Vitale, Rosa (2013) Developing metalloprotein models for diagnostic and sensing applications. [Tesi di dottorato]

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Abstract

Through the study of natural proteins and model systems, it is becoming increasingly clear that different proteins are able to optimize the environment surrounding the heme-cofactor for the acquisition of specific functions. In this respect, this PhD thesis was focused on the study of new models of heme-proteins, belonging to the Mimochrome family. These complexes are porphyrin-peptide conjugates, containing two peptides, covalently linked to deuteroporphyrin IX, through an amide bond between the propionic groups of the porphyrin and the ε-amino function of a lysine residue. The two peptide chains are in α-helical conformation and cover both planes of the heme, resulting in a helix-heme-helix sandwich. Through an iterative process of design, synthesis, characterization and re-design, a penta-coordinated model was developed, Fe(III) Mimochro-meVI, which exhibits a peroxidase-like activity. In order to gain deep insight on the role of the peptide chains in modulating the structural and functional properties of Mimochrome VI, some point amino acid substitutions in its peptide chains were introduced. Here, the rational design, the synthesis and the characterization of five analogs of Mimochrome VI is reported.The newly designed analogues were first screened for their catalytic properties. For this purpose, the oxidation reaction of ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), through the activation of hydrogen peroxide, was studied. This bottom-up approach allowed to select the Fe(III) Mimochrome VI E2L TD analog, whose catalytic efficiency is about three fold higher than the horseradish peroxidase (HRP). Moreover, Fe(III) Mimochrome VI E2L TD is able to perform about 6000 turn over, without bleaching. The spectroelectrochemical characterization of Fe(III)Mimochrome VI immobilized on indium tin oxide (ITO) tridimensional nanoporous films was performed in collaboration with the Laboratoire d’Electrochimie Moléculaire (LEM) in Paris. Nanoporous films of indium tin oxide (ITO) were used as highly sensitive transparent 3D-electrodes for time-resolved spectroelectrochemistry.The high film loading, due to the columnar structure of metal oxides in ITO matrix and Fe(III) Mimochrome VI small dimension, combined with the excellent conductivity of this metal oxide film, gave high sensitivity measurements. This allowed the simultaneous characterization of the heterogeneous one-electron transfer dynamics of the Fe(III)/Fe(II) redox couple by cyclic voltammetry and cyclic voltabsorptometry, up to a scan rate of few volts per second, with a satisfactory single-scan signal-to-noise ratio. The spectroelectroanalytical strategy presented here, opens new opportunities for characterizing redox-coupled chemical reactions, within redox biomimetic systems and catalysts. It might also be of great interest for the development and optimization of new spectroelectrochemical sensors and biosensors. Based on its small dimensions (3.5 kDa) and its highly efficient peroxidase-like catalytic activity, Fe(III)Mimochrome VI is here proposed as reporter enzyme for the functionalization of antibodies usable in immunochemical assays. Human polyclonal antibodies (h IgG) were functionalized with this synthetic enzyme, by the use of cross-linkers. Conjugates with an enzyme/h IgG ratio up to 13 were prepared. This represents a significant result, when compared to commercial immune-globulins, which are usually linked to 3-4 HRP molecules. The complete characterization of the conjugates is in progress, in order to determine their immunoreactivity and to develop a marketable immunochemical kit.

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