Metal Species in Biology: Bottom-Up and Top-Down LC Approaches in Applied Toxicological Research

Publisher’s version of article deposited according to Hindawi Publishing Corporation Author Guidelines Copyright information: http://www.hindawi.com/journals/isrn/guidelines/ August 27, 2015.Since the inception of liquid chromatography (LC) more than 100 years ago this separation technique has been developed into a powerful analytical tool that is frequently applied in life science research. To this end, unique insights into the interaction of metal species (throughout this manuscript “metal species” refers to “toxic metals, metalloid compounds, and metal-based drugs” and “toxic metals” to “toxic metals and metalloid compounds”) with endogenous ligands can be obtained by using LC approaches that involve their hyphenation with inductively coupled plasma-based element specific detectors. This review aims to provide a synopsis of the different LC approaches which may be employed to advance our understanding of these interactions either in a “bottom-up” or a “top-down” manner. In the “bottom-up” LC-configuration, endogenous ligands are introduced into a physiologically relevant mobile phase buffer, and the metal species of interest is injected. Subsequent “interrogation” of the on-column formed complex(es) by employing a suitable separation mechanism (e.g., size exclusion chromatography or reversed-phase LC) while changing the ligand concentration(s), the column temperature or the pH can provide valuable insight into the formation of complexes under near physiological conditions. This approach allows to establish the relative stability and hydrophobicity of metal-ligand complexes as well as the dynamic coordination of a metal species (injected) to two ligands (dissolved in the mobile phase). Conversely, the “top-down” analysis of a biological fluid (e.g., blood plasma) by LC (e.g., using size exclusion chromatography) can be used to determine the size distribution of endogenous metalloproteins which are collectively referred to as the “metalloproteome”. This approach can provide unique insight into the metabolism and the plasma protein binding of metal species, and can simultaneously visualize the dose-dependent perturbation of the metalloproteome by a particular metal species. The concerted application of these LC approaches is destined to provide new insight into biochemical processes which represent an important starting point to advance human health in the 21st century.Ye

The cisplatin/serum albumin system: A reappraisal

Since the first approval of cisplatin for cancer treatment in 1978, a lot of attempts have been carried out to characterize in detail its interactions with serum albumin, by far the most important and most abundant plasma protein. The state of the art of those studies was recapitulated by Keppler and coworkers in a comprehensive review article which appeared in Chem. Rev. in 2006. Yet, the general picture was still rather incomplete at that time due to the lack of conclusive structural data. We report here on the main achievements obtained on this system in the period 2006–2018 and try to describe what is now clearly ascertained and what are the still open issues. Remarkably, a detailed structural characterization of this metallodrug/protein system was recently gained thanks to the resolution of the crystal structure of a cisplatin/serum albumin adduct; crystallographic results are nicely complemented by independent MS data. Accordingly, detailed information is obtained on the number and the location of the platinum binding sites. In turn, metallomics investigations permitted to monitor platination of this serum protein in real blood samples. Thus, a rather complete molecular description of the system could be achieved. Conversely, the biological and pharmacological profiles of platinum drugs/serum albumin adducts were drafted in a couple of specific studies; however the results on theses issue are in our opinion still preliminary and controversial and more studies are needed, aimed in particular at establishing clear correlations between the nature of the various platinum/serum albumin derivatives and their biological actions. In any case, the relevance and the impact of cisplatin/serum albumin adducts are herein highlighted and future perspectives briefly depicted.Canadian Institutes of Health Research (CIHR)Alberta Innovates - Research Gran

Manduca sexta IRP1: molecular characterization and in vivo response to iron

Manduca sexta IRP1 was cloned and sequenced. The deduced amino acid sequence of Manduca IRP1 shows high similarity to other IRP1 proteins. The Cys residues required as ligands for the iron sulfur cluster, as well as all residues necessary for aconitase activity are conserved in the insect protein. Purified recombinant Manduca IRP1 binds specifically to transcripts of the iron responsive element (IRE) of Manduca or human ferritin subunit mRNA. Binding activity of the recombinant protein was not influenced by the presence of β-mercaptoethanol. However, IRP/IRE binding activity of cytoplasmic extracts from fat body was decreased by reducing agents in a dose-responsive manner. Fat body IRP1/IRE binding activity was reduced for Manduca sexta larvae injected with low doses of iron, while IRP1 mRNA and protein levels remained stable. At higher iron doses, binding activity increased and stabilized. Hemolymph ferritin levels showed an inverse relationship to IRP1/IRE binding activity. These data suggest that the Manduca IRP1 is likely involved in translational control of ferritin synthesis in a manner similar to that found in vertebrates. However, factors other than iron can influence IRP/IRE interaction and hemolymph ferritin levels in insects

Probing the interaction of bisintercalating (2,2':6',2"-terpyridine)platinum(II) complexes with glutathione and rabbit plasma

Platinum(II) complexes have demonstrated considerable success in the treatment of cancer, but severe toxic side effects drive the search for new complexes with increased tumour selectivity and better efficacy. A critical concept that has to be considered in the context of designing novel Pt complexes is their interactions with biomolecules other than DNA. To this end, here the interactions of 16 previously reported bisintercalating (2,2':6',2"-terpyridine)platinum(II) complexes, [{Pt(terpy)}2μ-(X)]n+ (where X is a linker) with glutathione (GSH) by means of 1H and 195Pt NMR spectroscopy were investigated. The GSH half-life (GSH t½) was determined following the incubation of each [{Pt(terpy)}2μ-(X)]n+ complex with GSH (8 mM). It was observed that complexes 1 – 7, 11, 12 and 14 – 16 reacted more rapidly than cisplatin, whereas complexes 8 – 10, 13 and 17 reacted more slowly (≥200 mins). There was no apparent correlation between linker length and the GSH t½. In order to understand these interactions, two complexes: 1 (t½ < 1 min) and a previously studied 17 [Pt(5,6-dimethyl-1,10-phenanthroline)(1S,2Sdiaminocyclohexane)] (56MESS) (GSH t½=4080 mins) were incubated with rabbit plasma. A “metallomics” approach was used to analyse plasma for all platinum species at the 5 and the 60 min time point and provided results that were congruent with the reaction of the selected Pt complexes with GSH. Our studies demonstrate that the combined application of NMR spectroscopy, cytotoxicity studies and a metallomics approach can contribute to better understand the interaction of [{Pt(terpy)}2μ-(X)]n+ complexes with biomolecules to better assess which compounds may be advanced to in vivo studies

Human cytosolic iron regulatory protein 1 contains a linear iron-sulfur cluster [12]

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