Liquid Chromatography Tandem With Mass Spectrometry Analysis Of Chinese Horseshoe Crab Tachypleus Tridentatus Hemocyanin And Its Antioxidant Activity

In this study,hemocyanin was extracted from the hemolymph of Tachypleus tridentatus and was purified with the AKTA pure protein purification system.The protein was identified by liquid chromatography-tandem mass spectrometry(LCMS/MS).Five in vitro chemical detection methods were applied for determining the antioxidant capacity of Tachypleus tridentatus hemocyanin and its enzymatic peptides.Mouse melanoma B16 cell line was selected to determine the effects of hemocyanin peptides on the proliferation,cell morphology,melanin synthesis and tyrosinase activity of B16 cells.Our results can be referred to further research of hemocyanin and active substances in the hemolymph of Chinese horseshoe crab Tachypleus tridentatus.1.Purification of hemocyanin in Tachypleus tridentatus and its purified product analysis using LC-MS/MSHemocyanin was extracted from the hemolymph of Tachypleus tridentatus and purified by AKTA pure protein purifier.The purified hemocyanin is qualitatively analyzed using liquid chromatography-tandem mass spectrometry(LC-MS/MS),and the result was used to search the target protein database using Maxquant(1.6.2.10).A comprehensive analysis was conducted based on the protein score,sequence coverage size,and the relative intensity of the protein.Finally,22 related protein sequences including Penaeus vannamei,Scylla serrata and four limulus animals were downloaded from the Uni Prot database,and 23 hemocyanin amino acids sequence were compared with multiple sequences using the Clustal W method.NJ phylogenetic tree was constructed with MEGA7 software.The results show that 12 types of hemocyanin subunits were identified from the purified Tachypleus tridentatus hemocyanin,including Hemocyanin subunit I,Hemocyanin subunit II,Hemocyanin subunit IIIa,Hemocyanin subunit IIIb,Hemocyanin subunit IV,Hemocyanin subunit V,Hemocyanin subunit VI etc.The Uniprot database shows that these subunits were first found in Carcinoscorpius rotundicauda and Limulus polyphemus.The N-J phylogenetic tree shows that the hemocyanin of the four limulus species has a closer evolutionary relationship.Secondly,the hemocyanin of the limulus species has a closer evolutionary relationship with the hemocyanin of the Penaeus vannamei and Scylla serrata.This suggests that we can infer the function of Tachypleus tridentatus hemocyanin based on the function of Penaeus vannamei and Scylla serrata hemocyanin.2.Comparative study on the antioxidant activities of Tachypleus tridentatus hemocyanin and its enzymatic hydrolyzed peptidesHemocyanin was extracted from the hemolymph of Tachypleus tridentatus,and its antioxidant activities were studied in vitro with its enzymolysis peptides(the enzymolysis time was 30 min,1h,3h,respectively).The scavenging ability to DPPH,hydroxyl free radicals,superoxide free radicals,hydrogen peroxide and the ability to inhibit lipid peroxidation were measured.Experimental results show that Tachypleus tridentatus hemocyanin has scavenging ability to DPPH,hydroxyl free radicals,superoxide free radicals,and hydrogen peroxide,except for its poor scavenging ability to DPPH(IC50 value is 14 mg/m L),It has a strong scavenging ability to hydroxyl radicals(IC50 value is 1.63 mg/mL),superoxide radicals(IC50 value is 0.42 mg/m L),and hydrogen peroxide(IC50 value is 0.89 mg/m L).Tachypleus tridentatus hemocyanin also has a strong anti-lipid peroxidation ability,with an IC50 value of 0.05mg/m L.Tachypleus tridentatus hemocyanin hydrolyzed peptide has a positive effect on DPPH(IC50 value greater than 50 mg/m L),hydroxyl radicals(IC50 value of 17-30mg/m L),superoxide radicals(IC50 value of 6.5-9.0 mg/m L)The scavenging ability is relatively weak,it has a strong scavenging ability for hydrogen peroxide(IC50 value0.56-0.91 mg/m L)and the ability to resist lipid peroxidation(IC50 value 0.047-0.071mg/m L)is also strong.Among the three groups of enzymatically hydrolyzed peptides,the peptides that were enzymatically hydrolyzed for 3 hours had the strongest ability to scavenge free radicals.It is worth mentioning that when the Tachypleus tridentatus hemocyanin reaches the preset maximum concentration,the scavenging ability of hydroxyl free radicals,superoxide free radicals and hydrogen peroxide are all close to the positive control Vc.The hydrogen scavenging ability is stronger than the positive control Vc.Experimental results show that Tachypleus tridentatus hemocyanin has good antioxidant activity,and the antioxidant activity of its enzymatically hydrolyzed peptide is slightly weaker than that of unhydrolyzed hemocyanin.3.Study on the whitening effect of Tachypleus tridentatus hemocyanin peptidesHemocyanin was extracted from the hemolymph of Tachypleus tridentatus and its enzymatic peptide was used to study the whitening effect.We prepared the hemocyanin peptides of 3h enzymatic hydrolysis into 62.5μg/m L,125μg/m L,250μg/m L,500μg/m L,1000μg/m L solutions,and explored the effects of different concentrations of hemocyanin peptides of melanin synthesis,cell morphology,cell proliferation and tyrosinase activity on mouse melanoma B16 cells.The experimental results show that the Tachypleus tridentatus hemocyanin polypeptide has no significant effect on the proliferation of B16 cells at low concentrations,and has an inhibitory effect on the proliferation of B16 cells when the concentration reaches 250μg/m L.Different concentrations of hemocyanin peptides had no significant effect on the morphology of B16 cells.The above two points indicate that Tachypleus tridentatus is non-toxic or has low toxicity to B16 cells.Tachypleus tridentatus hemocyanin polypeptide has a slight inhibitory effect on melanin synthesis and tyrosinase activity in B16 cells.When the concentration reaches 1000 μg/m L,Tachypleus tridentatus hemocyanin polypeptide has an effect on melanin synthesis and tyrosinase activity of B16 cells.The inhibition rates were 12% and 15%,respectively.In summary,the evolutionary relationship between Tachypleus tridentatus hemocyanin and the other three horseshoe crab hemocyanins is very close.Tachypleus tridentatus hemocyanin has a good ability to scavenge free radicals and has good antioxidant activity.However,Tachypleus tridentatus hemocyanin peptides have poor inhibitory effects on melanin synthesis and tyrosinase activity in B16 mouse melanoma cells.This may be due to the excessive molecular weight of the polypeptide hindering its entry into the cell.The research results of this subject lay the foundation for the study of Tachypleus tridentatus hemocyanin,and also explore new ways for the deep processing of Tachypleus tridentatus serum.