Screening, purification, and identification of a copper-dependent FITC-binding protein in human plasma: albumin.
In this study, a protein purified by fluorescein isothiocyanate (FITC)-affinity chromatography from human plasma was identified as albumin by MALDI-TOF-MS. Albumin was found to conjugate with FITC-labeled molecules through a copper-dependent reaction. The formation of this complex was confirmed by methods including a newly developed "charcoal-based fluorescence assay" (CFA), gel-filtration, affinity chromatography, and ultrafiltration. The binding was identified as disulfide bridge formation. This is the first to demonstrate that copper induces a covalent binding of FITC-labeled molecules with albumin. In addition, the developed CFA method facilitates the screening of small fluorescent dyes binding to macromolecules.
Wu
Yu-Wei
YW
Graduate Institute of Pharmacy, Taipei Medical University, Taipei, Taiwan.
Chen
Sung-Fang
SF
Yang
Charng-Bin
CB
Tsai
Yu-Hui
YH
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry; D002850:Chromatography, Gel; D003300:Copper / Q000097:blood / Q000737:chemistry*; D003619:Dansyl Compounds / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D001798:Blood Proteins; D003619:Dansyl Compounds; D004220:Disulfides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012709:Serum Albumin; D003300:Copper; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate