Structure, biological activity and interactions with proteins of phycocyanobilin and C-phycocyanin chromopeptides from cyanobacteria Arthrospira platensis

Arthrospira (Spirulina), fotosintetska filamentozna cijanobakterija, koristi se u ishrani čovjeka od davnina. To je jedan od najbogatijih izvora proteina i esencijalnih amino kiselina, kao i izvanredan izvor vitamina, makro- i mikro-elemenata, esencijalnih masnih kiselina, pigmenata, glikolipida i polisaharida. C-fikocijanin (C-PC), najzastupljeniji protein u Spirulini, je u vodi rastvoran heterodimerni molekul koji se odlikuje intezivnom fluorescencijom. Njegova plava boja potiče od kovalentno vezane (tioetarskom vezom) tetrapirolne hromofore fikocijanobilina. Jedan molekul PCB-a je vezan za α subjedinicu preko cisteinskog ostatka 84, dok β subjedinica vezuje dva molekula hromofore preko cisteinskih ostataka 82 i 153. Brojne studije su pokazale da C-PC ima značajne antoksidativne, anti-inflamatorne, imunomodulatorne i antikancerogene efekte. Štaviše, PCB per se ispoljava različite biološke aktivnosti koje mogu poboljšati zdravlje ljudi. U literaturi ne postoje podaci o strukturi i bioaktivnosti digestijom oslobođenih peptida sa vezanom hromoforom (hromopeptida), kao ni informacije o vezivanju PCB-a za humani (HSA) i goveđi (BSA) serum albumin, glavne proteine plazme i model sisteme za proučavanje protein-ligand interakcija. Prvi dio ove doktorske disertacije imao je za zadatak ispitivanje digestibilnosti (C-PC-a) pepsinom, kao i određivanje strukture i ispitivanje bioaktivnih svojstava hromopeptida dobijenih nakon digestije C-PC-a, izolovanog i prečiščenog iz komercijalnog praha Arthrospira platensis. SDS-PAGE pod redukujućim uslovima je pokazala da se hromoprotein brzo digestuje pepsinom u simuliranoj želudačnoj tečnosti. HPLC analiza digesta je pokazala postojanje pet dominantnih hromopeptidnih frakcija. Struktura oslobođenih hromopeptida je određena primjenom tandemske masene spektrometrije i peptidi, sa varijacijom u veličini od 2 do 13 aminokiselinska ostatka, su identifikovani u obje subjedinice C-PC-a. Utvrđeno je da sve hromopeptidne frakcije ispoljavaju odličnu antioksidativnu (ORAC esej i test ukupne redukujuće moći) i Cu2+-helirajuću aktivnost (studija gašenja fluorescencije), kao i značajne citotoksične efekte (MTT esej vijabilnosti) na ćelijskim linijama humanog cervikalnog adenokarcinoma i epitelnog kancera kolona. Zaključeno je da digestija pepsinom oslobađa biološki aktivne hromopeptide iz C-PC-a, čija je aktivnost u najvećoj mjeri zasnovana na njihovom antioksidativnom potencijalu. U drugom dijelu ove disertacije, najpre je okarakterisano vezivanje (prethodno izolovanog) PCB-a za HSA, a zatim je ispitan uticaj liganda na stabilnost proteina...Arthrospira (Spirulina), photosynthetic, filamentous cyanobacteria, has been used as food for centuries. It is one of the the richest known natural source of proteins and essential amino acids, excellent source of vitamins, macro- and micro-elements, pigments, essential fatty acids, glycolipids, and polysaccharides. C-phycocyanin (C-PC), the most abundant protein of Spirulina, is highly fluorescent and water-soluble heterodimeric protein. Its blue color arises from covalently attached (via tioether bond) tetrapyrrole chromophore phycocyanobilin (PCB). One PCB molecule is attached to α-subunit via Cys 84, while β-subunit binds two molecules of PCB via cysteines 82 and 153. Numerous studies have shown C-PC exhibit significant antioxidative and free radical-scavenging properties, anti-inflammatory, immunomodulatory and anti-cancer effects. Furthermore, PCB itself exhibits various strong health-promoting activities. There are no literature data evaluating structure and bioactivities of peptides with bound chromophore (chromopeptides) released in C-PC digest, nor information on the PCB binding to HSA and BSA, major plasma proteins and model systems for studying protein-ligand interactions. First part of this thesis examined the structures and bioactivities of chromopeptides obtained by pepsin digestion of C-PC, isolated from commercial Arthrospira platensis powder. SDS-PAGE under reducing conditions has shown that chromoprotein is rapidly digested by pepsin in simulated gastric fluid. HPLC analyses of digest revealed five major chromopeptide fractions. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-PC. It was shown that all chromopeptide fractions have excellent antioxidant (ORAC and reducing power test) and Cu2+-chelating (fluorescence quenching study) activities, and show significant cytotoxic effect (MTT viability assay) on human cervical adenocarcinoma and epithelial colonic cancer cell lines. Therefore, obtained results demonstrate that digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore. In the seacond part, binding of PCB to HSA was studied, as well as effects of ligand binding on protein stability. Based on a computational approach (molecular docking), we demonstrated two putative high-affinity binding pockets on HSA for PCB chromophore..

