Role of amino acid oxidation and protein unfolding in peroxyl radical and peroxynitrite-induced inactivation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO●) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROO● source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 µM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO●, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.

Recombinant actin-depolymerizing factor of the apicomplexan Neospora caninum (NcADF) is susceptible to oxidation.

Neospora caninum is a member of Apicomplexa Phylum and the causative agent of neosporosis, a disease responsible for abortions in cattle. Apicomplexan parasites have a limited set of actin-binding proteins conducting the regulation of the dynamics of nonconventional actin. The parasite actin-based motility is implicated in the parasite invasion process in the host cell. Once no commercial strategy for the neosporosis control is available, the interference in the parasite actin function may result in novel drug targets. Actin-depolymerization factor (ADF) is a member of the ADF/cofilin family, primarily known for its function in actin severing and depolymerization. ADF/cofilins are versatile proteins modulated by different mechanisms, including reduction and oxidation. In apicomplexan parasites, the mechanisms involved in the modulation of ADF function are barely explored and the effects of oxidation in the protein are unknown so far. In this study, we used the oxidants N-chlorotaurine (NCT) and H2O2 to investigate the susceptibility of the recombinant N. caninum ADF (NcADF) to oxidation. After exposing the protein to either NCT or H2O2, the dimerization status and cysteine residue oxidation were determined. Also, the interference of NcADF oxidation in the interaction with actin was assessed. The treatment of the recombinant protein with oxidants reversibly induced the production of dimers, indicating that disulfide bonds between NcADF cysteine residues were formed. In addition, the exposure of NcADF to NCT resulted in more efficient oxidation of the cysteine residues compared to H2O2. Finally, the oxidation of NcADF by NCT reduced the ability of actin-binding and altered the function of NcADF in actin polymerization. Altogether, our results clearly show that recombinant NcADF is sensitive to redox conditions, indicating that the function of this protein in cellular processes involving actin dynamics may be modulated by oxidation.

Comparative Study of Two Oxidizing Agents, Chloramine T and Iodo-Gen®, for the Radiolabeling of ß-CIT with Iodine-131: Relevance for Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to alteration of the integrity of dopaminergic transporters (DATs). In recent years, some radiopharmaceuticals have been used in the clinic to evaluate the integrity of DATs. These include tropane derivatives such as radiolabeled ß-CIT and FP-CIT with iodine-123 (123I), and TRODAT-1 with metastable technetium-99 (99mTc). Radiolabeling of ß-CIT with radioactive iodine is based on electrophilic radioiodination using oxidizing agents, such as Chloramine T or Iodo-Gen®. For the first time, the present work performed a comparative study of the radiolabeling of ß-CIT with iodine-131 (131I), using either Chloramine T or Iodo-Gen® as oxidizing agents, in order to improve the radiolabeling process of ß-CIT and to choose the most advantageous oxidizing agent to be used in nuclear medicine. Both radiolabeling methods were similar and resulted in high radiochemical yield (> 95%), with suitable 131I-ß-CIT stability up to 72 h. Although Chloramine T is a strong oxidizing agent, it was as effective as Iodo-Gen® for ß-CIT radiolabeling with 131I, with the advantage of briefer reaction time and solubility in aqueous medium.

Use of chloramine-T and two dyes in the sensitive determination of stavudine in pharmaceuticals

Three new methods are described for the assay of stavudine (STV) in bulk drug and in dosage forms using chloramine-T (CAT) and two dyes, methyl orange and indigocarmine, as reagents. Titrimetry involves treating STV with a measured excess of CAT in hydrochloric acid medium, and after the oxidation of STV is judged to be complete, the unreacted oxidant is determined iodometrically. Spectrophotometric methods entail the addition of a known excess of CAT to STV in hydrochloric acid medium followed by determination of residual oxidant by reacting with a fixed amount of either methyl orange and measuring the absorbance at 520 nm (Method A) or indigo carmine and measuring the absorbance at 610 nm (Method B). In all the methods, the amount of CAT reacted corresponds to the amount of STV. In titrimetric method, the reaction follows 1:1 stoichiometry (STV: CAT), and is applicable over the range 1.5-10 mg of STV. In spectrophotometric methods, the absorbance is found to increase linearly with concentration of STV. The systems obey Beer's law for 0.2-2.0 and 1.0-10.0 mg/mL for method A and method B, respectively. The apparent molar absorptivities are calculated to be 5.7x10(4) and 1.5x10(4) L/mol/cm for method A and method B, respectively, and the corresponding Sandell sensitivity values are 0.004 and 0.015 µg/cm². The limits of detection and quantification are reported for both methods. Intra-day and inter-day precision and accuracy of the developed methods were evaluated as per the current ICH guidelines. The methods were successfully applied to the assay of STV in tablet and capsule formulations and the results were compared with those of a reference method by applying Student's t-test and F-test. No interference was observed from common tablet adjuvants. The accuracy and reliability of the methods were further ascertained by performing recovery experiments via standard-addition method.

Descrevem-se três novos métodos para o ensaio de estavudina (STV) na matéria-prima e nas formulações utilizando-se clroamina-T (CAT) e dois corantes, alaranjado de metila e índigo carmim como reagentes. A titulação envolve o tratamento de STV com excesso medido de CAT em meio de ácido clorídrico, e, quando a oxidação se completar, o oxidante que não reagiu é determinado iodometricamente. Os métodos espectrofotométricos compreendem a adição de excesso conhecido de CAT ao STV em ácido clorídrico, seguida da determinação do oxidante residual por meio da reação com quantidade fixada de alaranjado de metila, medindo-se a absorvância a 520 nm (Método A) ou índigo carmim, medindo-se a absorvância a 610 nm (Método B). Em todos os métodos, a quantidade de CAT que reagiu corresponde à quantidade de STV. No método titulométrico, a reação segue a estequiometria 1:1 (STV:CAT) e é aplicável na faixa de 1,5 a 10 mg de STV. Nos métodos espectrofotométricos, a absorvância aumenta linearmente com a concentração de STV. Os sistemas obedecem a lei de Beer nos intervalos de 0,2 a 2,0 mg/mL e 1,0 a 10,00 mg/mL para os métodos A e B, respectivamente, e os valores de sensibilidade de Sandell correspondentes são 0,004 e 0,015 µg/cm². Os limites de detecção e de quantificação são apresentados para ambos os métodos. A precisão e a exatidão intra-dia e inter-dia dos métodos desenvolvidos são avaliadas de acordo com as normas ICH. Os métodos foram aplicados com êxito aos ensaios de STV em comprimidos e em cápsulas e os resultados foram comparáveis com aqueles obtidos com o método de referência, utilizando-se o teste t de Student e o teste F. Não se observou interferência dos adjuvantes comuns em comprimidos. A exatidão e a confiabilidade dos métodos foram ajustadas por meio de experimentos de recuperação via método de adição de padrão.