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Native And Chimeric Metal-Binding Lactate Dehydrogenase As Detection And Protection Tools For Oxidative Stress Induced By Fenton'S Reaction

Tangkosakul, Teerawit LU ; Tantimongcolwat, Tanawut; Isarankura-Na-Ayudhya, Chartchalerm; Mejare, Malin; Bülow, Leif LU and Prachayasittikul, Virapong (2009) In EXCLI Journal 8. p.1-11
Abstract
In the present study, a simple and reliable antioxidant screening technique based on lactate dehydrogenase (LDH) oxidation by Cu2+-mediated Fenton's reaction has successfully been developed. Oxidation of LDH by hydroxyl radical consequently leads to enzymatic inactivation, while addition of antioxidants can protect and regain enzyme activity. This method demonstrated a high feasibility on detecting of antioxidative activity of lipophilic (e.g. alpha-Tocopherol and beta-Carotene) and hydrophilic compounds (e. g. glutathione, mannitol and thiourea) in a single assay. Results from linear correlation curves revealed that the IC50 were in the order of beta-carotene (3.45 mu g/ml) > alpha-Tocopherol (52.31 mu g/ml) > Mn(II)-bacitracin... (More)
In the present study, a simple and reliable antioxidant screening technique based on lactate dehydrogenase (LDH) oxidation by Cu2+-mediated Fenton's reaction has successfully been developed. Oxidation of LDH by hydroxyl radical consequently leads to enzymatic inactivation, while addition of antioxidants can protect and regain enzyme activity. This method demonstrated a high feasibility on detecting of antioxidative activity of lipophilic (e.g. alpha-Tocopherol and beta-Carotene) and hydrophilic compounds (e. g. glutathione, mannitol and thiourea) in a single assay. Results from linear correlation curves revealed that the IC50 were in the order of beta-carotene (3.45 mu g/ml) > alpha-Tocopherol (52.31 mu g/ml) > Mn(II)-bacitracin (109.37 mu g/ml) > glutathione (122.63 mu g/ml). Detailed investigations revealed that oxidation of LDH resulted in enzyme degradation, which was metal- and time-dependent mechanism. Therefore, further experiments were conducted to determine whether extension of the N-terminus of LDH with metal- binding regions possesses protective effect against the inactivation process. Genetic construction of chimeric LDH carrying two and four repetitive sequences of cadmium binding peptide (CdBP), designated as CdBP2LDH and CdBP4LDH, has been carried out. From our findings, the CdBP2LDH and the CdBP4LDH exhibited protective action and enzyme activity regained 20-30 % and 70 % higher than that of the native LDH, respectively. Two possible mechanisms have been proposed to play important role in protection against metal- mediated Fenton's reaction: i) changing in redox potential of Cu2+ in metal-peptide complex, and ii) taking away of Cu2+ ion from the crucial amino acids by metal saturation at the cadmium-binding peptides. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
metal binding, Fenton's reaction, antioxidants, lactate dehydrogenase, peptides
in
EXCLI Journal
volume
8
pages
1 - 11
publisher
Univ Mainz-Med Dept
external identifiers
  • wos:000263240900001
ISSN
1611-2156
language
English
LU publication?
yes
id
123cc8bb-be08-4b34-8efa-abbc064adeef (old id 1375004)
date added to LUP
2009-05-08 15:36:16
date last changed
2016-04-16 01:24:26
@article{123cc8bb-be08-4b34-8efa-abbc064adeef,
  abstract     = {In the present study, a simple and reliable antioxidant screening technique based on lactate dehydrogenase (LDH) oxidation by Cu2+-mediated Fenton's reaction has successfully been developed. Oxidation of LDH by hydroxyl radical consequently leads to enzymatic inactivation, while addition of antioxidants can protect and regain enzyme activity. This method demonstrated a high feasibility on detecting of antioxidative activity of lipophilic (e.g. alpha-Tocopherol and beta-Carotene) and hydrophilic compounds (e. g. glutathione, mannitol and thiourea) in a single assay. Results from linear correlation curves revealed that the IC50 were in the order of beta-carotene (3.45 mu g/ml) > alpha-Tocopherol (52.31 mu g/ml) > Mn(II)-bacitracin (109.37 mu g/ml) > glutathione (122.63 mu g/ml). Detailed investigations revealed that oxidation of LDH resulted in enzyme degradation, which was metal- and time-dependent mechanism. Therefore, further experiments were conducted to determine whether extension of the N-terminus of LDH with metal- binding regions possesses protective effect against the inactivation process. Genetic construction of chimeric LDH carrying two and four repetitive sequences of cadmium binding peptide (CdBP), designated as CdBP2LDH and CdBP4LDH, has been carried out. From our findings, the CdBP2LDH and the CdBP4LDH exhibited protective action and enzyme activity regained 20-30 % and 70 % higher than that of the native LDH, respectively. Two possible mechanisms have been proposed to play important role in protection against metal- mediated Fenton's reaction: i) changing in redox potential of Cu2+ in metal-peptide complex, and ii) taking away of Cu2+ ion from the crucial amino acids by metal saturation at the cadmium-binding peptides.},
  author       = {Tangkosakul, Teerawit and Tantimongcolwat, Tanawut and Isarankura-Na-Ayudhya, Chartchalerm and Mejare, Malin and Bülow, Leif and Prachayasittikul, Virapong},
  issn         = {1611-2156},
  keyword      = {metal binding,Fenton's reaction,antioxidants,lactate dehydrogenase,peptides},
  language     = {eng},
  pages        = {1--11},
  publisher    = {Univ Mainz-Med Dept},
  series       = {EXCLI Journal},
  title        = {Native And Chimeric Metal-Binding Lactate Dehydrogenase As Detection And Protection Tools For Oxidative Stress Induced By Fenton'S Reaction},
  volume       = {8},
  year         = {2009},
}