Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry

Abstract

A sample preparation strategy for the determination of the Fe-containing enzyme catalase (CAT) by Fe specific monitoring in human erythrocytes has been optimized. For this purpose, the combined use of elemental mass spectrometry (via inductively coupled plasma, ICP-MS), molecular mass spectrometry (via MALDI-TOF) and enzymatic activity measurements has been required. The procedure involved haemoglobin precipitation from cell lysate with a solution of ethanol–chloroform and preconcentration of the supernatant by using a Speed-Vac concentrator. Catalase recoveries of about 88 ± 15% could be measured by monitoring the protein enzymatic activity before and after precipitation. Further fractionation of Fe-containing proteins from the preconcentrated extract was achieved by size exclusion chromatography (Superdex 200) with a mobile phase of ammonium acetate (0.05 M, pH 7.4) coupled to ICP-MS (Fe monitoring) and UV/VIS detection (specific absorption of the heme-group at 408 nm). A second dimensional chromatography of the CAT-positive activity fraction was carried out by anion-exchange chromatography (Mono Q 5/50) using for elution a linear gradient of ammonium acetate (0–0.750 M in 15 min). This second step revealed a single Fe-containing species in the chromatogram and permitted the unambiguous characterization of the CAT in such fractions by MALDI-TOF. Column recoveries were evaluated and were quantitative, in terms of Fe bound to protein and CAT activity.

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Introduction

Human catalase (CAT; E.C. 1.11.1.6) is a tetrameric heme containing enzyme, consisting of four identical subunits of about 60 kDa each with four ferriprotoporphyrin (heme) prosthetic groups per molecule and so CAT molecular mass is about 240 kDa.¹ This enzyme is widely distributed within our body, with a high concentration in the liver, kidneys and erythrocytes (more than 98% of blood CAT is present in the cytosol of erythrocytes). CAT detoxifies H₂O₂, a potent oxidative agent that could damage any cell, by catalyzing its breakdown to water and divalent oxygen.² Therefore, CAT has long been recognized as a defence for oxidative stress (cellular imbalance of the pro-oxidant/antioxidant ratio)^3–5 which is ubiquitously associated with many pathologic conditions including cancer, diabetes, Alzheimer's and other important neurodegenerative diseases.^6,7 CAT has also been implicated, as a relevant factor, in ethanol metabolism,⁸ inflammation,⁹ mutagenesis,¹⁰ prevention of apoptosis,¹¹ stimulation of a wide spectrum of tumours¹² and aging.¹³ Loss of CAT leads to the human genetic disease known as acatalasemia.¹⁴ Blood CAT is also decreased in alcoholism, certain cancers, and psychiatric diseases while it is increased in pancreatic, liver, hemolytic, renal, skin, respiratory, acquired immunodeficiency, and oxidant-mediated vascular diseases.¹⁵ Thus, the accurate determination of CAT in blood and tissues has become increasingly important due to its significance, not only as marker of oxidative stress, but also for clinical diagnosis and physiological research.

At present most of the studies have been focused on measuring the CAT activity changes under certain health disorders. In these cases the enzyme activity is generally determined using assays that are based on either the decrease in absorbance of hydrogen peroxide at λ = 240 nm or by measuring oxygen release with Clark-type electrodes.^16,17 The activity measurements have some limitations, for instance, the inability to assay catalase at saturating substrate (H₂O₂) concentrations, as is conventionally done in standardized enzyme assays, which results in the absence of an official reference method and a wide variation in the reaction conditions. Thus, estimates of catalase activity for different tissues may vary widely, depending on the activity method, concentration of H₂O₂ and units of measurement. For instance, human plasma activity of catalase is reported to be 70 ± 29 U/L by using a peroxidatic method, 1 mM H₂O₂, and catalase standards, but approximately 200-fold or 700-fold higher in catalatic methods based on measurement of the decrease in absorbance of 10 and 54 mM H₂O₂, respectively. Therefore, the activity measurements are only useful for comparison purposes (relative measurement). However in order to gain insight into the pathophysiological relevance of CAT, direct quantitative data of its concentration level in biological samples is demanded. In this regard, very few strategies have been developed over the years for the direct quantification of CAT in red blood cells. A recent method has been developed for the determination of catalase by means of electrospray-MS using multiple reaction monitoring (MRM) strategies after tryptic digestion of the protein into the corresponding peptides. However, the authors reported a discrepancy between the protein concentrations as determined from two different peptides. They claim that most likely these differences arise from inadequacy in the sample preparation and handling procedures, in particular, incomplete tryptic digestion of the protein or sample losses due to unspecific adsorptions.¹⁸ Another approach for quantification of CAT levels in transfected cells is proposed by using competitive ELISA, with rabbit anti-human CAT antibody.¹⁹ Plates are read in an ELISA reader at 492 nm and the total catalase levels are determined by reference to a standard curve, constructed with serial dilutions of human catalase. However, this procedure involves several preparation steps that make it prone to errors due to protein losses obtaining final inaccurate results.