R-Phycocyanin from red algae Porphyra spp: Binding of selected heavy metal ions

Phycobiliproteins are major photosynthetic accessory pigments in cyanobacteria and red algae. Their vivid colours arise from covalently attached tetrapyrrole chromophores. The exciting characteristic of tetrapyrrole chromophores is the ability to bind metal ions. Heavy metals are among the most abundant and most dangerous environmental pollutants, and their removal from the environment is a crucial challenge. Therefore, utilizing PBPs-metal binding properties could be helpful in heavy metal detection and/or removal. The main aim of this study is to characterize the binding of selected heavy metal ions (Hg2+, Pb2+, Cd2+) to R-phycocyanin (R-PC) isolated and purified from red algae Porphyra spp.The protein fluorescence quenching approach revealed the strong binding affinity of R-PC to Hg2+ (Kd~0.1 μM), while protein binding to Pb2+ and Cd2+ is lower (Kd~3 μM) but still in the high to moderate range. Circular dichroism spectroscopy demonstrated the ability of Hg2+, Pb2+ and Cd2+ to slightly -helical content) in R-PC. Our results indicatethat R-PC could beexploited as a potential biosensor for heavy metal ions detection (especially Hg2+) in aquatic systems as well as in their removal from the environment (e.g. waste-water management)

Sugar-mediated thermal stabilisation of C-phycocyanin from Arthrospira platensis

C-phycocyanin (C-PC), the major protein of cyanobacteria Arthrospira platensis, is a blue pigment that primarily transfers energy during photosynthesis.It has diverse biotechnological applications since it can be used in nutraceutical, cosmetics, pharmaceutical industries, and biomedical research.Its intensive blue colour and strong antioxidant activity give C-PC significant potential to replace synthetic colourants in the food industry. However, thermal treatment of food has detrimental effects on C-PC colour due to sensitivity to higher temperatures; therefore,the application of this natural colourant in food and other products is limited. Hence, improving C-PC stability is the major challenge for successful application in food and beverages colouring. In the light of this,we aim to investigate the thermal stability of C-PC in the presence of selected sugars (glucose, fructose and sucrose), commonly used in the food industry. Ex-situabsorption spectrophotometryshowed that 18% solution (w/v) of glucose, sucrose and fructose at pH 7,upon incubation at 65℃, exhibit 91.4, 52.9 and 52.5% of colour preservation, respectively.In situ fluorescence measurements revealed that free C-PC has a melting point of 55.4°C, while the presence of glucose and sucrose increases the melting point of C-PC to 64.4 and 61.4°C, respectively. On the other hand, fructose does not significantly influence the C-PC melting point. These results show that the thermal stability ofthe CPCsolution is substantially increased in the presence of sugars, while the type of sugar significantly determines the extent of the stabilisation effect. Overall, our study provides the strategy for enhancing the application potential of C-PC as natural food colourant, providing a food product with vivid blue colour and substantial antioxidant activities

