Purification, Solid-phase Synthesis and Structural Differentiation of Casein Phosphopeptides Active Monomers
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Abstract:
In this paper, the preliminary purification of casein phosphopeptides(CPPs) was investigated using preparative reverse phase high performance liquid chromatography. Four components were collected according to the peak shape of the chromatogram. The F2 component was further separated and purified by analytical RP-HPLC to obtain sub-component P3 The casein phosphopeptides active monomers P3 powder was collected by reduced pressure distillation and lyophilization to remove the solvent. The casein phosphopeptide monomers P3 were synthetically synthesized based on the amino acid sequence of P3. The molecular weight of synthesized P3 was uniform with the natural monomer and the puritywas 98.13%, which was determined by Mass Spectrometry and High Performance Liquid Chromatography. The retention time of the natural and synthetic casein phosphopeptides monomer P3 was consistent, and the ultraviolet absorption of the synthesized monomer was much higher than that of the natural monomer, which could be related to the diversity of protein tertiary structure. There were α-helix, β-turn, β-sheet and random coil in the secondary structure of the natural and synthetic CPPs monomer P3 according to the analysis of Fourier Transform Infrared Spectroscopy (FT-IR) and circular dichroism. The synthesized and natural monomers were mainly in the form of antiparallel and random coil, but there were difference in the ratio of the α-helix, β-turn and random curl of natural and synthetic CPPs monomer P3. The second structural change of natural monomer P3 changed small with the concentration of monomers from 2 mg/mL up to 4 mg/mL, which the α-helix and random coil were transformed to the antiparallel. The second structural of synthesized monomer P3 had a significant variation, which mainly transformed the α-helix and β-turn to the antiparallel and random coil.