In this work we propose an integrated speciation approach based on the complementary use of elemental mass spectrometry (ICP-MS) for the sensitive Fe detection, UV/VIS absorption for selective monitoring of the heme-group (408 nm) and the CAT activity measurements. The use of ICP-MS also allows the accurate quantification of heteroelements in biomolecules by isotope dilution analysis (IDA).²⁰ The quantitative determination of such metals and/or metalloids in combination with the knowledge of the heteroelement-protein stoichiometry provides an elegant methodology to obtain absolute protein concentrations.^21,22 However, since the quantification of heteroatom-containing biomolecules via HPLC-ICP-MS is only based on the detection of the corresponding heteroelement, extreme care must be taken to preserve the native compositional and coordinative structure of the metallo- or heteoatom-containing protein during the whole analytical procedure. Moreover, protein column recovery for the biomolecule must be always taken into account since quantification errors will still occur if the metal containing protein does not elute completely from the column.²¹

Thus, in this study we propose the use of complementary techniques to asses the recovery of every step necessary for the determination of CAT in real samples, by using post-column IDA-HPLC-ICP-MS strategies. The different steps during the required sample preparation and species isolation will be quantitatively controlled aiming at developing a reliable alternative analytical procedure for the determination of CAT via elemental detection of the iron present in the hemo groups of the protein.

Experimental

Reagents, materials and samples

Analytical reagent grade chemicals were used throughout unless otherwise stated. All solutions and dilutions were made with high-purity deionized water (>18 MΩ Milli-Q water, Millipore, Bedford, MA, USA). For protein separation, Tris(hydroxymethyl)-aminomethane (Tris) from Merck (Darmstadt, Germany), acetic acid and ammonium acetate from Panreac Quimica SAU (Barcelona, Spain), were used. Trifluoroacetic acid (TFA) was purchased by Sigma-Aldrich (St. Louis, MO, USA), sinapinic acid was obtained from Broker Daltonics, (Bremen, Germany). HPLC grade ethanol, acetonitrile and chloroform were supplied by Teknocroma S.L. (Barcelona, Spain). Cut-off membrane filters (100 kDa) were from Amicon-Ultra (Millipore, Iberica, Madrid). The standards used to size-calibrate the size exclusion column included cytochrome c (13 kDa, bovine), haemoglobin (64 kDa, bovine), ferritin (450 kDa, bovine) and catalase (240 kDa, bovine liver) were all purchased from Sigma. Human serum samples from healthy volunteers were kindly provided by Hospital Central of Asturias (Spain), which complies with the relevant laws and institutional guidelines for handling this type of samples. The work is conducted in the frame of a Project between both institutions (Hospital and University) and has been approved by the ethics committee of the Hospital.

Instrumentation

Two analytical columns were used for the isolation of catalase: a size exclusion Superdex™ 200 10/300GL (300 × 10 mm i.d, GE Healthcare Bio-Sciences Uppsala, Sweden) and an anion-exchange Mono-Q™ HR 5/50 GL (50 × 5 mm i.d., Pharmacia, Amershan Bio-Sciences, Uppsala, Sweden) columns. The columns were connected to a dual piston liquid chromatographic pump (Shimadzu LC-10AD, Shimadzu Corporation, Kyoto, Japan) equipped with a sample injection valve Rheodyne, Model 7125 (Cotati, CA, USA) fitted with a 50 μL injection loop. A scavenger column (25 × 0.5 mm i.d.) packed with Kelex-100 (Schering, Spain) impregnated silica C₁₈ material (20 μm particle size, Bondapack, Waters Corp., MA) was placed between the pump and the injection valve in order to eliminate possible metal traces present as contamination in the mobile phases. UV/VIS detection was performed with a Diode Array Detector (DAD) from Agilent Technologies (1100 Series, Waldbron, Germany). The detection of Fe in the column effluent was carried out using ICP-MS model 7500 from Agilent Technologies (Agilent, Tokyo, Japan) equipped with a collision cell system (ICP-(ORS)-MS). In this work He was used as collision gas (4.0 mL min⁻¹) in order to reduce the ⁴⁰Ar¹⁶O⁺ interference on ⁵⁶Fe⁺.

