Covalent modifications of globular proteins with phycocyanobilin and characterization of obtained conjugates

[M. Radomirović]

155 str.

Hemijske nauke - Biohemija / Chemistry - Biochemistry

C-fikocijanin (C-PC) je glavni hromoprotein cijanobakterije Arthrospira platensis, poznate kao Spirulina, koju odlikuje niz pozitivnih efekata po zdravlje ljudi. Predmet ove doktorske disertacije bilo je ispitivanje kovalentne modifikacije odabranih globularnih proteina fikocijanobilinom (PCB), bioaktivnom, fluorescentnom hromoforom C-PC-a.Goveđi β-laktoglobulin (BLG) modifikovan je PCB-om direktno preko slobodne sulfhidrilne (SH) grupe Cys119. U albumin goveđeg seruma (BSA), IgG antitela i goveĊi α-laktalbumin (ALA) SH grupe su uvedene modifikacijom lizinskih ostataka Trautovim reagensom (TR), nakon čega su modifikovani PCB-om. Modifikacijom BLG-a PCB-om dobija se neohromoprotein izmenjene strukture, poboljšanih antioksidativnih osobina, koji je stabilniji na oksidaciju, termički tretman i dejstvo proteolitiĉkih enzima, a uz to ima neizmenjene IgE-vezujuće osobine. BSA modifikovan PCB-om ima značajno povećanu antioksidativnu aktivnost i oksidativnu stabilnost, uz neizmenjenu sposobnost vezivanja malih bioaktivnih molekula. Antitela IgG klase modifikovana PCB-om zadržavaju specifiĉnost prepoznavanja antigena, dok osetljivost fluorescentne detekcije zasnovane na njima zahteva dalju optimizaciju. Modifikacija PCB-om nakon uvođenja SH grupa u ALA, koji sam ne sadrži slobodnu SH grupu, dovodi do značajnih promena strukture i oligomerizacije proteina.Dobijeni rezultati pružaju nova saznanja o efektima modifikacije PCB-om na strukturne i funkcionalne osobine proteina, čime se otvaraju nove mogućnosti za primenu PCB-a, a ujedno pružaju uvid u efekte tiolovanja. Optimizacijom koncentracije TR-a potrebno je napraviti balans između stepena modifikacije PCB-om i negativnog efekata na strukturne i funkcionalne osobine proteina.

C-Phycocyanin (C-PC) is the main chromoprotein of the cyanobacterium Arthrospira platensis, known as Spirulina, renowned for its numerous positive health effects on humans. This thesis investigates the covalent modification of selected globular proteins with phycocyanobilin (PCB), a bioactive fluorescent chromophore of C-PC. Bovine β-lactoglobulin (BLG) was modified with PCB directly through the free sulfhydryl (SH) group of Cys119. In bovine serum albumin (BSA), IgG antibodies, and bovine α-lactalbumin (ALA), SH groups were introduced by modifying lysine residues using Traut’s reagent (TR), followed by PCB modification. Modification of BLG with PCB resulted in a novel chromoprotein with an altered structure and enhanced antioxidant properties, showing increased stability against oxidation, thermal treatment, and proteolytic enzymes, while maintaining unaltered IgE-binding properties. PCB-modified BSA exhibited significantly improved antioxidant activity and oxidative stability, with its ability to bind small bioactive molecules remaining unchanged. PCB-modified IgG antibodies retained their specificity in antigen recognition, though further optimization is required to improve the sensitivity of fluorescent detection. The modification of ALA with PCB, following the introduction of SH groups into ALA, led to significant structural changes and protein oligomerization. The obtained results provide new insights into the effects of PCB modification on the structural and functional properties of proteins, opening up new possibilities for PCB applications while also shedding light on the impact of thiolation. Optimizing TR concentration is essential to balance the degree of PCB modification with potential adverse effects on the structural and functional properties of proteins.

fikocijanobilin, kovalentna modifikacija, β-laktoglobulin, Trautov reagens, albumin goveđeg seruma, IgG antitela, α-laktalbumin, konjugat, funkcionalna hrana, fluorescentno obeležavanje

phycocyanobilin, covalent modification, β-lactoglobulin, Traut’s reagent, bovine serum albumin, IgG antibodies, α-laktalbumin, conjugate, functional food, fluorescent labelling

577.1(043.3)

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