Investigation of the mechanism of bivalent iron binding by casein phosphopeptides
AbstractThe article presents the results of investigations of mechanism of binding (chelation) of iron by casein phosphopeptides (CPPs). Molecular mass distribution of peptides in the form of the aqueous solutions of casein phosphopeptides, obtained by enzymatic hydrolysis of sodium caseinate by various proteinases has been studied. The greatest number of low-molecular peptides with molecular weight less than 2.8 kDa has been detected in the trypsin hydrolysate. Peptic and chymotryptic hydrolysates have similar qualitative composition and contain peptides with higher molecular weight. Under chymosin hydrolysis of sodium caseinate, mainly peptides with high molecular weight were formed, and fragments with molecular weight less than 2.8 kDa were not detected. In the study of chelating ability of CPPs it has been found that the maximum amount of iron was bound by peptide fraction of trypsin hydrolysate. Iron content in peptide complexes of trypsin hydrolysate was 18.8 mg/g of protein, and in those of himotrypsin hydrolysate and pepsin hydrolysate – 13.2 and 11.3 mg/g protein, respectively. It was noted that with increasing doses of iron, the degree of chelation of this micronutrient in all hydrolysates reduced. Chromatographic studies have shown that more than 70% of chelated iron in pepsin and trypsin hydrolysates, was found in peptide fractions with a molecular weight of 0.5–1.4 kDa and only 30% in fractions with a molecular weight of 1.4–4.5 kDa. Amino acid profiles of CPPs peptides have been studied in trypsin hydrolysate by using mass-spectroscopy. It was found that after the interaction of amino acids with iron the value of molecular ion peaks on the plots -Val-Ser-Ser-Glu-Glu-, and -Ala-Glu-Ser-Ser-Ser-GluGlu- increased by 15–25%, indicating the binding of these amino acids with the metal. The data obtained showed that most of the iron was associated with low molecular weight fraction of relatively short peptides of molecular weight from 1.4 to 4.5 kDa. It was noted that enzymatic hydrolysis of sodium caseinate by trypsin produced the greatest amount of low molecular weight peptides that bond iron and increased its bioavailability. It should be noted that chelated complexes are easy to penetrate into the cells, freeing the metal exactly where it is needed, protecting it from oxidation and interaction with other elements in the gastrointestinal tract. The results obtained allowed to optimize the nutrient medium and to provide high chelation of iron.
Keywords:casein phosphopeptide, iron, solubilisate, chelation, molecular weight distribution, hydrolysis