C-phycocyanin, the major protein of cyanobacteria Spirulina, possesses significant antioxidant, anti-cancer, anti-inflammatory and immunomodulatory effects, ascribed to covalently attached linear tetrapyrrole chromophore phycocyanobilin. There are no literature data about structure and biological activities of released peptides with bound chromophore in C-phycocyanin digest. This study aims to identify chromopeptides obtained after pepsin digestion of C-phycocyanin and to examine their bioactivities. C-phycocyanin is rapidly digested by pepsin in simulated gastric fluid. 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-phycocyanin. Following separation by HPLC, chromopeptides were analyzed for potential bioactivities. It was shown that all five chromopeptide fractions have significant antioxidant and metal-chelating activities and show cytotoxic effect on human cervical adenocarcinoma and epithelial colonic cancer cell lines. In addition, chromopeptides protect human erythrocytes from free radical-induced hemolysis in antioxidative capacity-dependant manner. There was a positive correlation between antioxidative potency and other biological activities of chromopeptides. Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore.
Spirulina has a documented history of use as a food for more than 1000 years, and has been in production as a dietary supplement for 40 years. Among many of Spirulina bioactive components, blue protein C-phycocyanin and its linear tetrapyrrole chromophore phycocyanobilin occupy a special place due to broad possibilities for application in various areas of food technology. The subject of this chapter is up-to-date food applications of these Spirulina components, with a focus on their use as food colorants, additives, nutriceuticals, and dietary supplements. Their other actual and future food application possibilities will also be briefly presented and discussed.
Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.
Human serum albumin (HSA) is an important regulator of the pharmacokinetic properties of bioactive compounds. Phycocyanobilin is a blue tetrapyrrole chromophore of Cphycocyanin with proven health-promoting activities. Despite its structural similarity to bilirubin, the conformation it adopts in aqueous solution is different and the pigment is more soluble than bilirubin. The aim of our study was to examine binding of phycocyanobilin for HSA and to investigate its competition with bilirubin. Based on a computational approach, we demonstrated two putative high-affinity binding pockets on HSA of virtually identical energies for the neutral and anion forms of bilirubin, but with slightly favorable predictions for anion forms of phycocyanobilin. Computational prediction of phycocyanobilin pK a values suggested a monoanion form to be the most stable form at physiological conditions. The computationaly predicted binding sites for phycocyanobilin were identical to the two previously identified binding sites for bilirubin (subdomains IB and IIA). Results obtained by protein and pigment fluorescence measurements, circular dichroism, and competition experiments confirmed high affinity (binding constant of 2.2 x 10 6 M -1 at 25°C), stereoselective binding of phycocyanobilin M-conformer to HSA and its competition with bilirubin, warfarin and hemin. Our experimental data confirm that phycocyanobilin binds to IB and IIA binding site of HSA with an affinity similar to bilirubin. In conditions characterized by an increased bilirubin plasma concentration, or intake of drugs binding to IB or IIA binding site, pharmacokinetics of phycocyanobin may also be changed.
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