Quantitative Analysis and Simultaneous Activity Measurements of Cu, Zn-Superoxide Dismutase in Red Blood Cells by HPLC−ICPMS

Ordóñez, Yoana Nuevo; Montes-Bayón, Marı́a; Blanco-González, Elisa; Sanz‐Medel, Alfredo

doi:10.1021/ac902624b

Cited by 51 publications

(27 citation statements)

References 30 publications

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“…Metal content of the protein was measured by ICP-MS at SGS India Pvt. Ltd. Gurgaon, India and further analyzed following Zincon and Bathocuproine protocols [ 46 - 48 ]. In all the cases, metal content were analyzed from proteins that were desalted and buffer exchanged with 5 mM K-PO 4 buffer (pH-7.2).…”

Section: Methodsmentioning

confidence: 99%

Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum

et al. 2015

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BackgroundCuZn-Superoxide dismutase (SOD) is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research.ResultsHigh-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl β-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble (enzymatically active) form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu (CuSO4) and Zn (ZnSO4). Yield of the purified recombinant protein was ~ 4 mg L−1 of culture volume and the bacterial biomass was ~ 4.5 g L−1. The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage.ConclusionsOverexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0117-0) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum

et al. 2015

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“…They were then lysed with 6 ml of cold water. Hemoglobin was removed by treatment with EtOH and CHCl 3 . The temperature was maintained at 0°C by means of an ice‐water bath, while 2.7 ml of cold EtOH was added to the lysate under adequate stirring.…”

Section: Methodsmentioning

confidence: 99%

Characterization of superoxide dismutase 1 (SOD‐1) by electrospray ionization‐ion mobility mass spectrometry

et al. 2013

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In this paper, we report nano-electrospray ionization-ion mobility mass spectrometry (nano-ESI-IM-MS) characterization of bovine superoxide dismutase (SOD-1) and human SOD-1 purified from erythrocytes. SOD-1 aggregates are characteristic of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in humans that could be triggered by dissociation of the native dimeric enzyme (Cu(2),Zn(2)-dimer SOD-1). In contrast to ESI-MS, nano-ESI-IM-MS allowed an extra dimension for ion separation, yielding three-way mass spectra (drift time, mass-to-charge ratio and intensity). Drift time provided valuable structural information related to ion size, which proved useful to differentiate between the dimeric and monomeric forms of SOD-1 under non denaturing conditions. In order to obtain detailed structural information, including the most relevant post-translational modifications, we evaluated several parameters of the IM method, such as sample composition (10 mM ammonium acetate, pH 7) and activation voltages (trap collision energy and cone voltage). Neutral pH and a careful selection of the most appropriate activation voltages were necessary to minimize dimer dissociation, although human enzyme resulted less prone to dissociation. Under optimum conditions, a comparison between monomer-to-dimer abundance ratios of two small sets of blood samples from healthy control and ALS patients demonstrated the presence of a higher relative abundance of Cu,Zn-monomer SOD-1 in patient samples.

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“…Human SOD‐1 was purified from blood combining and adapting different parts of several methods described in the literature . Four milliliters of blood were processed at a time.…”

Section: Methodsmentioning

confidence: 99%

Separation and characterization of superoxide dismutase 1 (SOD‐1) from human erythrocytes by capillary electrophoresis time‐of‐flight mass spectrometry

Borges-Alvarez¹,

Benavente²,

Barbosa³

et al. 2012

Electrophoresis

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Cu, Zn‐superoxide dismutase (SOD‐1) is a homodimeric metalloenzyme that has been related to ALS (amyotrophic lateral sclerosis). The majority of ALS cases are sporadic while approximately 10% are inherited (familial ALS, FALS). Mutations in the amino acid sequence of human SOD‐1 cause only 25% of the FALS cases, while the explanation for the rest is not clear yet. In this way, several authors have suggested the importance of posttranslational modifications or dimer dissociation on formation of the characteristic fatal intraneuronal SOD‐1 aggregates. In this paper, we used capillary electrophoresis‐electrospray mass spectrometry with an accurate mass and high‐resolution time‐of‐flight mass spectrometer (CE‐TOF‐MS) for separation and characterization of standard bovine SOD‐1 and human SOD‐1 purified from erythrocytes. Two background electrolytes (BGEs) were used for CE‐TOF‐MS experiments in positive ion mode. An acidic BGE allowed detection of apo‐monomer SOD‐1, because the metal ions were completely released during the electrophoretic separation. The better sensitivity at acidic pH was especially interesting to detect different isoforms of human SOD‐1. In contrast, a neutral BGE provided enhanced conditions for detection of the fully metalated dimeric and monomeric enzyme, but selecting an appropriate fragmentor voltage value in the TOF analyzer was critical to obtain reliable quantitative information. Anyway, only the metalated forms involving the main isoform of human SOD‐1 were detected due to the lower sensitivity. Hence, the combination of both methodologies resulted necessary to obtain detailed structural information from the enzyme.

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Quantitative Analysis and Simultaneous Activity Measurements of Cu, Zn-Superoxide Dismutase in Red Blood Cells by HPLC−ICPMS

Cited by 51 publications

References 30 publications

Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum

Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum

Characterization of superoxide dismutase 1 (SOD‐1) by electrospray ionization‐ion mobility mass spectrometry

Separation and characterization of superoxide dismutase 1 (SOD‐1) from human erythrocytes by capillary electrophoresis time‐of‐flight mass spectrometry

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