Identification of platination sites on human serum transferrin using 13C and 15N NMR spectroscopy

Abstract

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.

A Mass Spectrometric and Molecular Modelling Study of Cisplatin Binding to Transferrin

“…[18] By contrast, binding of platinum(ii) compounds, including cisplatin, does not induce this spectroscopic change. [19] The binding of cisplatin to Tf has been investigated by using UV/Vis spectrophotometry and the results suggest that, in contrast to the two high-affinity sites per Tf monomer observed for iron(iii) binding, [20] cisplatin preferentially occupies a single binding site, [21] in line with the binding of ruthenium(iii) complexes to the protein, although binding of additional cisplatin units elsewhere on the protein cannot be discounted. Accordingly, mass spectrometry (MS) has been used to provide further information and the results show that several cisplatin units can bind to Tf.…”

Shape changes induced by N-terminal platination of ubiquitin by cisplatin

“…A further possibility that was considered was that upon chelation at the Met site, the promoted loss of one of the ammonia ligands could allow for an interaction of a deprotonated amide NH site along the peptide backbone. Platinum-induced deprotonation of backbone amide NH sites has been proposed to occur in peptides and proteins e.g., in the product after reaction of cisplatin with the protein transferrin [46], but tends to occur in X-Met sequences where 6-membered rings can be formed and not at isolated amide NH sites.…”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[36] Of note, a gelbased bottom-up approach using nHPLC-ESI-LTQ MS/MS on purified transferrin rather concluded that the Met382 residue was the most favourable hTf platination site, [113] in agreement with previously reported NMR studies. [160] Competitive experiments using HPLC-ICP-MS and reacting cisplatin with several plasma proteins determined a preference of the metallodrug for HSA after 24 h incubation (complete complexation versus 50% in the case of transferrin) and for haemoglobin. [33,161] Haemoglobin (Hb), the iron-containing oxygentransport metalloprotein found in red blood cells, has also been considered as potential target for cisplatin.…”