Direct Evidence for Catalase as the Predominant H₂O₂ -Removing Enzyme in Human Erythrocytes

HYDROGEN PEROXIDE (H₂O₂ ) and other reactive oxygen species (ROS) are increasingly recognized as toxic intermediates in a wide variety of pathologies.1,2 H₂O₂ is considered a key metabolite because of its relative high stability, its diffusion,3,4 and its involvement in cell signaling cascades.5-8 

For these reasons the erythrocyte has been a traditional target for studying the metabolism of H₂O₂ . Since the first description of glutathione peroxidase (GPO) in 1957, an intense debate was created on whether catalase or GPO was the primary enzyme in the removal of H₂O₂ .9-20 A major role of GPO in decomposing H₂O₂ was especially derived from studies on erythrocytes with a reduced capability to generate nicotinamide adenine dinucleotide phosphate (NADPH).10 Conversely, experiments with acatalasic erythrocytes provided more evidence that catalase is a first line of defense against H₂O₂ .11 Using a mixture of GPO and catalase in a cell-free system with corresponding activities to erythrocytes, it was previously concluded that catalase accounts for more than 50% of the removal of H₂O₂ .14 20 

Most drawbacks in studies of H₂O₂ decomposition within erythrocytes stem from the lack of sensitive, direct and nontoxic H₂O₂ assays. Furthermore, a determination of the activity of GPO and catalase was only possible at high, unphysiological H₂O₂ concentrations.

We have previously described a sensitive H₂O₂ assay that is based on the oxidation of luminol (5-dihydro-1,4-phthalazinedione) by NaOCl21-23 and capable of detecting the catalase-mediated decomposition of H₂O₂ at submicromolar H₂O₂ concentrations.24,25 This assay had promising potential for the study of other H₂O₂ metabolizing enzymes.24 26 

In this study, we show that the luminol/hypochlorite assay allows the determination of purified erythrocyte GPO activity within the 10⁻⁷ mol/L H₂O₂ range at glutathione concentrations normally found in erythrocytes. Furthermore, it is shown that catalase and GPO activities can be determined individually in a titrated mixture of purified enzymes without influencing the respective activities. The method is applied to fresh human hemolysate using these enzymes in their original quantitative ratio. These direct measurements showed a clear prevalence of catalase even at very low H₂O₂ concentrations.

Reagents. Luminol, catalase, GPO, glutathione reductase, reduced glutathione (GSH), hydrogen peroxide, aminotriazole, NADPH, sodium hypochlorite, and sodium azide were from Sigma (Deisenhofen, Germany).

Solutions. Stock solutions of luminol were prepared in 140 mmol/L sodium chloride and 20 mmol/L phosphate buffer and adjusted to pH 7.4. Stock solutions of NaOCl and H₂O₂ were prepared in water. Their concentrations were determined spectrophotometrically (ε₂₉₀ = 350 mol/L × cm⁻¹ at pH 12 [Morris²⁷] and ε₂₃₀ = 74 mol/L × cm⁻¹ [Beers and Sizer²⁸] for NaOCl and H₂O₂ , respectively). Solutions of NaOCl and H₂O₂ were freshly prepared daily.

Preparation of human erythrocytes and hemolysates. Fresh human red blood cells were prepared as previously described29 with the following modifications: Blood from healthy volunteers was collected in tubes containing EDTA as anticoagulant. Blood cells were washed at 800g in four volumes of saline solution (Hanks' solution, pH 7.4) and the supernatant, buffy coat, and upper 15% of the packed cells were removed by aspiration after three additional washes. Hemolysates were obtained on hypotonic lysis. Cell number and hemoglobin content were determined with a Celldyn 1700 (Abbott, Maidenhead, UK). The activities of catalase and GPO obtained from these erythrocyte preparations by using conventional assays30,31 matched those reported by others.14 

Determination of catalase activity. Catalase activity was determined as previously described.25 This enzyme does not show Michaelis-Menten kinetics. Catalase-mediated H₂O₂ decomposition follows an exponential rate law and is described by the rate constant k:

where dt is the measured time interval and S₁ and S₂ are H₂O₂ concentrations at time t₁ and t₂ , respectively.25 30 Therefore, catalase activity was expressed as the rate constant k of the exponential decay of H₂O₂ and calculated by linear regression analysis. Solutions of purified catalase, diluted hemolysates, or cell suspensions were aspirated via a peristaltic pump (4 mL/min). Luminol (10⁻⁴ mol/L) and hypochlorite (10⁻⁴ mol/L) were continuously added by a perfusion pump (12 mL/h) to the sample solution closed to the light detector, thus monitoring the actual H₂O₂ concentration in real time. The luminescence integral was determined over 5 seconds. After the addition of H₂O₂ (10⁻⁵ mol/L) to the catalase solution, the breakdown of H₂O₂ concentration could be assessed. A magnetic stirrer was used to ensure a rapid mixing of the enzyme-substrate solution. In experiments with purified catalase, the enzyme activity in the sample solution is expressed as the exponential rate constant k in s⁻¹ (see preceding equation). In experiments with hemolysate, catalase activity k (s⁻¹) was related to hemoglobin concentration. Therefore, catalase activity is expressed by (s⁻¹ × g⁻¹ × L). Luminescence measurements were performed using a luminometer AutoLumat LB 953 at 37°C (Fa. Berthold, Wildbad, Germany).

