Isolation, Purification and Structure Identification of a Calcium-Binding Peptide from Sheep Bone Protein Hydrolysate

To isolate a novel peptide with calcium-binding capacity, sheep bone protein was hydrolyzed sequentially using a dual-enzyme system (alcalase treatment following neutrase treatment) and investigated for its characteristics, separation, purification, and structure. The sheep bone protein hydrolysate (SBPH) was enriched in key amino acids such as Gly, Arg, Pro, Leu, Lys, Glu, Val, and Asp. The fluorescence spectra, circular dichroism spectra, and Fourier-transform infrared spectroscopy results showed that adding calcium ions decreased the alpha-helix and beta-sheet content but significantly increased the random and beta-turn content (p < 0.05). Carboxyl oxygen and amino nitrogen atoms of SBPH may participate in peptide-calcium binding. Scanning electron microscopy and energy dispersive spectrometry results showed that SBPH had strong calcium-chelating ability and that the peptide-calcium complex (SBPH-Ca) combined with calcium to form a spherical cluster structure. SBPH was separated and purified gradually by ultrafiltration, gel filtration chromatography, and reversed-phase high-performance liquid chromatography. Liquid chromatography-electrospray ionization/mass spectrometry identified the amino acid sequences as GPSGLPGERG (925.46 Da) and GAPGKDGVRG (912.48 Da), with calcium-binding capacities of 89.76 +/- 0.19% and 88.26 +/- 0.25%, respectively. The results of this study provide a scientific basis for the preparation of a new type of calcium supplement and high-value utilization of sheep bone.

Automated summary

Using a dual-enzyme system, sheep bone protein was hydrolyzed to isolate a novel peptide with strong calcium-chelating ability. The fluorescence, circular dichroism, and infrared spectroscopy results showed changes in protein structure upon calcium binding. Two calcium-binding peptides were identified in the sheep bone protein hydrolysate.