title: Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin.
abstract: Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4' Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4' Cl-tpy)(dach)Cl](+) (2) (where 4' Cl-tpy=4'-chloro-2,2':6',2″-terpyridine, en=1,2-diaminoethane and dach=1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77±0.14 and 6.95±0.43mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue.
authors.affiliation: Laboratory of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia. Electronic address: marija.nisavic@vin.bg.ac.rs.
authors.forename: Marija
authors.initials: M
authors.lastname: Nišavić
authors.affiliation: Laboratory of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: Romana
authors.initials: R
authors.lastname: Masnikosa
authors.affiliation: Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia.
authors.forename: Ana
authors.initials: A
authors.lastname: Butorac
authors.affiliation: Centre for Proteomics and Mass Spectrometry, Division of Molecular Medicine, Ruđer Boškovic Institute, Zagreb, Croatia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Perica
authors.affiliation: Faculty of Science, University of Kragujevac, Kragujevac, Serbia.
authors.forename: Ana
authors.initials: A
authors.lastname: Rilak
authors.affiliation: Laboratory of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: Lela
authors.initials: L
authors.lastname: Korićanac
authors.affiliation: Centre for Proteomics and Mass Spectrometry, Division of Molecular Medicine, Ruđer Boškovic Institute, Zagreb, Croatia.
authors.forename: Amela
authors.initials: A
authors.lastname: Hozić
authors.affiliation: Laboratory of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia.
authors.forename: Marijana
authors.initials: M
authors.lastname: Petković
authors.affiliation: Centre for Proteomics and Mass Spectrometry, Division of Molecular Medicine, Ruđer Boškovic Institute, Zagreb, Croatia.
authors.forename: Mario
authors.initials: M
authors.lastname: Cindrić
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D013058:Mass Spectrometry; D012428:Ruthenium / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*
chemical_list: D012710:Serum Albumin, Bovine; D012428:Ruthenium
keywords: Anticancer drug; MALDI MS; Meridional geometry; Metallodrug protein binding; Protein binding site; Ruthenium complex