GPO activity. GPO activity was determined on the basis of recent preliminary results.24,26 Details are described in the Results section. A conventional assay using tert-butyl hydroperoxide as a substrate was used for reference measurements.31 The units are expressed as U/mL.

Determination of purified erythrocyte catalase and GPO activity at physiological H₂O₂ concentrations. Figure 1 shows the decomposition of H₂O₂ down to nanomolar concentrations after the addition of purified erythrocyte catalase. The decomposition follows an exponential decay (r² = .997) typical for catalase, because the rate of catalase-mediated H₂O₂ decomposition depends linearly on H₂O₂ concentration according to the following equation:

where k′ is the specific catalase activity.30 Catalase is completely inhibited by 1 mmol/L NaN₃ as can be shown after a second addition of H₂O₂ . At these low substrate concentrations no enzyme inactivation or gaseous oxygen release is observed.24 25 

The effect of purified GPO on H₂O₂ was studied at a GSH concentration of 2 mmol/L, which corresponds to the level found in erythrocytes and which permits determination of submicromolar H₂O₂ concentrations without significant interference with the assay method. Figure 2 shows that the addition of GPO elicits a rapid degradation of H₂O₂ . Under saturating conditions, the decomposition follows a zero order kinetics and the degradation rate depends linearly on GPO concentrations (not shown). After a second addition of H₂O₂ , GPO activity is not altered at these low H₂O₂ concentrations (Fig 2). Heat inactivation of the enzyme (boiling for 10 minutes) prevented H₂O₂ degradation. Because GSH concentration requires maintenance at a constant level, we studied the effect of a GSH-regenerating system (NADPH and glutathione reductase31 ) on GPO activity. GSH regeneration did not change GPO-mediated H₂O₂ degradation. It may be surmised that GSH concentration is not significantly changed under these conditions, most likely because of the high [GSH]/[H₂O₂ ] ratio (>200). The small decrease in GSH concentration during the reaction does not affect H₂O₂ decomposition rate. Conversely, experiments performed with [GSH]/[H₂O₂ ] ratio (<10) are associated with a significant GSH depletion and a consequent decrease in GPO activity (data not shown).

The rate of H₂O₂ degradation by purified erythrocyte GPO as a function of GSH concentration is shown in Fig 3. The reaction rate depends linearly on GSH concentration within the concentrations examined. These findings are consistent with ping-pong mechanisms, inherent in the GPO activity^31-33: 2 GSH + ROOH → GSSG + ROH + H₂O.

In summary, the luminol/hypochlorite assay allows a measurement of H₂O₂ removal by GPO at submicromolar concentrations of H₂O₂ and at concentrations of GSH normally found in erythrocytes. Under these conditions, regeneration of glutathione via glutathione reductase is not a requisite condition.

Determination of individual GPO and catalase activities in a mixture of purified enzymes at physiological H₂O₂ concentrations. Catalase and GPO are present in the erythrocyte cytoplasm. An experimental model was designed to determine the individual catalase and GPO activity in a mixture containing both enzymes and mimicking the ability of a hemolysate or erythrocyte cytoplasm to decompose H₂O₂ .

Addition of a mixture of purified GPO and catalase to a 10⁻⁵ mol/L H₂O₂ solution (Fig 4A) resulted in an exponential decay of H₂O₂ , which was ascribed to catalase activity based on the sensitivity of the signal to NaN₃ (Fig 4B) and the subsequent lack of removal of a second addition of H₂O₂ (Fig 4C). Catalase activity calculated from these data tallies with the activity calculated from data in Fig 1. The addition of 2 mmol/L GSH (Fig 4D) and 10⁻⁴ mol/L H₂O₂ (Fig 4E) to the above mixture permits to evaluated GPO-dependent H₂O₂ degradation: The rates obtained matched those calculated from data in Fig 2. These experiments were performed with GPO activities higher than those in erythrocytes (with respect to catalase) to improve the kinetic characterization of both enzymes.

Determination of catalase and GPO activity in erythrocytes at physiological H₂O₂ concentrations. We applied the procedure described previously to fresh human hemolysate to study H₂O₂ removal in human erythrocytes. Because, in contrast with other cells (eg, hepatocytes), catalase and GPO are freely distributed in the cytoplasm of erythrocytes,34 a hemolysate reflects intracellular conditions in respect to H₂O₂ decomposition.

H₂O₂ removal by fresh human hemolysate is shown in Fig 5: After the addition of H₂O₂ (Fig 5A), an exponential decay of H₂O₂ is observed (r² = .995) ascribed to catalase activity as inferred by the effect of NaN₃ (Fig 5B) and the lack of H₂O₂ consumption when added subsequently (Fig 5C). Of note, negligible GSH levels were present in this highly diluted hemolysate (1/1,000) preventing any detectable GPO activity. Further addition of 2 mmol/L GSH and H₂O₂ (as described for Fig 4) permitted evaluation of GPO activity, which exhibited a kinetic pattern different from catalase. This degradation pattern was similar to that observed with purified GPO. The H₂O₂ degradation rate depended linearly on GSH concentration (as shown in Fig 3 for the purified enzyme).

