Separation And Purification Of Cyclolinopeptide And Metal Binding Analysis

Cyclolinopeptide(CLs) are anti-inflammatory orbitides with highly effective bioactivity, consisting of 8 to 10 amino acid residues and molecular masses of approximately 1 k Da. The first part of this study were focusing on the composition and oxidation property and reduction of crude cyclolinopeptides extract. HPLC was employed to detect the content of cyclolinopeptide C, E, D, A in the extract.Reduction was used to aid the separation of singular peptides as it can reduce the types of cyclolinopeptide in the mixture effectively. Furthermore, Fluorescence spectrophotometer,1H-NMR, and ESI-MS were used to explore the properties of binding metals with cyclolinopeptides. Dilution experiments were conducted to simulate the concentration of heavy metals inside human body, in order to analysis the possibility of developing one kind of functional food component. Results were as follows:(1) It was indicated that the cyclolinopeptides were separated well with a good linear correlation with their concentration ranging from 10 to 150 μg/m L where the correlation coefficients were 0.9990 ~ 0.9997. The content of peptides were: CLC 8.1wt.%,CLE 7.7 wt.%,CLD 0.6 wt.%,and CLA 35.2 wt.%.(2) Crude peptides may be transformed by oxidation or reduction. According to the results of qualitative HPLC analysis, 6 peptides were left after oxidation, while only 4peptides were left after reduction and were separated better than the oxidized ones. By comparing with the HPLC analysis of the standard peptides, it confirmed that these 4peptides were CLB, CLL, CLA, CLM. Based on this, singular peptides were separated effectively by preparative HPLC using Microsorb 100-5 C18 column(240mm × 21.4 mm) at an elution concentration of 70% acetonitrile.(3) It is not reasonable to detect the chelation between crude cyclolinopeptide and lead salt by using fluorescence spectra, as the impurity constituent contained in CLs may influence the fluorescent absorbance in a large degree, leading to a confused result.(4) By the detection of 1H-NMR, it was obvious to see the chemical shifts in all case,suggesting that mixture CLs-CE and CLs-JKA,as well as pure CLE and CLJ, hold a strong binding ability with four metal salts, that were Zn(OAc)2, Pb(OAc)2, Zn SO4,Cd(NO3)2 with the gradually rising trend. What’s more, after the oxidation of the cyclolinopeptide, its coordinating capacity with metal ions tended to be weaker.(5) Under the condition of ESI-MS, singly charged [LOs+M–2H]+ and doubly charged [LOs+M–2H]2+, [2LOs+M]2+ were produced in peptide metal solution(10-2 M, methanol). Cd(NO3)2 exhibited stronger binding at 10-2 M when compared to other metals and Zn(OAc)2 least. Metal complexes were observed forming at 10-2 M to 10-4 M but not at lower concentrations(10-5 M to 10-8 M).