The Antioxidant Activities Of Six Recombinant Allophycocyanins

Phycobiliproteins are important light-harvesting pigment proteins in some alga. Theseproteins are widely used as food additives, dye, medical diagnose, medicine, fluorescenceprobes and so on, because of their strong fluorescence and many bioactivities, such asanti-oxidative, anti-inflammatory, anti-tumor or cell-growth promoting. In our lab, therecombinant allophycocyanin (r^APC) as a fusion protein with maltose binding protein (MBP)was expressed and developed as a bioactivity protein. The apo-allophycocyanin and itssubunits were expressed by pMAL-p2X and pET28a in Escherichia coli.In this work, recombinant allophycocyanin fused with 6×His tag ---Hβ^APC was purifiedby Ni⁺ affinity chromatography gel. After Ni⁺ affinity chromatography, the purity of Hβ^APCreached 95%.We have also studied and optimized the method of isolation and purification ofphycobiliproteins. We got phycocyanin with absorption ratio A620/A280=4.221 andallophycocyanin absorption ratio A650/A280=4.102 after hydroxylapatite chromatography andDEAE anion exchanged chromatography.The antioxidant activity of different subunits of hybrid protein and nativeallophycocyanin were studied in different vitro experimental models.1. Hydroxyl radical scavenging capacity of different recombinantallophycocyanin have been assayed by the inhibition of damage to 2-deoxyribose. Inthis system, recombinant allophycocyanin fused with MBP tag had the ability toscavenge hydroxyl radicals, and the scavenging effects increased with the increasingconcentration.2. By evaluated competition kinetics of crocin bleaching,we found that onlyrecombinant allophycocyanin fused with 6×His-tag and their α or β subunits canscavenge peroxyl radical successfully. And the separated β subunits had a higherinhibition effect on peroxyl radical than others, with an IC50 value of 27.2μg/mL and therate constant ratio of 1.24, higher than the native allophycocyanin with an IC50 value of57.5μg/mL. We also observed that recombinant allophycocyanin fused with MBP-tagand their α or β subunits have no remarkable scavenging ability on peroxyl radical.3. By the O2-scavenging capacity assay, we found that recombinantallophycocyanin and native allophycocyanin can also scavenging the O2-radical, exceptMβAPC and MαAPC.4. Recombinant allophycocyanin and native allophycocyanin can not alleviatehemolysis of RBC.5. RAPC and HβAPC could inhibit ·OH-induced lipid peroxidation (LPO) in ratliver microsomes remarkably, and the IC50 values were 230.50 and 217.35μg/mL.HAPC and Native APC have weak inhibited effect. And MαAPC, MβAPCand HαAPC haveno remarkable inhibited effect.Those assays showed that HβAPC could scavenge the hydroxyl radical, peroxylradicaland superoxide radical successfully and it could alleviate hemolysis of RBC andinhibit ·OH-induced lipid peroxidation (LPO) in rat liver microsomes.