The H₂O₂ degradation rate of catalase and GPO was plotted against H₂O₂ concentration (Fig 6). These data showed a prevalence of catalase at low H₂O₂ concentrations. GPO reaches only approximately 8% of the rate at which catalase simultaneously decomposes H₂O₂ . H₂O₂ degradation by catalase depends linearly on H₂O₂ concentration, whereas GPO becomes saturated at concentrations of H₂O₂ greater than 10⁻⁶ mol/L. At H₂O₂ concentrations above 10⁻⁵ mol/L, catalase contributes almost exclusively to the overall turnover of H₂O₂ . The relative activities of catalase and GPO at different H₂O₂ concentrations are listed in Table 1. In H₂O₂ concentrations less than 10⁻⁶ mol/L, GPO becomes unsaturated: Under these conditions, the decomposition rate of H₂O₂ depends linearly on H₂O₂ concentration. At 2 mmol/L GSH, the K_M of GPO for H₂O₂ is calculated as 2 × 10⁻⁵ mol/L H₂O₂ in agreement with previous reports.14 35 

H₂O₂ degradation in erythrocytes has been studied for several decades in connection with the high oxygen turnover of these cells and the toxic properties of ROS derived from H₂O₂ metabolism.11,36,37 Interest in this area was renewed by articles describing a role for H₂O₂ in signal transduction.5 38 

In the present study, we have determined both catalase and GPO activities in hemolysate at physiological H₂O₂ concentrations by using a novel H₂O₂ assay.23,25 Because erythrocyte catalase and GPO are apparently not compartmentalized,34 the studies performed on hemolysates may reflect a situation similar to that in the erythrocyte. The luminol/hypochlorite method may be used to determine H₂O₂ degradation by GPO in the 10⁻⁷ mol/L H₂O₂ range and at GSH concentrations normally found in erythrocytes.

The assumption that the glutathione-GPO system has greater affinity for its substrate led both Keilin and Hartree39 and Cohen and Hochstein10 to suggest that at H₂O₂ concentrations below 10⁻⁷ mol/L, catalase played almost no role in H₂O₂ detoxification. However, the studies herein indicated a clear predominance of catalase in removing H₂O₂ and they provided understanding of the increasing data underlying the function of catalase in erythrocytes.11,13,14,17-19,34 Considering these and the data herein, catalase needs to be regarded as the major H₂O₂ -decomposing enzyme in normal erythrocytes. It decomposes H₂O₂ without generation of free radicals by minimizing one-electron-transfers. Hence, a protective role against free radicals may be the main physiological function in these cells. The existence of healthy acatalasic individuals cannot serve as an argument against the physiological importance of this enzyme. Most patients do not show a real “acatalasemia” but a hypocatalasemia.40,41 Additionally, Japanese acatalasic patients show evidence for increased oxidative damage, and Swiss acatalasic variants show, at least in vitro, an enhanced sensitivity to a number of H₂O₂ -generating agents.40 41 According to our data, a remaining activity of 1% catalase would still contribute largely to the overall H₂O₂ removal (approximately 50% at 10⁻⁵ mol/L H₂O₂ ).

Otherwise, the hexosemonophosphate shunt in erythrocytes lacking detectable catalase activity is significantly increased because of glutathione use via GPO.11 Because the steady-state levels of H₂O₂ in tissues are considered to be between 10⁻¹⁰ and 10⁻⁷ mol/L⁴² and were recently estimated to be approximately 2 × 10⁻¹⁰ mol/L H₂O₂ in erythrocytes,29 GPO is not supposed to be saturated under these conditions. It may be surmised that GPO can undertake a substantial part of catalase activity in acatalasic erythrocytes. The contribution of hemoglobin to H₂O₂ degradation is minimal because of the low reaction rates.29 34 A major function of GPO in normal erythrocytes may be the disposal of organic peroxides and the maintenance of protein thiols in their reduced state.

It is worth mentioning that some discrepancies in the literature may be bridged by considering effects of azide and cyanide on erythrocyte metabolic pathways other than a unique inhibition of catalase.11 Additionally, it was long thought that GPO was the sole H₂O₂ -removing enzyme in erythrocytes depending on NADPH. The discovery of the role of catalase-bound NADPH unified the concept of two different mechanisms for disposing of H₂O₂ (catalase and the glutathione reductase/peroxidase pathway) by showing that both mechanisms are dependent on NADPH.12 18 

In summary, these data strengthen the notion that H₂O₂ removal in normal erythrocytes is mainly the domain of catalase. On a methodological note, the luminol/hypochlorite assay may be a useful tool to study H₂O₂ degradation in other cells and tissues, which is a notion supported by previous preliminary studies with hepatocytes.43 

We are indebted to Dr E. Cadenas of the University of Southern California, Los Angeles, CA, for critical reading of the manuscript.