]_red gives the pseudo first-order rate constant that characterizes the consumption of H₂O₂ by glutathione peroxidase. Under in vivo conditions, most of the enzyme is in the reduced form [7] (99.8% according to a mathematical model [12]) and, therefore, the term k ₂×[GPx]_total represents a good estimation of the desired parameter (k ₂×[GPx]_red=6.0±0.1×10⁻³ s⁻¹ per 10⁶ cells).

Glutathione peroxidase is mainly present in cytosol and mitochondria [13], but also in the nucleus [14]. As mentioned above, glutathione peroxidase requires GSH to complete the catalytic cycle. GSH is not transported into cells [15] and, hence, titration of glutathione peroxidase activity with digitonin is not as useful as for catalase, because the latency of glutathione peroxidase will be determined by both H₂O₂ and GSH. Because most of the glutathione peroxidase activity is present in cytosol, an approximation of the cytosolic activity was inferred by considering the value found in the cell lysate: 6.0×10⁻³ s⁻¹ per 10⁶ cells. H₂O₂ consumption values in intact and disrupted Jurkat T-cells are listed in Table 1.

Table Table 1. Parameters of H₂O₂ catabolism and gradients across biomembranes in Jurkat T-cells

Parameters measured
H2O2 consumption by intact cells
k cell (bolus addition)	1.0×10−3 s−1 per 106 cells
k cell (continuous flow)	1.0×10−3 s−1 per 106 cells
H2O2 consumption by disrupted cells
Catalase pathway
disrupted peroxisomal membranes	1.65×10−3 s−1 per 106 cells
intact peroxisomal membranes	0.58×10−3 s−1 per 106 cells
Glutathione peroxidase pathway	6.0×10−3 s−1 per 106 cells
Parameters estimated
H2O2 catabolism
Turnover time	0.2 s
Glutathione peroxidase pathway	4.1 s−1 (91%)
Catalase pathway	0.4 s−1 (9%)
H2O2 profile and gradients
P s	2×10−4 cm s−1
Equilibrium [H2O2]in/[H2O2]out	0.9 s
[H2O2]cytosol/[H2O2]peroxisomes	3
[H2O2]out/[H2O2]in	7

3.3 H₂O₂ catabolism

The information gathered on the activities of catalase and glutathione peroxidase above serves two purposes: on the one hand, it is essential for the calculation of an H₂O₂ gradient and, on the other hand, it makes it possible to characterize the catabolism of H₂O₂.

The turnover time of H₂O₂ may be calculated from the sum of the pseudo first-order rate constants for H₂O₂ consumption. Assuming that glutathione peroxidase and catalase represent the main H₂O₂ sinks, the capacity of Jurkat T-cells to consume H₂O₂ is 6.6×10⁻³ s⁻¹ per 10⁶ cells, which, after conversion to units referred to the cell volume 2 , corresponds to 4.5 s⁻¹, thus yielding a turnover time of 0.2 s (1/4.5) (Table 1). This turnover time is slower than that estimated for hepatocytes (3×10⁻³ s) [12], an observation that may be explained by the very high activities of glutathione peroxidase and catalase present in the liver. Nevertheless, a turnover time of 0.2 s indicates that when Jurkat T-cells are exposed to extracellular H₂O₂ or to an agent that increases intracellular H₂O₂ production, an intracellular steady state will be reached in a few seconds.

The relative importance of catalase and glutathione peroxidase for the elimination of H₂O₂ can be calculated from the pseudo first-order rate constants obtained for these enzymes. Glutathione peroxidase activity (6×10⁻³ s⁻¹ per 10⁶ cells=4.1 s⁻¹) is responsible for most of the H₂O₂ consumption in cytosol (91%), whereas catalase (0.58×10⁻³ s⁻¹ per 10⁶ cells=0.4 s⁻¹) appears to play a minor role (9%), as also observed in other cell types, such as hepatocytes and endothelial cells [17, 18]. Therefore, most of the H₂O₂ added to Jurkat T-cells is probably removed via glutathione peroxidase with the concomitant change of the thiol/disulfide status inside the cell within a few seconds.

3.4 Profile of H₂O₂ in Jurkat T-cells

The ratio between the concentration of H₂O₂ inside (cytosol) and outside the cells (R; Eq. 1) may be inferred from the consumption of H₂O₂ by intact cells over the sum of activities that consume H₂O₂ in disrupted cells:

This estimated gradient is a lower limit, because only the glutathione peroxidase and catalase pathways were considered for H₂O₂ removal; if other enzymes consume H₂O₂, the gradient will be steeper.

To determine how fast the gradient is established, the permeability coefficient (P _s) of the plasma membrane of Jurkat T-cells for H₂O₂ must be obtained. A P _s value of 2×10⁻⁴ cm s⁻¹ may be estimated from Eq. 7 [8]:

((7))

(where A is the area of the plasma membrane, V _in is the intracellular volume, k ₃ is the pseudo first-order rate constant for H₂O₂ consumption) and by considering that (a) Jurkat T-cells are spheres with an area of 627±21 μm² [16], (b) a gradient R of 0.15, and (c) a k ₃ of 4.5 s⁻¹. This P _s is smaller than that obtained for erythrocytes [8] (6×10⁻⁴ cm s⁻¹), which is expected in view of the higher water permeability of erythrocytes compared with other cell types [19]; in particular, erythrocytes show a P _s for water that is more than one order of magnitude higher than those of lymphocytes [20]. For a spherule of the size of a Jurkat T-cell, a P _s of 2×10⁻⁴ cm s⁻¹ indicates that the time scale for the equilibration with H₂O₂ outside the cell is 0.9 s (Table 1). Therefore, when Jurkat T-cells are exposed to an external source of H₂O₂, the intracellular components sense the presence of H₂O₂ within 1 s. This fast diffusion of H₂O₂ is important for the postulated signaling roles that have been attributed to this molecule.

In addition to the gradient across the plasma membrane, a gradient is expected across the membrane of cellular organelles that consume significant amounts of H₂O₂, like peroxisomes [5] (which contain catalase), nucleus [14], mitochondria [13], and endoplasmic reticulum [13] (all of which contain glutathione peroxidase). For peroxisomes, results shown in Fig. 3 allow the estimation of the gradient across the peroxisomal membrane: in fact, the ratio measured between catalase activities in intact and in lysed peroxisomes was 0.35, which, according to Eq. 1, is the gradient between cytosolic and peroxisomal H₂O₂ concentrations upon incubation with an external source of H₂O₂. H₂O₂ gradients were not estimated for organelles containing glutathione peroxidase, but upon exposure to an external source of H₂O₂, the concentration of H₂O₂ in these organelles may be anticipated to be lower than in the cytosol. In organelles like lysosomes, that do not contain H₂O₂-consuming enzymes, a concentration similar to that in cytosol may be expected. Fig. 5 shows the H₂O₂ profile that is expected to form in Jurkat T-cells upon incubation with an external source of H₂O₂. Because H₂O₂ permeation and catabolism are fast, this profile will form, within a few seconds, whether the source is a steady state or a bolus addition.