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

Abstract

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.

Metallothioneins in Earthworms: The Journey So Far

“…The improvement of fl uorescent technique for MT detection resulted in detecting trace amount of MTs where fl uorescent agents like ammonium-7fl uorobenzo-2-oxa-1, 3-diazole-4-sulfonate (SBD-F) [96] and monobromobimane (mBBr) [97] are derived. Geng, et al [98] further improved on the fl uorimetric method for MT quantifi cation; it was sensitive to a wide range of MT concentrations and gave a relatively accurate estimation of MT. It however required tandem column system to separate the derived compound to eliminate interference or require a prior MT purifi cation before derivation.…”

Cellular Response of Adapted and Non-Adapted <em>Tetrahymena thermophila</em> Strains to Europium Eu(III) Compounds

“…In this latter study, fluorescence analysis indicates that La(III) binds to both metallothioneins via the oxygen atoms of aspartic or glutamic acid residues. A fluorimetric method of quantifying MTs is based on the use of lomefloxacin-europium(III) complex as a fluorescent probe, since MT reacts with the LMLX-Eu(III) system forming a stable ternary complex (LMLX-Eu(III)-MT) [65]. Therefore, in the case of both La(III) and Eu(III) it is shown that these trivalent cations can interact with these metal chelating proteins.…”

Cellular Response of Adapted and Non-Adapted Tetrahymena thermophila Strains to Europium Eu(III) Compounds

“…In the latter study, fluorescence analysis indicates that La(III) binds to both metallothioneins via the oxygen atoms of aspartic or glutamic acid residues. A fluorimetric method for quantification of MTs is based on the use of lomefloxacin-europium(III) complex as a fluorescent probe, since MT reacts with the LMLX-Eu(III) system to form a stable ternary complex (LMLX-Eu(III)-MT) [65]. Thus, in the case of both La(III) and Eu(III), it is shown that these trivalent cations can interact with these metal chelating proteins.…”