Intact protein analysis by MALDI-TOF-MS was conducted using a Voyager-DETM STR BiospectrometryTM Workstation (Applied Biosytems, Langen, Germany) instrument equipped with nitrogen pulsed laser (337 nm). All spectra were recorded in linear, positive ion mode. Spectra were collected as a sum of 100 shots across a spot. External calibration was performed for molecular assignments using a standard of albumin from bovine serum. Sample preparation was performed on a stainless steel hydrophobic target (Voyager 96 × 2 Sample Plate P/N V700813) using the dried-droplet technique. For that, an aliquot (0.5 μL) of the sample solution and an equal aliquot of the matrix solution were mixed on the target in the given order and dried at room temperature. The matrix solution was prepared with sinapinic acid (5 mg mL⁻¹) in 70% (v/v) water, 29.9% (v/v) acetic acid and 0.1% (v/v) TFA. Operational conditions and data acquisition parameters are summarized in Table 1.

Table 1 Operational conditions and data acquisition parameters for ICP-(ORS)-MS and MALDI-TOF

ICP-(ORS)-MS
Instrument	Agilent 7500 CE (ORS)
RF power	1500 W
External flow	15 L min^−1
Nebuliser gas flow rate	1.1 L min^−1
m/z monitored	^54Fe, ^56Fe, ^57Fe
Integration time per isotope	0.1 s
Collision/reaction gas	He
Flow rate	4 mL min^−1
Octapole-bias	−18 V
QP-bias	−14 V
MALDI-TOF
Instrument	Voyager-DETM STR BiospectrometryTM
Scan type	Positive
Instrument mode	Linear
External calibration	Albumin of bovine serum
Matrix	5 mg mL^−1 Sinapinic acid, 70% H2O, 29.9% acetic acid, 0.1% TFA
Laser	2600 V
Spectrum acquisition
Shoots	150
Scan range	6000–35000

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Procedures

Sample preparation. Human blood samples (3.0 mL) from healthy volunteers were centrifuged at 3000 g, 4 °C, for 5 min to separate red blood cells from plasma. The upper plasma phase and the buffer coat on top of the erythrocyte layer (probably ascribed to white cells) were carefully removed. The cells were then haemolysed by addition of ice cold distilled water (9.0 mL per 1.0 mL of cells) and centrifuged (3000 g, 5 min) at room temperature. Then, a solution containing ethanol–chloroform (1 : 1), 2 mL per 8 mL of cell lysate was added in order to accomplish haemoglobin precipitation. After manual shaking for 5 min, the mixture was centrifuged (12000 g, 4 °C, 1 h) and the supernatant was preconcentrated 10 times using a Speed-Vac concentrator (Vacufuge, Eppendorf, Germany).

Size exclusion chromatography. Fractionation of proteins from the cell lysate supernatant after haemoglobin precipitation and preconcentration was performed by size exclusion chromatography (SEC). An aliquot of 50 μl of the sample was injected in the Superdex 200 column (fractionation range of 10–600 kDa) and eluted under isocratic conditions with a mobile phase of 0.05 M ammonium acetate buffer (pH 7.4) at a flow rate of 0.6 mL min⁻¹. The Superdex column was calibrated using different iron containing proteins with known molecular weights ranging 13–450 kDa. Detection was carried out by UV/VIS absorption at 280 nm (generic for all proteins) and at 408 nm (specific for the proteins containing a heme group). Fe was also monitored by ICP-(ORS)-MS using the conditions of Table 1.

Anion exchange chromatography. The fraction corresponding to CAT elution from the SEC column was collected and pre-concentrated by ultrafiltration through a 100 kDa cut-off filter and then injected in the Mono-Q anion exchange column (50 μL). Separation was performed by means of a linear gradient of ammonium acetate (0–750 mM in 15 min) buffered by 20 mM Tris-acetic acid (pH 7.4) solution at a flow rate of 1 mL min⁻¹. The eluent from the HPLC column was introduced into the ICP-(ORS)-MS detector for specific iron detection (conditions Table 1). The eluent was also passed to a UV/VIS detector.

