Quantification of bindings of organometallic ruthenium complexes to GSTπ by mass spectrometry.

Quantification of bindings of organometallic ruthenium complexes to GSTπ by mass spectrometry.
J Inorg Biochem. 2015 Mar 2;146:44-51
Authors: Lin Y, Huang Y, Zheng W, Wu K, Luo Q, Zhao Y, Xiong S, Wang F
Abstract
Electrospray ionization mass spectrometry (ESI-MS) has been widely used to identify binding sites of metal complexes to proteins. However, the MS quantification of the metal-protein coordination remains a challenge. We have recently demonstrated by ESI-MS analysis that organometallic ruthenium complexes [(η(6)-arene)Ru(en)Cl](+) (arene=p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en=ethylenediamine) bound to human glutathione-S-transferase π (GSTπ) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GSTπ dimer, showing inhibitory potency against the enzyme (J. Inorg. Biochem., 128 (2013) 77-84). Herein, we developed a mass spectrometric method to quantify the binding stoichiometry of the three complexes to GSTπ. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GSTπ complexes and the respective light-labelled peptides in the tryptic digest of equimolar GSTπ were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GSTπ with its substrate GSH significantly reduced the bindings of the ruthenium complexes at Met92 and Cys102, but had little impa...

Source: Journal of Inorganic Biochemistry - March 2, 2015 Category: Biochemistry Authors: Lin Y, Huang Y, Zheng W, Wu K, Luo Q, Zhao Y, Xiong S, Wang F Tags: J Inorg Biochem Source Type: research