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Light‐Driven Horseradish Peroxidase Cycle by Using Photo‐activated Methylene Blue as the Reducing Agent
Author(s) -
Soares Vanessa A.,
Severino Divinomar,
Junqueira Helena C.,
Tersariol Ivarne L. S.,
Shida Cláudio S.,
Baptista Maurício S.,
Nascimento Otaciro R.,
Nantes Iseli L.
Publication year - 2007
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2007.00158.x
Subject(s) - chemistry , photochemistry , horseradish peroxidase , reaction rate constant , electron paramagnetic resonance , hydrogen peroxide , methylene blue , peroxidase , flash photolysis , dissociation constant , absorbance , visible spectrum , nuclear chemistry , photocatalysis , kinetics , chromatography , organic chemistry , enzyme , catalysis , optoelectronics , biochemistry , physics , receptor , nuclear magnetic resonance , quantum mechanics
In this work, the regeneration of native horseradish peroxidase (HRP), following the consecutive reduction of oxo‐ferryl π‐cation (compound I) and oxo‐ferryl (compound II) forms, was observed by UV–visible spectrometry and electron paramagnetic resonance (EPR) in the presence of methylene (MB + ), in the dark and under irradiation. In the dark, MB + did not affect the rate of HRP compound I and II reduction, compatible with hydrogen peroxide as the solely reducing species. Under irradiation, the dye promoted a significant increase in the native HRP regeneration rate in a pH‐dependent manner. Flash photolysis measurements revealed significant redshift of the MB + triplet absorbance spectrum in the presence of native HRP. This result is compatible with the dye binding on the enzyme structure leading to the increase in the photogenerated MB ˙ yield. In the presence of HRP compound II, the lifetime of the dye at 520 nm decreased ∼75% relative to the presence of native HRP that suggests MB ˙ as the heme iron photochemical reducing agent. In argon and in air‐saturated media, photoactivated MB + led to native HRP regeneration in a time‐ and concentration‐dependent manner. The apparent rate constant for photoactivated MB + ‐promoted native HRP regeneration, in argon and in air‐saturated medium and measured as a function of MB + concentration, exhibited saturation that is suggestive of dye binding on the HRP structure. The dissociation constant ( K MB ) observed for the binding of dye to HRP was 5.4 ± 0.6 μ m and 0.57 ± 0.05 μ m in argon and air‐saturated media, respectively. In argon‐saturated medium, the rate of the conversion of HRP compound II to native HRP was significantly higher, k 2argon = (2.1 ± 0.1) × 10 −2 s −1 , than that obtained in air‐equilibrated medium, k 2air = (0.73 ± 0.02) × 10 −2 s −1 . Under these conditions the efficiency of photoactivated MB + ‐promoted native HRP regeneration was determined in argon and air‐equilibrated media as being, respectively: k 2 / K MB = 3.9 × 10 3 and 12.8 × 10 3 m −1 s −1 .