Studying the distribution pattern of selenium in nut proteins with information obtained from SEC-UV-ICP-MS and...

In this work, size exclusion chromatography (SEC) with UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used to study the association of selenium to proteins present in Brazil nuts (Bertholletia excelsa) under five different extraction conditions. As expected, better solubilization of proteins was observed using 0.05molL(-1) sodium hydroxide and 1% sodium dodecylsulfate (SDS) in Tris/HCl buffer (0.05molL(-1), pH 8) as compared to 0.05molL(-1) HCl, 0.05molL(-1) Tris/HCl or hot water (60 degrees C). Due to non-destructive character of Tris-SDS treatment, this was applied for studying molecular weight (MW) distribution patterns of selenium-containing nut proteins. Three different SEC columns were used for obtaining complete MW distribution of selenium: Superdex 75, Superdex Peptide, and Superdex 200 were tested with 50mmolL(-1) Tris buffer (pH 8), 150mmolL(-1) ammonium bicarbonate buffer (pH 7.8), phosphate (pH 7.5), and CAPS (pH 10.0) mobile phases. Using Superdex 200 column, the elution of at least three MW fractions was observed with UV detection (200-10kDa) and ICP-MS chromatogram showed the co-elution of selenium with the two earlier fractions. The apparent MWs of these selenium-containing fractions were respectively about 107 and 50kDa, as evaluated from the column calibration. For further characterization of individual selenium species, the defatted nuts were hydrolyzed with proteinase K and analyzed by capillary electrophoresis (CE) with ICP-MS detection. The suitability of CE for the separation of selenite, selenate, selenocystine and selenomethionine in the presence of the nut sample matrix is demonstrated. Complete separation of the above mentioned selenium species was obtained within a migration time of 7min. In the analysis of nut extracts with CE-ICP-MS, selenium was found to be present mainly as selenomethionine.