Activity measurements. CAT activity was determined in erythrocytes lysates spectrophotometrically using the UV assay method of Aebi.¹⁶ CAT catalyses the decomposition of H₂O₂ that can be directly monitored by measuring the decrease in absorbance at 240 nm with time, at 37 °C. Catalase activity unit (U) was calculated as the amount of sample required to hydrolyze 1 μmol of H₂O₂ per minute, based on the molecular absorbance of 0.04 × 10⁶ for H₂O₂. Assays were performed at least twice on each lysate and the mean value determined.

Results and discussion

Separation and characterization of catalase in red blood cells

Catalase determination is conducted in the soluble fraction of lysed red blood cells where haemoglobin is the most abundant protein present which contains also a heme group (accounting for more than 90% of the total protein content). That is, haemoglobin is a potential interfering protein in the detection of CAT by both UV/VIS absorption at 408 nm (heme group) and ICP-MS (detection of Fe). Admittedly, most of the haemoglobin from the sample can be removed by precipitation during sample preparation with ethanol–chloroform (next section), the remaining haemoglobin is still present and may interfere. Therefore, in order to separate CAT from other Fe-proteins in the sample extract we used a SEC column (Superdex 200).

For this purpose, ammonium acetate was chosen as eluent providing adequate compatibility with ICP-MS detection and the separation was carried out at the physiological pH of 7.4. A group of iron containing protein standards including cytochrome c (13 kDa), haemoglobin (63 kDa), ferritin (450 kDa) together with CAT from bovine liver (240 kDa) were used for calibration (data not shown). The chromatogram obtained with UV/VIS detection (280 and 408 nm) (Fig. 1A) and ICP-MS detection of Fe (Fig. 1B) for a bovine CAT standard revealed the presence of an important number of unexpected peaks. The UV trace at 280 nm shows that the protein standard is not pure and different species absorb at this wavelength at different retention times. However, only the species eluting at about 21 min showed absorbances at 280 nm and 408 nm, which corresponds to the main Fe-signal (see Fig. 1B). Therefore, such fractions can be ascribed to CAT. The Fe trace (Fig. 1B) shows also another species at about 18 min that corresponds, according to the different injected Fe-protein standards, to the retention time of ferritin that could be present as impurity. In order to rule out that this Fe-signal is due to possible CAT agglomerates, the peak associated to CAT at 21 min was collected and re-injected in the same column. The inset of Fig. 1B reveals a pure peak at 21 min, confirming the previously observed results of ferritin impurities present in the commercial CAT standard.

Fig. 1 SEC chromatogram of the CAT standard from bovine liver obtained by: (A) UV/VIS detection at 280 nm and 408 nm; and (B) ICP-(ORS)-MS detection of Fe. The shadowed fraction of Fig. 1A was collected and re-injected and the corresponding chromatogram is shown in the inset. Experimental conditions in the text. A.u. (absorbance arbitrary units).

Fig. 2 shows the separation obtained for a human blood sample processed as described in the experimental section. As can be seen in the chromatogram of Fig. 2A, several Fe-containing species are still present after haemoglobin precipitation. The main Fe and heme containing peak corresponds to the retention time of the haemoglobin standard (≈ 27 min) which reveals that, as anticipated, a small percentage of this protein is still present in solution after precipitation. Additionally, both the UV/VIS and the ICP-MS signals (see Fig. 2A) reveal the presence of two peaks in the fraction corresponding with the elution of the standard of CAT (20–25 min, see Fig. 1). In order to evaluate if the sought protein was present in any of the two observed peaks (around 21 min and 24 min) different fractions of the eluate from the SEC column were collected every 30 s (0.3 ml fractions) and the enzymatic activity of CAT was measured in each fraction as described in the procedures section. The chromatographic profile obtained is shown in Fig. 2B superimposed to the ⁵⁶Fe iron chromatographic profile. As can be observed, the fraction presenting, almost exclusively, CAT activity, Fe-signal and absorbance at 408 nm occurs at 20–22 min. This finding confirms the presence of CAT in the Fe containing sought fraction although it does not provide any information about species purity. The peak at about 31 min (Fig. 2B) could be due to the presence of other peroxidases, primarily glutathione peroxidase that could be present in the extract and could cause overestimation of catalase activity if these species are not chromatographically resolved, as reported by other authors.¹⁵

Fig. 2 SEC chromatogram of a sample of human erythrocytes after sample treatment obtained by: (A) UV/VIS detection at 408 nm (red trace) and ICP-(ORS)-MS on the ⁵⁶Fe (black trace); and (B) same Fe signal that in Fig. 2A but superimposed to the enzymatic activity profile (green trace). Experimental conditions in the text. A.u. (absorbance arbitrary units).

