Isolation, characterization and biological activity of R-phycoerythrin from red macroalgae Porphyra spp.

Abstract

Red algae Porphyra spp. are traditionally used in cuisine and medicine of Eastern Asia countries. Porphyra algae, popularly known as “nori”, are rich in proteins, dietary fiber, pigments, inorganic elements, vitamins, polyunsaturated fatty acids and mycosporine-like amino acids. In addition to exceptional nutritional value, a number of studies have shown beneficial physiological effects of these compounds, such as immunomodulating, anticancer, antihyperlipidemic and antioxidative activities, which is why nori has gained recognition as a superfood.1 R-phycoerythrin (R-PE) is the most abundant pigment in Porphyra spp. It is a water-soluble, intensely pink to red colored phycobiliprotein with yellow fluorescence. It's composed of apoprotein portion and covalently bound open-chain tetrapyrrole chromophores, red phycoerythrobilins and yellow-orange phycourobilins. Commercially, R-PE is mostly used as a fluorescent probe, with emerging application as an industrial dye. This protein gets increased recognition as a nutraceutical with pronounced antioxidative, anticancer, immunomodulatory and anti-aging potential.2 In this study, we firstly isolated and purified R-PE from commercially available nori algae dried flakes, by the procedure optimised in our laboratory (Fig. 1). By the same protocol, we also isolated Porphyra's less abundant purple phycobiliprotein R-phycocyanin (R-PC), which was used for comparison purposes. Identities of isolated proteins were confirmed by SDS-PAGE (14% gel; not shown) and by standard spectroscopic methods (Fig. 2), based on positions of the peaks in the UV-visible absorption and fluorescence emission spectra. Obtained purity index was 3.8 for R-PC (A620/A280) and 5.7 for R-PE (A565/A280), suggesting analytical/standard purity for both proteins. Furthermore, results of secondary structures analysis (from far-UV CD spectra data) suggest a high content of α-helices in R-PE (72%) and R-PC (66%), in accordance to literature data.3 Thermal stability monitoring (by CD spectroscopy) and melting point (Tm) determination results indicate that R-PE (Tm~76,0°C) is notably more stable than R-PC (Tm~55°C), which makes it a good candidate for application in food industry. Finally, we evaluated R-PE bioactivity in terms of its ability to bind physiologically important, redox active Cu2+ and Zn2+ ions, and protein antioxidant and free radicals scavenging activities. UV-VIS and CD spectroscopy data revealed binding of metal ions to R-PE, without significant impact on protein secondary structure. Binding constants determined by fluorescence quenching method were: 7.4 x 105 M–1 (Cu2+) and 1.2 x 103 M–1 (Zn2+). Results from in vitro assay systems [DPPH–, ABTS–, hydroxyl radical–, and superoxide anion radical–scavenging activity, ferric ion reducing ability of plasma (FRAP) assay, ferrous ion-chelating activity (FICA), and reducing power (RP) assay] showed that R-PE exhibit concentration-dependent antioxidant potential similar to, if not better than that found in R-PC. Our results support observed health-related benefits and importance of further research on this phycobiliprotein. References 1. Fleurence J, Levine I (eds) Seaweed in health and disease prevention. 1st ed. Cambidge: Academic Press, 2016. 2. Sonani RR, Rastogi RP, Patel R, Madamwar D. Recent advances in production, purification and applications of phycobiliproteins. World J Biol Chem 2016;7(1):100-9. 3. D'Agnolo E, Rizzo R, Paoletti S, Murano R. R-phycoerythrin from the red alga Gracilaria longa. Phytochemistry 1994;35(3):693-6.