Determination of the activity of catalase using a europium(III)-tetracycline-derived fluorescent substrate.
A one-step method is described for the fluorometric determination of the activity of the enzyme catalase (EC 1.11.1.6.), based on the finding that H(2)O(2) in the europium (III)-tetracycline-hydrogen peroxide system is consumed by catalase. This is accompanied by a large decrease in both fluorescence intensity and decay time. The limit of detection (LOD; at S/N=3) for catalase at 30 degrees C for a 10-min kinetic assay is 1.0 unit/mL, with a linear range from 1.0 to 10 unit/mL. At an incubation time of 30 min at 37 degrees C for a one-point assay, the LOD is 0.046 unit/mL, with a linear range from 46 to 400 munit/mL. The assay was performed on microtiterplates and is fully compatible with existing plate readers. It is a one-step, simple, and sensitive method suitable for both continuous kinetic and one-point detections, does not require the addition of other substrates, and works best at neutral pH (with an optimum at pH 6.9). The reagent has the typical spectral features of a europium-ligand complex including a large Stokes shift (210 nm), a red line-like emission (centered at 616 nm), and a decay time in the microsecond domain. It is also the first europium-based probe that is compatible with the 405-nm diode laser. In summary, the new assay provides distinct advantages over direct ultraviolet detection and over the two-reagent (peroxidase) method.
Wu
Meng
M
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
Lin
Zhihong
Z
Wolfbeis
Otto S
OS
D002138:Calibration; D002374:Catalase / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D015203:Reproducibility of Results; D013752:Tetracycline / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D009942:Organometallic Compounds; C475854:europium(III) chelate-2,2',2'',2'''-((4-((4-isothiocyanatophenyl)ethynyl)pyridine-2,6-diyl)bis(methylenenitrilo))tetrakis(acetic acid); D002374:Catalase; D013752:Tetracycline