In order to evaluate species purity, the fraction 1 eluting from the SEC column between 20–22 min and containing CAT (see shadowed area of Fig. 2A) was collected, desalted and preconcentrated (using a 100 kDa cut-off filter) and then analyzed by anion exchange chromatography. For this purpose, different ammonium acetate gradients buffered at pH 7.4 were tested for elution of the CAT standard in this column and a linear gradient of acetate (0–0.750 M in 15 min) was chosen. As can be seen in Fig. 3, the chromatogram obtained by anion exchange of the fraction 1 from Fig. 2A shows a single species at a retention time of about 7 min using both ICP-MS (⁵⁶Fe) and UV/VIS (408 nm) detection. This retention time matches the CAT standard retention time (data not shown) and confirms the presence of the sought protein in fraction 1 (additionally, the small ICP-MS peak observed at 10 min appeared, with the same intensity, in the gradient blank). Therefore, adequate species purity seems to be achieved for separation of CAT from human erythrocytes by sequential fractionation by size exclusion (Superdex 200) followed by anion-exchange chromatography (Mono Q 5/50).

Fig. 3 Anion-exchange chromatogram of the fraction collected from 20 to 22 min in the chromatogram of Fig. 2A, obtained by UV/VIS detection at 408 nm (red trace) and with ICP-(ORS)-MS for detection of Fe. Experimental conditions in the text.

For complete confirmation of the above described results, the fraction eluting at 7 min in the chromatogram of Fig. 3 was collected, cleaned-up and analysed by MALDI-TOF-MS using sinapinic acid as matrix and the conditions described in the experimental section. The mass spectrum obtained is depicted in Fig. 4. The most intense signal observed at m/z 59616.39 Da could correspond to the singly charged ion of the apo-subunit of CAT (one catalase chain without the heme group) which has a theoretical molecular mass of 59585 Da (Δm = 0.05%). This result confirms the presence of CAT in the fraction analyzed, since it is well known from the literature that analysis of quaternary structure of proteins by MALDI-TOF using typical acidic matrix solutions such as sinapinic acid/trifluoroacetic acid yields spectra of monomeric protein subunits rather than the intact protein.²³ This is due to the fact that the acid conditions for MALDI leads to dissociation of the non-covalent subunits assemblies and, in the case of metaloproteins, often to the lost of the metal bound to the protein (unless special MALDI matrices are used).^20,24 In fact, only a very limited number of publications reported the observation of the intact holohomotetramer of beef liver catalase (assembly composed of four catalase chains and four heme groups) by MALDI-MS using less acid matrix substances, such as hidroxyacetophenones derivatives in organic solvents with and without the addition of MALDI-compatible buffer salts.^23,24

Fig. 4 MALDI-TOF-MS spectrum of the fraction collected from 6 to 8 min in the chromatogram of Fig. 3. Experimental conditions in the text.

Finally, in order to prove the suitability of the developed multidimensional chromatographic method for future quantification studies, it was necessary to ensure that the recovery of both Fe and CAT along the separation procedure is quantitative. To evaluate this recovery, the eluate from the entire chromatographic run (50 min for SEC-HPLC, 30 min for anion-exchange-HPLC) was collected (n = 3) and Fe was quantified (5 replicates) by ICP–MS with direct calibration using Ga as internal standard. The amount of Fe found was compared with that measured in the sample before injection. The results of this mass-balance for Fe were quantitative from both columns, SEC (79 ± 12%) and anion-exchange (87 ± 10%). Moreover, the integrity of CAT during the multidimensional chromatographic separation procedure was also evaluated by measuring its enzymatic activity in the sample and in the eluate collected from the column after sample separation. The recovery for activity obtained was again quantitative (90 ± 20%). Since the heme group is essential for the enzyme activity, this result indicates no loss of the enzyme-bound heme during chromatographic separation.

