Purification And Characterization Of Bioactive Peptides From Phycoerythrin

In recent year, marine algae was attracted increasing interest based on beneficial health effects, including reduction of risk for various diet-related chronic diseases such as cardiovascular disease(CVD), antioxidant activity and anticancer activity. Angiotensin I-converting enzyme(ACE, EC 3.4.15.1) catalyzes the conversion of angiotensin I to angiotensin II, a stronger vasopressor. It also inactivates bradykinin, a vasodilatory peptide. Moreover, various oxygen free radicals, generated in excess or not eradicated after formation, can cause dysregulated cell signaling and oxidative DNA damage. Nowadays, cancer is one of the worldwide public health problems, including silver cancer, colon cancer, breast cancer and leukemia. Recent studies have focused on the preparation of natural bioactive peptides from various sources with the inhibition of ACE to decrease blood pressure, the elimination of oxygen free radicals to reduce oxidative damage and the effect on the cancer viability. Red algae Bangia fusco-purpurea is a unique consumable seaweed which distributes mainly near the southeast coast of Fujian province, China. R-phycoerythrin is produced predominately by red algae, and its content in Bangia fusco-purpurea is 4-5 times higher than that in Porphyra umbilicalis. R-phycoerythrin from red algae could be used as an interesting source for investigating bioactive peptides.In this study, R-phycoerythrin was purified from red algae Bangia fusco-purpurea after 35-50% ammonium sulfate fractionation followed by ion-exchange column chromatography on DEAE-Sepharose, resulted in a purity(A565/A280) of 5.1. The Circular Dichroism(CD) spectroscopy result suggested that the structure of R-phycoerythrin predominately consists of helix conformation. Circular dichroism chromatograms, ultraviolet-visible absorption spectra and fluorescence emission spectra of R-phycoerythrin revealed its conformational changes at different temperatures and p Hs. After in vitro simulated gastrointestinal(GI) digestion of R-phycoerythrin, the ACE inhibitory activity of the final GI digest with IC50 value of 187.0 μg/m L was determined, and its scavenging activity of ABTS radical(EC50, 769.9 μg/m L), DPPH radical(EC50, 421.9 μg/m L), hydroxyl radical(EC50, 32.4 μg/m L), and reducing power(A700=0.5, 625.8 μg/m L) were obtained. Our present results indicated that digestion resistance bioactive peptides of R-phycoerythrin could be obtained by in vitro gastrointestinal proteinases including pepsin and trypsin degradation. Pepsin followed by trypsin was selected to hydrolyze R-phycoerythrin to produce bioactive peptides. Gel filtration chromatography analysis showed that the molecular weight distribution of the final hydrolysate of R-phycoerythrin was below 2 k Da, which occupied a proportion of 89.9%.Peptides with bioactivities were purified respectively from the final hydrolysate of R-phycoerythrin by chromatographies including Sephadex G-15 and reversed-phase high performance liquid chromatogram(RP-HPLC). Two peptides with potent ACE inhibitory activity and other three peptides with scavenging activity of ABTS radical were obtained. The sequences of two purified peptides were further identified as ALLAGDPSVLEDR and VVGGTGPVDEWGIAGAR by automated Edman degradation method. ALLAGDPSVLEDR was 92.3% identical(12/13) to β-subunit of R-phycoerythrin from Polysiphonia urceolata, while VVGGTGPVDEWGIAGAR was 94.1% identical(16/17) to α-subunit of R-phycoerythrin from Polysiphonia urceolata. Both of them were then synthesized, and exhibited corresponding ACE inhibitory activity with IC50 value of 70.1 μg/m L and 65.5 μg/m L respectively. Furthermore, the effects of these synthesized peptides on liver cancer(Hep G2 cells, SMMC 7721 cells) and colon cancer(Caco-2 cells) were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide(MTT) assay. ALLAGDPSVLEDR exhibited a slight decrease of cancer cells viability in a dose dependent manner, indicating inhibitory activity with IC50 value of 7.89 mg/m L for Hep G2 cells growth, 7.48 mg/m L for SMMC 7721 cells growth and 4.28 mg/m L for Caco-2 cells growth respectively. Our present study suggested that bioactive peptides derived from R-phycoerythrin hydrolysate may be used as an ideal nutrient for exploitation of functional foods.