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1.
Talanta ; 199: 32-39, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952265

RESUMO

The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.


Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Técnicas Eletroquímicas , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Antineoplásicos/química , Bortezomib/química , Inibidores Enzimáticos/química , Humanos , Microscopia de Força Atômica , Estrutura Molecular , Oligopeptídeos/química , Inibidores de Proteassoma/química , Técnicas de Microbalança de Cristal de Quartzo
2.
Anal Chem ; 91(3): 1920-1927, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30574784

RESUMO

The interaction of proteins with free radicals leads, among other types of damages, to the production of stable carbonyl groups, which can be used as a quantification of oxidative stress at proteins level. The aim of this study was the development of an electrochemical sensor for the detection of carbonyl groups in proteins oxidized by reactive oxygen species. Its working principle is based on the redox properties of dinitrophenylhydrazine (DNPH). BSA was used as a model protein and its oxidation achieved through Fenton reactions. Using differential pulse voltammetry at glassy carbon electrode, the electrochemical behavior of DNPH and of the native and oxidized BSA was investigated in solution. It has been shown that the hydrazine moiety of the DNPH is the electroactive center and is responsible for carbonyl complexation. Special attention was paid to the immobilization of the DNPH in order to retain its redox properties, and this was achieved on a mixed 4-styrenesulfonic acid-nafion matrix. The sensor's surface characterization and the detection of carbonyl groups in oxidized protein were performed by voltammetry, Fourier-transformed infrared spectroscopy and scanning electron microscopy while the voltammetric results were confirmed by surface plasmon resonance measurements. It has been shown that upon interaction with carbonyl groups of the oxidized protein, the oxidation peak of the hydrazine moiety of DNPH decreases as a function of incubation time and protein concentration. The sensor sensitivity was 0.015 nmol carbonyl per mg of oxidized protein and detection limits of 50 µg oxidized BSA and 0.75 pmol carbonyls.

3.
Bioelectrochemistry ; 107: 50-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26523506

RESUMO

Danusertib is a kinase inhibitor and anti-cancer drug. The evaluation of the interaction between danusertib and dsDNA was investigated in bulk solution and using the dsDNA-electrochemical biosensor. The dsDNA-danusertib interaction occurs in two sequential steps. First, danusertib binds electrostatically todsDNA phosphate backbone through the positively charged piperazine moiety. The second step involved the pyrrolo-pyrazolemoiety and led to small morphological modifications in the dsDNA double helix which were electrochemically characterised through the changes of guanine and adenine residue oxidation peaks and confirmed by electrophoretic and spectrophotometric measurements. The nitrenium cation radical product of danusertib amino group oxidation was electrochemically generated in situ on the dsDNA-electrochemical biosensor surface. The danusertib nitrenium cation radical redox metabolite was covalently attached to the C8 of guanine residues preventing their oxidation. An interaction mechanism of dsDNA-danusertib is proposed and the formation of the danusertib redox nitrenium radical metabolite-guanine adduct explained.


Assuntos
Antineoplásicos/química , Benzamidas/química , Técnicas Biossensoriais/métodos , DNA/química , Nitrogênio/química , Pirazóis/química , Adenina/química , Eletroquímica , Radicais Livres/química , Guanina/química , Estrutura Molecular , Conformação de Ácido Nucleico , Oxirredução
4.
Phys Chem Chem Phys ; 13(12): 5227-34, 2011 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-21359288

RESUMO

An in situ evaluation of the dsDNA-methotrexate (MTX) interaction was performed by voltammetry using a DNA-electrochemical biosensor and characterized by atomic force microscopy (AFM) at a highly oriented pyrolytic graphite (HOPG) surface. Electrochemical experiments in incubated solutions showed that the interaction of MTX with dsDNA leads to modifications to the dsDNA structure in a time-dependent manner. The AFM images show reorganization of the DNA self-assembled network on the surface of the HOPG electrode upon binding methotrexate and the formation of a more densely packed and slightly thicker MTX-dsDNA lattice with a large number of aggregates embedded into the network film. The intercalation of MTX between complementary base pairs of dsDNA lead to the increase of purine oxidation peaks due to the unwinding of the dsDNA. The dsDNA-electrochemical biosensor and the purinic homo-polynucleotide single stranded sequences of guanosine and adenosine, poly[G] and poly[A]-electrochemical biosensors, were used to investigate and understand the interaction between MTX and dsDNA.


Assuntos
Antineoplásicos/química , Técnicas Biossensoriais , DNA/química , Metotrexato/química , Eletroquímica , Microscopia de Força Atômica , Estrutura Molecular
5.
J Nucleic Acids ; 20102010 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-20798847

RESUMO

The adsorption and the redox behaviour of thrombin-binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K(+), and voltammetric behaviour of TBA and eTBA. In the presence of K(+), only a few single-stranded sequences present adsorption, while the majority of the molecules forms stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K(+) ions, TBA and eTBA fold into a quadruplex, causing the decrease of guanine oxidation peak and occurrence of a new peak at a higher potential due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the greater difficulty of electron transfer from the inside of the quadruplex to the electrode surface than electron transfer from the more flexible single strands.

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