Quantification of Metallothionein-like Proteins in the Mussel <i>Mytilus galloprovincialis</i> Using RP-HPLC Fluorescence Detection

Ghazi, Issam El; Menge, Sieglinde; Miersch, Juergen; Chafik, A.; Benhra, Ali; Elamrani, M.; Krauss, Gerhard

doi:10.1021/es035093+

Cited by 14 publications

(4 citation statements)

References 39 publications

(43 reference statements)

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“…The idea for this method was based on earlier results obtained from uorescence probes for proteins and our proposed uorescence system. 20,21 We also show that earthworm MT are responsive to metals in a concentration-and timedependent manner. Cadmium, lead, zinc, and copper (all as ions) were used in this study because they have been shown to induce the production of MT in earthworms.…”

Section: Introductionmentioning

confidence: 51%

Quantification of metallothioneins in the earthworm by lomefloxacin–europium(iii) fluorescent probe

Geng

Liang

Liu

et al. 2014

Environ. Sci.: Processes Impacts

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A new fluorimetric method was established for the determination of trace amounts of metallothioneins (MT) in earthworm, using a lomefloxacin-europium(iii) (LMLX-Eu(3+)) complex as a fluorescent probe. In a pH 6.5 Tris-HCl buffer solution, MT can markedly decrease the fluorescence intensity of LMLX-Eu(3+) at λ = 613 nm, and the magnitude of the decrease in this intensity was in direct proportion to the concentration of MT. The linear range was 0.08-20 mg L(-1) with a detection limit of 0.022 mg L(-1), and the recovery was in the range of 91.9-104.4%. The results show that the fluorimetric method is relatively accurate and sensitive to measurements of concentration for MT over a wide range. This method has been successfully applied to the determination of the concentration of MT induced by heavy metal ions (Cd(2+), Pb(2+), Cu(2+), Zn(2+)) in Eisenia andrei. The amount of MT increased significantly in a dose-dependent manner to the heavy-metal exposure, and these proteins can be used as biomarkers to assess the impact of heavy-metal contamination in soils. The method offered high sensitivity as well as accuracy with simple instrumentation and is suitable for direct quantification of total MT in Eisenia andrei.

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Section: Introductionmentioning

confidence: 51%

Quantification of metallothioneins in the earthworm by lomefloxacin–europium(iii) fluorescent probe

Geng

Liang

Liu

et al. 2014

Environ. Sci.: Processes Impacts

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“…Since MTs have a much higher Cys content than most proteins, an assessment of the sulfhydryl groups would facilitate the development of a highly selective assay whose sensitivity would be enhanced by a reaction with a fluorogenic compound, 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium salt (SBD-F) [ 41 , 42 ]. An analysis by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector (FLD) is shown in Figure 4 .…”

Section: Resultsmentioning

confidence: 99%

Structural Characterization and In Vitro Antioxidant Activity of Metallothionein from Oratosquilla oratoria

Mei

Zhang

et al. 2022

Molecules

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We report here the purification of a novel metal-binding protein from Oratosquilla oratoria (O. oratoria MT-1) by gel and ion-exchange chromatography. SDS-PAGE and MALDI-TOF analyses demonstrated that isolated O. oratoria MT-1 was of high purity with a molecular weight of 12.4 kDa. The fluorescence response to SBD-F derivatives revealed that O. oratoria MT-1 contained a large number of sulfhydryl groups, which is a general property of metallothioneins. Zn and Cu metal stoichiometries for O. oratoria MT-1 were 3.97:1 and 0.55:1, respectively. The proportion of cysteine (Cys) residues in the amino acid composition was 32.69%, and aromatic amino acids were absent. The peptide sequence coverage with Macrobrachium rosenbergii calmodulin (accession AOA3S8FSK5) was 60%. Infrared spectroscopy of O. oratoria MT-1 revealed two obvious peaks at absorption frequencies for the amide I band and the amide II band. CD spectra revealed that the secondary structure was mainly composed of random coil (57.6%) and β-sheet (39.9%). An evaluation of in vitro antioxidant activity revealed that isolated O. oratoria MT-1 has strong reducing activities, exhibiting scavenging rates for DPPH and OH of 77.8% and 75.8%, respectively (IC50 values 0.57 mg/mL and 1.1 mg/mL). O. oratoria MT-1 may be used as a functional additive in cosmetics, health foods, and medical products, as well as a reference material for quantitative analysis of metallothionein in such products.

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“…A number of analytical protocols for the isolation and quantification of total MTs and MT isoforms have been developed. Reported methods include the use of sulfhydryl labeling fluorophores (SBD-F) followed by tandem-column HPLC-fluorescence quantification [21], indirect assays for the quantification of bound metals [22], high-performance liquid chromatography (HPLC) assays [23], chronopotentiometric stripping analysis [24], capillary electrophoresis (CE) [25], and enzyme-linked immune sorbent assays [26]. Contemporary hyphenated CE/ICP-MS and HPLC/ICP-MS techniques for improved characterization of MT isoforms and the determination of stoichiometric formulas of MT metal complexes have also been reported [27][28][29][30].…”

Section: Model Applications and Experimental Conditionsmentioning

confidence: 99%

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening

et al. 2012

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This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use.

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Quantification of Metallothionein-like Proteins in the Mussel Mytilus galloprovincialis Using RP-HPLC Fluorescence Detection

Cited by 14 publications

References 39 publications

Quantification of metallothioneins in the earthworm by lomefloxacin–europium(iii) fluorescent probe

Quantification of metallothioneins in the earthworm by lomefloxacin–europium(iii) fluorescent probe

Structural Characterization and In Vitro Antioxidant Activity of Metallothionein from Oratosquilla oratoria

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening

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