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

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

Physicochemical characterisation of dihydro-alpha-lipoic acid interaction with human serum albumin by multi-spectroscopic and molecular modelling approaches

The binding of a popular food supplement and well-known antioxidant, dihydro-alpha-lipoic acid (DHLA) to human serum albumin (HSA) was characterised. The binding was monitored by several spectroscopic methods together with the molecular docking approach. HSA was able to bind DHLA with moderate affinity, 1.00±0.05×104 M-1. Spectroscopic data demonstrated that the preferential binding site for DHLA on HSA is IIA (Sudlow I). Both experimental and molecular docking analysis identified electrostatic (salt bridges) and hydrogen bonds as the key interactions involved in DHLA binding to HSA. Molecular docking confirmed that the Sudlow I site could accommodate DHLA and that the ligand is bound to the protein in a specific conformation. The molecular dynamic simulation showed that the formed complex is stable. Binding of DHLA does not affect the structure of the protein, but it thermally stabilises HSA. Bound DHLA had no effect on the susceptibility of HSA to trypsin digestion. Since DHLA is a commonly used food supplement, knowledge of its pharmacokinetics and pharmacodynamic properties in an organism is very important. This study further expands it by providing a detailed analysis of its interaction with HSA, the primary drug transporter in the circulation

Antipsychotic clozapine binds catalase and preserves its activity in oxidative environment

Oxidative stress undoubtedly accompanies mental disorders, and the pleiotropic effects of atypical antipsychotics, recommended drugs in the treatment of psychosis, are not clarified at the molecular level. Catalase is one of the key enzymes of the primary antioxidant protection system. This work studied the binding of second-generation antipsychotic drug Clozapine to commercial bovine liver catalase. Using various spectroscopic methods under simulated physiological conditions, we found moderate binding affinity of clozapine for catalase (Ka ~ 2x105 M-1), the binding influenced the secondary and tertiary structure of protein (according to UV-VIS and CD spectroscopy) and it managed to slightly increase its thermal stability. In AAPH induced oxidation experiments, we found that clozapine efficiently protects catalase from free-radicals oxidation and preserves its activity. Clozapine affects catalase activity in dose dependant manner, having no significant effect at lower concentrations but significantly inhibiting enzyme at saturating concentrations. In conclusion, our results indicate that the effect of direct binding of clozapine to catalase can be both beneficial and harmful and that this effect is dose dependent.The Biochemistry Global Summit, 25th IUBMB Congress, 46th FEBS Congress, 15th PABMB Congress, July 9-14, 2022, Lisbon, Portuga

Stability of food proteins at high pressure conditions

High pressure (HP) is particularly suited to study protein folding/unfolding, revealing subtle structural rearrangements not accessible by other types of denaturation. HP also has many industrial-scale advantages over heat treatments, including “greener” processing and preservation of nutritional values, colors, and flavors of foods. We have combined in situ HP with small-angle (X-ray and neutron) scattering (SAS) and spectrophotometry to follow the structure in solution of proteins of interest for the food industry. SAS is an essential technique for obtaining structural, but low-resolution, information about proteins, when conventional high-resolution structural biology methods are not possible. I will illustrate this approach with two studies on proteins of food interest: (i) bovine β-lactoglobulin (BLG), a whey protein with a high propensity to bind to various bioactive molecules. We probed by SANS1 and absorbance the effects on pressure stability and reversibility of BLG of the binding of retinol (vitamin A), resveratrol (polyphenol), and biliverdin (linear tetrapyrrole chromophore) to different sites on the protein2, 3. (ii) C-phycocyanin (CPC), a phycobiliprotein from cyanobacteria, to which tetrapyrrole chromophores are covalently attached and which can be used as a natural blue dye in the food industry. We studied by SAXS and absorbance HP-induced CPC unfolding and reversibility from two oligomeric states of the protein as a function of pH. Acknowledgements LLB, SOLEIL, and I2BC facilities are acknowledged for beamtime and proteomic expertise. This work was also supported by ANSO Project No. ANSOCR-PP-2021- 01. References 1. Annighöfer B, et al. High pressure cell to investigate protein unfolding up to 600 MPa by small-angle neutron scattering. Rev Sci Instr 2019;90:025106. 2. Minic S, et al. Effect of ligands on HP-induced unfolding and oligomerization of β-lactoglobulin. Biophysical J 2020;119:2262-74. 3. Minic S, et al. Structure of proteins under pressure: covalent binding effects of biliverdin on beta-lactoglobulin. Biophysical J 2022;121:2514-25