Optimization of the sample preparation procedure

Once the chromatographic separation was optimized and the specific recovery was well established, the next step was the optimization of the sample preparation procedure. Sample preparation involves first the precipitation of haemoglobin from erythrocyte lysates with a solution of ethanol–chloroform. This step has to be carefully optimized in order to obtain efficient removal of haemoglobin without lost of CAT. Therefore, optimization of the ratio cell lysate/ethanol–chloroform volumes was carried out in order to obtain quantitative recovery of CAT from the sample. Based on the literature recommendations, two different ratios were investigated, 8/1/1 and 8/4/2. After application of each protocol of haemoglobin precipitation, the sample aqueous extracts were analysed by SEC-UV/VIS and SEC-ICP(ORS)-MS. Fig. 5 shows the results obtained for the two different ratios (8/1/1 and 8/4/2). As expected, the haemoglobin is more efficiently removed by using a major volume of ethanol–chloroform (volume ratio of 8/4/2) but unfortunately the CAT is almost completely adsorbed in the precipitate. Therefore, a final ratio of 8 ml cell lysate/1 ml ethanol/1 ml chloroform was chosen for further experiments. After haemoglobin precipitation the aqueous layer was preconcentrated 10 times before injection into the HPLC-UV/ICP-MS system in order to increase the sensitivity. This preconcentration was carried out by using a Speed-Vac concentrator or by passing the supernatant through a 100 kDa cut-off filter. Similar results (data not showed) were obtained with both preconcentration devices.

Fig. 5 Chromatographic profile of Fe obtained by SEC-ICP-(ORS)-MS after the sample treatment in human erythrocytes using a relation cell lysate volume /ethanol volume /chloroform volume of 8/1/1 (black trace) and 8/4/2 (red trace).

Finally the recovery of the whole sample treatment procedure described (haemoglobin precipitation and preconcentration using a Speed-Vac concentrator) was evaluated by measuring the specific activity of CAT in the erythrocyte lysate before and after treatment. Recoveries of about 88 ± 15% (mean±SD, n = 3) of the enzymatic activity of the CAT remained after sample treatment. Since the CAT enzymatic activity depends on the presence of the heme group in the molecule, quantitative recoveries calculated through activity measurements imply species stability throughout the precipitation step. As consequence, Fe (target element for further ICP-MS quantification of CAT) remains present within the molecule and can be directly quantified after separation of the remaining Fe-proteins by SEC (which provides also quantitative recoveries). Such results should permit the direct correlation between the enzymatic activity with the metal measurements in the ICP-MS (Fe in this case), previously demonstrated for Cu, Zn-SOD.²⁰ This would be an elegant way to obtain, via elemental mass spectrometry simultaneous data about CAT concentration and activity and has to be now proved in several samples.

Conclusions

Sample preparation is one of the critical steps in quantitative metaloprotein analysis, particularly by HPLC-ICP-MS since the accuracy of the final determination relies on the stability of the metal association to the protein. In this work we have tried to establish a quantitative protocol for isolation, purification and characterization of catalase from red blood cells aiming at developing an ICP-MS robust method for the protein determination. For this purpose, complementary information has been sought using typical spectroscopic methods (ICP-MS, UV/VIS and MALDI-MS) and biochemical strategies (activity measurements). The different steps necessary for protein isolation from blood, starting with haemoglobin precipitation with ethanol–chloroform followed by preconcentration of supernant by speed-vac have been found to be critical to obtain quantitative results. Under optimized conditions good CAT extraction efficiency (88 ± 15%) without noticeable chemical alteration of the protein, was obtained. Moreover, anion-exchange HPLC turned out to be a convenient method for orthogonal separation of CAT after their prior fractionation by size-exclusion chromatography as demonstrated by the MALDI-TOF spectra obtained. Good resolution and quantitative recoveries of both Fe present in the protein (heme group) and CAT activity were obtained by the proposed methodologies. All in all, our results indicate that the native compositional and coordinative structure of CAT is preserved during the whole analytical procedure (including sample preparation and chromatographic separation). Therefore, the described robust methodology of sample preparation opens the way to robust quantitative studies of CAT levels and its small changes by post-column HPLC-IDA-ICP-MS in clinical diagnosis and CAT-related oxidative stress research.

Acknowledgements

The authors gratefully acknowledge the Hospital Central of Asturias (Laboratory for Clinical Analysis, Asturias, Spain) for providing the human blood samples. Financial support provided by the Ministry of Education and Science of Spain and Fondo Social Europeo (FEDER) under the Project CTQ2007-60206/BQU is also acknowledged.

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