Abstract

A method based on the coupling of high resolution size-exclusion liquid chromatography using a polymer stationary phase with inductively coupled plasma mass spectrometry was developed to study the interactions of two metallodrugs - cisplatin and RAPTA-T - with the serum proteins albumin and transferrin. In contrast to previous approaches, the technique allowed the total recovery of the metals from the column and was able to discriminate between the different species of the metallodrugs and their complexes with the proteins at femtomolar detection levels. Metal binding was found to be dependent on the protein concentration and on the incubation time of the sample. Cisplatin was found to bind the serum proteins to the same extent, whereas RAPTA-T showed marked preference for transferrin. The affinity of the ruthenium complex for holo-transferrin was higher than for the apoform suggesting a cooperative iron-mediated metal binding mechanism. RAPTA-T binding to holotransferrin was further investigated by electrospray mass spectrometry using both the intact protein and a model peptide mimicking the iron-binding pocket.

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