Arthrospira platensis and Porphyra sp. – prospective serum- substitute in HEK293T cell culture

Both Arthrospira platensis (Spirulina) and Porphyra sp. (Nori) are algae known for their richness in vitamins, minerals, antioxidants, proteins, such as phycobiliproteins (PBPs). Тhanks to their exceptional nutritional properties they have potential to be considered as a high-quality substitute for fetal bovine serum (FBS) in cellular cultivation, which has numerous drawbacks since it can be involved in contamination development and its composition is still unclear. In this study we investigated the influence of Spirulina and Nori extracts on HEK293T cell line growth and viability in serum-reduced conditions. In DMEM/F12 medium supplemented with 0–10% FBS, the concentration-dependent effects of PBPs on cell proliferation were investigated. Cell viability and cytotoxicity were evaluated by MTT assay. During 3-day observation prior to MTT assay and MTT assay itself showed that HEK293T exhibited improvement in viability at lower PBP concentrations, while presence of higher concentrations resulted in inhibition of growth and change in morphology as a consequence of their cytotoxicity at higher concentrations. These findings suggest that PBPs have a positive outcome on cell growth at relevant doses. In general, in this study were obtained results proving the potential advantages of PBPs at lower doses on cell proliferation in serum-reduced conditions, but also HEK293T cells ability to adapt in non-standard cultivation set-up

Supplementary data for the article: Gligorijević, N.; Minić, S. L.; Robajac, D. B.; Nikolić, M.; Ćirković-Veličković, T.; Nedić, O. Characterisation and the Effects of Bilirubin Binding to Human Fibrinogen. International Journal of Biological Macromolecules 2019, 128, 74–79. https://doi.org/10.1016/j.ijbiomac.2019.01.124

Supplementary material for: [https://www.sciencedirect.com/science/article/pii/S014181301835880X?via%3Dihub]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2824]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/2825

Antioxidant potential of R-phycoerythrin, red protein isolated from macroalga Porphyra spp.

Algae have been consumed as food and medicine for centuries. Their benefits are so pronounced, due to high concentrations of vitamins, minerals, antioxidants and proteins, that they are commonly referred to as superfoods. R-phycoerythrin (R-PE) is a red protein-pigment complex from the light-harvesting phycobiliprotein family, present in large quantities in red algae. It contains covalently bound open chain tetrapyrolle pigments: red phycoerythrobilin and yellow-orange phycourobilin. This study aims to evaluate the antioxidant potential of R-PE from commercially available red alga Porphyra spp. ("nori"). Two other phycobiliproteins (with dark blue phycocyanobilin pigment): R-phycocyanin (also isolated from Porphyra spp.) and C-phycocyanin (from Hawaiian Spirulina Pacifica), were used for comparison purposes. Purified phycobiliproteins were evaluated for antioxidant and metal ion chelating activity by various in vitro antioxidant 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. The results showed R-PE exhibit concentration-dependent antioxidant potential similar to, if not better than that found in phycocyanins, confirming the exceptional value of R-PE as a nutraceutical (dietary supplement, functional food and pharmaceutical)