Covalent binding of food-derived blue pigment phycocyanobilin to bovine β-lactoglobulin detected by mass spectrometry

Abstract

Objective. Phycocyanobilin (PCB) is a blue tetrapyrrole chromophore of C-phycocyanin (C-PC), the major chromoprotein of cyanobacteria Spirulina platensis. It is covalently attached to cysteine residues of C-PC via thioether bond. β-lactoglobulin, the major whey protein, is frequently used as an additive in food products due to its various techno-functional properties. This study aimed to investigate covalent binding of bioactive PCB to bovine β-lactoglobulin.

Material and Methods. Combination of fluoresence spectroscopy, mass spectrometry and electrophoretic techniques was employed in order to examine covalent binding of PCB to BLG. Effects of PCB binding on secondary and tertiary structure of BLG were studied by CD spectroscopy.

Results. SDS-PAGE with Zn2+ staining and fluorescence spectroscopy have revealed that PCB covalently binds to BLG via free cysteine residue, with binding constant of 4 x 105 M-1. BLG-PCB covalent adduct has been detected by mass spectrometry, with both isoforms of BLG being modified to similar extent. Binding of PCB influences secondary and tertiary structure of BLG, while BLG-PCB adduct has altered secondary and tertiary structure in comparison to native BLG.

Conclusions. Our results indicate that BLG, modified by PCB, could serve as suitable oral delivery system of bioactive tetrapyrrole chromophore. Covalent modification of BLG at the same time represents a feasible strategy to impart new functionalities to this important food protein.