@conference{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Minić, Simeon and Nikolić, Milan and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Phycocyanobilin (PCB) is an open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from the cyanobacterium Arthrospira platensis. Our group has previously demonstrated the potential of PCB to covalently modify free cysteine residue of proteins using food protein β-lactoglobulin as a model protein. Relying on the proven ability of PCB to be attached to sulfhydryl groups of proteins, we propose a new method for covalent attachment of PCB to potentially any protein. We used Traut’s reagent (TR, 2-iminothiolane) to introduce free sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its lysine residues. All tested molar ratios of TR per mole of protein successfully modified BSA. A higher degree of modification by TR induced more profound alterations of BSA structure, as evidenced by near-UV and far-UV circular dichroism spectroscopy. At the same time, minor changes in BSA oligomerization and aggregation profile occurred with increasing TR molar ratio. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold molar ratio of PCB per mole of protein. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal choice for balancing a satisfactory signal amplification level and the negative effect on protein structure. BSA covalently modified with PCB has higher antioxidative activity than free BSA. The proposed method thus serves as a proof of concept for labeling virtually any protein with PCB as means of either functionalization through covalent attachment of bioactive PCB or obtaining fluorescent probes for application in fluorescence-based techniques.",
publisher = "Federation of European Biochemical Societies, Wiley",
journal = "The Biochemistry Global Summit, 9th-14th July, 2022. In: FEBS Open Bio",
title = "Covalent modification of bovine serum albumin with phycocyanobilin using Traut’s reagent",
volume = "12",
number = "Suppl. 1",
pages = "302-303",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6029"
}