Regeneration of the column was achieved with 50 mM EDTA, overnight. All chromatographic experiments were carried out at room temperature (24 °C). Iron-chelating peptide fractions isolated by IMAC were concentrated under nitrogen gas and lyophilized. Finally, they were characterized by amino acid profiling. Analyses were performed in triplicate. Data were expressed as means ± standards deviations (SD) and compared using analysis of variance (ANOVA) and the Tukey test. Statistical analysis was performed using the STATISTICA 7 software package for Windows (StatSoft, Inc., Tulsa, OK, USA). Differences were considered statistically significant check details at P < 0.05. The yeast extract utilised in this study consisted of 3.35%
moisture, 13.88% ash, 62.82% proteins (conversion factor 5.78), 0.15% lipids and 19.80% carbohydrates. The best conditions for hydrolysing the yeast extract were defined by performing a 22 RCCD experiment whose results are shown in Table 1. The peptide elution profile obtained by using IMAC-Fe(III)
technique HDAC inhibitor confirms that there is a fraction of peptides from yeast extract hydrolysates with high ability to chelate iron. The elution profile of Viscozyme hydrolysate is shown in Fig. 1. Peptides without affinity eluted with the equilibration buffer (F1). The peptides with affinity were retained in the column matrix, bound to the immobilized iron, and were eluted with the 100 mM NH4H4PO4 buffer. Similar profiles were obtained for the other two enzymes (Alcalase and Protex). The amino acid profile for each of the yeast extract hydrolysates smaller than 5 kDa and the respective iron-chelating fractions isolated by IMAC were obtained by RP-HPLC (data not shown). Matching the amino acidic composition of peptides in hydrolysates
and chelated peptides Janus kinase (JAK) for the same enzymatic treatment, we observed that chelated peptides from Alcalase hydrolysates were rich in His, Lys and Arg residues, whilst those from Protex and Viscozyme hydrolysates were rich in His, Lys, Arg, and Gly residues. Table 2 shows the enrichment of amino acids in the chelated fractions related to original hydrolysates, total fraction. The iron chelating capacity of residues His, Lys and Arg in peptides has been reported by other researchers (Choi et al., 1998, Kim et al., 2007 and Swain et al., 2002). In the present work it is not so well understood why the presence of these residues favours the chelation of iron in IMAC-Fe(III) preferentially to Asp or Glu. Iron solubility is considered one of the main requirements for promoting a high availability of iron. The solubility of iron plus yeast extract was 20.2 ± 0.8%. This value was lower than that obtained for enzymatic hydrolysates (Table 3), indicating that peptides have a greater number of binding sites for iron than the original yeast extract material. Iron solubility in yeast extract hydrolysates (fractions <5 kDa) ranged from 34% to 40% (Table 3).