High voltage atmospheric cold plasma modification of bovine serum albumin

Abstract While atmospheric cold plasma has successfully inactivated biohazardous proteinaceous molecules (unwanted enzymes, prions, and allergens) and modified proteins with improved functionality, few studies have characterized plasma-protein interactions. This study investigates the physicochemical interactions, structural alteration, and reaction mechanisms of bovine serum albumin (BSA) subjected to high voltage atmospheric cold plasma (HVACP) generated by a dielectric barrier discharge in sealed bags packed with air or modified air (65% O2, 30% N2, 5% CO2). Treating 10 mL of BSA solution (50 mg/mL) with HVACP for 60 min changed the sample from transparent to yellow and induced protein precipitation. HVACP also induced protein unfolding, altered secondary structure (27% loss of α-helix), and increased disorder structure (10% increase of random coil). HVACP-treated protein increased in average size from 10 nm to 113 μm, with a broader size distribution after 60 min of HVACP treatment. SDS-PAGE and mass spectrometry showed the formation of new peptides from 1 to 10 kDa, indicating plasma-triggered peptide bond cleavage. Chemical analysis and mass spectrometry demonstrated oxidation and deamidation in HVACP-treated samples. This study illustrates that interactions between HVACP-induced reactive species and proteins may introduce structural alterations, protein aggregation, peptide cleavage, and side-group modifications to proteins in aqueous conditions.