Tyrosine was explored as a cross-linking agent to form cross-linked bovine serum albumin
(BSA) using laccase as a catalyst. Liquid chromatography-mass spectrometry (LC-MS) and
fluorescence spectra indicated that tyrosine can be mainly oxidized to be dityrosine. Spectra
analysis and molecular weight were used to characterize the BSA treated with tyrosine and laccase.
Both SDS-PAGE and size exclusion chromatography confirmed the formation of cross-linked
BSA, while most of the protein products existed as BSA–tyrosine conjugates. The MALDI-TOF
analysis revealed that five tyrosine units were grafted on one BSA monomer, however one
cross-linked BSA consists of two BSA monomers and 18 tyrosine. Furthermore, the content of the
amino acid of BSA was identified using amino acid analysis, among those the percentage of lysine
presented a visible decline from 12.36% to 11.43%, corresponding to 4-5 lysine residues. The pure
and modified BSA were hydrolyzed by trypsin and the corresponding peptides were obtained.
Different mass of five peptides from LC-MS spectra after hydrolysis indicated that tyrosine could
react with Lys-136, Lys-204, Lys-224, Lys-322 and Lys-537 in BSA, promoting the formation of
BSA–tyrosine conjugates and cross-linked BSA.This study was supported by Chinese Government Scholarship under China Scholar Council (NO. 201906790043) and “the Fundamental Research Funds for the Central Universities (NO. JUSRP52007A). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte