Characterization of the H- and L-Subunit Ratios of Ferritins by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis

Grady, John K.; Zang, Jiachen; Laue, Thomas M.; Arosio, Paolo; Chasteen, N. Dennis

doi:10.1006/abio.2001.5561

Cited by 19 publications

(21 citation statements)

References 21 publications

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“…CE experiments were therefore undertaken to determine whether GFP undergoes oxidative/ reductive damage elsewhere on the protein following treatment with O 2 •− . SDS-CGE has been shown to be an excellent tool for studying the integrity of proteins following exposure to oxidative reagents [32]. The electropherograms of GFP samples obtained under either native or denaturing conditions showed no change in the protein peak when GFP was exposed to onlỹ 1-2 O 2 •− per GFP (data not shown) which is consistent with the retention of SOD-like activity as observed above.…”

Section: Resultssupporting

confidence: 84%

Quenching of superoxide radicals by green fluorescent protein

Bou-Abdallah

Chasteen

Lesser

2006

Biochimica et Biophysica Acta (BBA) - General Subjects

162

144

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Green fluorescent protein (GFP) is a widely used in vivo molecular marker. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O 2 •− ) and exhibits SOD-like activity by competing with cytochrome c for reaction with O 2 •− . When exposed to high amounts of O 2 •− the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.

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Section: Resultssupporting

confidence: 84%

Quenching of superoxide radicals by green fluorescent protein

Bou-Abdallah

Chasteen

Lesser

2006

Biochimica et Biophysica Acta (BBA) - General Subjects

162

144

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“…At 2.0 mmol?L 21 SDS only a small peak with remarkably increased plate numbers is induced by SDS and similar peak-sharpening has been described for MEKC systems [83], whereas at 10.0 mmol?L 21 SDS some additional minor peaks are induced (Fig. 1), probably representing subunits of Ft or fragments of them [82].…”

Section: Ferritin and Superoxide Dismutasementioning

confidence: 60%

“…Reasons for robustness against SDS are manifold and are related to distinct protein structures. According to Steinhardt et al [52] initial SDS affinity of Ft is low, which is related to the complex quaternary structure of Ft where a highly symmetrical 24-mer is formed by association of two types of subunits, L-and H-chain [82]. To commence unfolding of subunits, they have to be released from the polypeptide associate.…”

Section: Ferritin and Superoxide Dismutasementioning

confidence: 98%

Detection of coexisting protein conformations in capillary zone electrophoresis subsequent to transient contact with sodium dodecyl sulfate solutions

et al. 2005

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Non-native conformations of proteins were generated by temporary contact with aqueous solutions of sodium dodecyl sulfate (SDS) and separated from the native state with capillary zone electrophoresis (CZE) in alkaline borate buffer deficient of SDS. Nine proteins at concentrations of 2.0 or 3.0 mg.L(-1) were compared in terms of their susceptibility to SDS. For superoxide dismutase and ferritin the tendency of unfolding was modest with < 25% of the protein being transformed to the non-native state at 10 mmol.L(-1) SDS. Highest susceptibility was observed for albumin, myoglobin (Mb), and hemoglobin with > 75% in the non-native state even at 2.0 mmol.L(-1) SDS. The influence of varying SDS concentrations on the conformational state of Mb was tested. Increasing the SDS concentration, circular dichroism revealed a reduction in alpha-helix, an increase in random coil, and an introduction of beta-sheet, which is absent in native structure. Modifications in the secondary structure were in agreement with distinct changes in the shape of the non-native Mb peak in CZE and make a gradual unfolding/refolding process with several coexisting molten globules instead of two-state transition of conformations most plausible for Mb. CZE was found to contribute to a further understanding of holo-Mb transformation towards a population of non-native conformations (i) by means of calculated peak area ratios of native to non-native states, which showed sigmoid transition, (ii) by detecting the release of the prosthetic heme group, and (iii) by changes in the effective electrophoretic mobility of the Mb-SDS peaks. Reconstituted holo-Mb forms differed in the Soret band around 410 nm, indicating diversity in the conformation of the heme pocket.

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“…1,2 The approximately twofold slower reaction accounting for two-thirds of the iron, also presumably at the ferroxidase site ( Figure 6), may be due to heterogeneity in the heteropolymer protein from oxidatively damaged subunits, as observed in horse spleen ferritin by sodium dodecyl sulfate/ capillary gel electrophoresis. 28 Whether a peroxo complex is formed in the slower A/C 0 pathway but is not observed experimentally remains an open question. The fact that H 2 O 2 is produced quantitatively in HoSF, 23 that the oxidation stoichiometry is two Fe(II)/O 2 and that superoxide is not detected (Results) all support the idea of peroxo complex formation but does not prove it.…”

Section: Discussionmentioning

confidence: 99%