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

[S. Minić]

189, VII listova

Hemija - Biohemija / Chemistry - Biochemistry

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...

C-fikocijanin, fikocijanobilin, hromopeptidi, humani serum albumin, goveđi serum albumin, protein-ligand interakcije, stabilnost proteina, spektrofluorimetrija, masena spektrometrija, antioksidativna aktivnost, antikancerogena aktivnost

C-phycocyanin, phycocyanobilin, chromopeptides, human serum albumin, bovine serum albumin, protein-ligand interactions, protein stability, fluorescence spectroscopy, mass spectrometry, antioxidant activity, anticancer activity

547.9:577.1(043.3)

Tekst