Sample preparation strategies for quantitative analysis of catalase in red blood cells by elemental mass spectrometry.
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.
Department of Physical and Analytical Chemistry, University of Oviedo, C/Julián Clavería 8, Oviedo, Spain. eblancog@uniovi.es
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Mudarra Rubio
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Montes-Bayón
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Blanco-González
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Sanz-Medel
D000837:Anion Exchange Resins; D002374:Catalase / Q000097:blood*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D000837:Anion Exchange Resins; D002374:Catalase