Calcium-binding properties, stability, and osteogenic ability of phosphorylated soy peptide-calcium chelate

Abstract

Introduction: Bioactive peptides based on foodstuffs are of particular interest as carriers for calcium delivery due to their safety and high activity. The phosphorylated peptide has been shown to enhance calcium absorption and bone formation.

Method: A novel complex of peptide phosphorylation modification derived from soybean protein was introduced, and the mechanism, stability, and osteogenic differentiation bioactivity of the peptide with or without calcium were studied.

Result: The calcium-binding capacity of phosphorylated soy peptide (SPP) reached 50.24 ± 0.20 mg/g. The result of computer stimulation and vibration spectrum showed that SPP could chelate with calcium by the phosphoric acid group, carboxyl oxygen of C-terminal Glu, Asp, and Arg, and phosphoric acid group of Ser on the SPP at a stoichiometric ratio of 1:1, resulting in the formation of the complex of ligand and peptide. Thermal stability showed that chelation enhanced peptide stability compared with SPP alone. Additionally, in vitro results showed that SPP-Ca could facilitate osteogenic proliferation and differentiation ability.

Discussion: SPP may function as a promising alternative to current therapeutic agents for bone loss.

Keywords: calcium supplement; characterization; osteogenic differentiation; peptide-calcium chelate; phosphorylation; thermal stability.

Figure 1

Illustration of molecular dynamic analysis simulation for SPP-Ca. (A) RMSD stability analysis, changes of RMSD of SPPs with simulated time during molecular docking; (B) snapshots of molecular dynamics simulation of SPP-calcium chelate at (0, 20, 50, and 100 ns), the green ball represents Ca²⁺, the gray ball represents carbon atom on amino acid, the blue ball represents nitrogen atom on amino acid, the red ball represents oxygen atom on amino acid, and the white ball represents hydrogen atom on amino acid; (C) molecular dynamic analysis simulation chelation of SPP-Ca complex based on density functional theory calculation in the Materials Studio 6.0 software package at 100 ns.

Figure 2

ITC analyses of the SPP reacting with calcium ions. The upper panel exhibits a representative calorimetric titration curve. CaCl₂ (50 mM) was titrated into 2.5 mM of the peptide solution at 25°C. The lower panel shows the integrated areas corresponding to each titration, plotted as a function of the Ca²⁺/peptide molar ratio. The solid line represents the best curve fit obtained by using an independent binding site model.

Figure 3

UV spectra (A), FTIR spectra (B), and circular dichroism spectra (C) of SPP and SPP-Ca.

Figure 4

TG-DSC analysis of SPP (A) and SPP-Ca (B).

Figure 5

Proliferation of osteoblast response to SPP (A) and SPP-Ca (B). MC3T3-E1 was treated with different concentrations (0.7, 7, and 70 μM) of SPP and SPP-Ca (70 μM) for 72h. Changes in the ALP activity of MC3T3-E1 treated with different concentrations of SPP and SPP-Ca (C). n = 5. Data are presented as means ± SEMs and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. vehicle. ^##Indicated significant differences, p < 0.05.