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Protection of Biomolecules by Antioxidants - Mechanisms and Applications

Grey, Carl LU (2006)
Abstract (Swedish)
Popular Abstract in Swedish

Reaktiva syreföreningar s.k. ROS uppstår naturligt i den syrerelaterade metabolismen eller i andra processer och den orsakar skador på essentiella föreningar i cellen såsom DNA, proteiner och fetter. Denna oxidation ligger till grund för många allvarliga sjukdomar hos människor som t.ex. viss typ av cancer och Alzheimers och man tror även att den ligger bakom andra åkommor som t.ex. själva åldrandet. Förutom detta orsakar ROS-relaterad oxidation flera andra sorters oönskade effekter som härskning av mat och inaktivering av enzymer. Hydroxylradikalen anses vara en relevant radikal in vivo och den är dessutom mycket reaktiv, den kan i stort sett oxidera alla sorters organsiska molekyler. Den kan... (More)
Popular Abstract in Swedish

Reaktiva syreföreningar s.k. ROS uppstår naturligt i den syrerelaterade metabolismen eller i andra processer och den orsakar skador på essentiella föreningar i cellen såsom DNA, proteiner och fetter. Denna oxidation ligger till grund för många allvarliga sjukdomar hos människor som t.ex. viss typ av cancer och Alzheimers och man tror även att den ligger bakom andra åkommor som t.ex. själva åldrandet. Förutom detta orsakar ROS-relaterad oxidation flera andra sorters oönskade effekter som härskning av mat och inaktivering av enzymer. Hydroxylradikalen anses vara en relevant radikal in vivo och den är dessutom mycket reaktiv, den kan i stort sett oxidera alla sorters organsiska molekyler. Den kan bildas i en reaktion mellan väteperoxid, som är en naturligt förkommande metabolit, och en övergångsmetall såsom järn eller koppar, i en s.k. Fenton reaktion. För att försvara sig mot oönskad radikaloxidation har aeroba organismer utvecklat flera olika försvarsmekanismer. En viktig sådan utgörs av antioxidanter, vars uppgift är att reagera med fria radikaler före andra för cellen mer viktiga molekyler tar skada.



Denna sammanställningsavhandling är baserad på flera artiklar som handlar om forskning om antioxidanter och oxidation av DNA och protein. Angående DNA utfördes en mekanistisk studie som omfattade undersökandet av bildningen och identifieringen av flera oxidationsprodukter när nukleosiden dG utsattes för hydroxylradikaloxidation som genererades med hjälp av en järndriven Fenton reaktion. Ett tydligt resultat i studien var att den populära markören 8-oxodG som bildas är mycket känslig för vidare oxidation. Den uppmätta halten 8-oxodG var högst beroende av reaktionsförhållandena och reaktionstiderna som användes. Tillsatser av två stycken antioxidanter, den metallbindande catechin och den starka radikalfångande askorbinsyra (vitamin C) prövades, där catechin visade sig vara den mest effektiva antioxidanten. Återigen påvisades problem med 8-oxodG, där det var tydligt att det fanns stora fördelar med att använda sig av flera oxidationsproduktioner när man utvärderade antioxidanternas effektivitet. I en annan artikel användes en annan strategi för att utvärdera antioxidanter. Denna gång användes ett cellulärt test baserat på Ames-test-stammen Salmonella typhimurium TA102. Oxidationen inducerades med antingen väteperoxid eller den organiska hydroperoxiden tert-butylhydroperoxid. Precis som i den tidgare artikeln visade sig att metallbindande antioxidanter, denna gång quercetin och caffeic acid, fungerade klart bättre än de antioxidanter som saknar denna förmåga. De två sista artiklarna i avhandlingen handlar om proteinoxidation, där enzymet kloroperoxidas (CPO) från Caldariomyces fumago valdes ut som lämpligt målprotein. Detta enzym är av kommersiellt intresse, för att den kan katalysera stereo- och regio-selektiva oxidationsreaktioner, som är användbara i bl.a. tillverkningen av läkemedel, med hjälp av väteperoxid istället för tungmetallsalter som annars är brukligt. Det största problemet med CPO är att den är känslig för oxidativ inaktivering, som orsakas av väteperoxid i den katalytiska reaktionen. I en av studierna fanns ett samband mellan inaktivering och oxidation av en specifik aminosyra, en cysteine som är essentiell för CPOs aktivitet på grund av dess funktion som axiell ligand till järnet i hemgruppen i det aktiva sätet. Dessutom så kunde inte hemgruppen detekteras i de inaktiverade proverna, vilket tydde på att hemgruppen antagligen förstördes. I den andra studien med CPO upptäcktes att tillsatsen av antioxidanter signifikant motverkade inaktiveringen, upp till tio gånger fler reaktioner kunde katalyseras innan enzymet förstördes när antioxidanten caffeic acid tillsattes. Åter igen var det en metallbindande antioxidant som visade sig vara den mest effektiva.



Kontentan av dessa artiklar är att de metallbindande antioxidanterna visade sig klart överlägsna de enbart radikalfångande antioxidanterna. Vidare så indikerar resultaten att Fenton reaktionen är relevant in vivo och därför är lämplig att använda i in vitro test. Dessutom verkar möjligheten för antioxidanter att metallbinda vara en högst relevant för den antioxidativa effekten både in vitro och in vivo. (Less)
Abstract
Reactive oxygen species (ROS) consisting of various oxygen-based free radicals as well as other reactive non-radical species produced in O2-related metabolism or through other processes are involved in the oxidation of such vital biomolecules as DNA, proteins and lipids. The types of oxidation represented here are known to cause many different diseases and disorders in human beings, such as cancer, Alzheimer's disease and ageing. In addition, oxidation by ROS causes deterioration of food and is sometimes involved in the deactivation of enzymes. A particularly important source of ROS is believed to be the reaction of hydrogen peroxide with transition metal ions, mostly iron and copper, resulting in the formation of the highly deleterious... (More)
Reactive oxygen species (ROS) consisting of various oxygen-based free radicals as well as other reactive non-radical species produced in O2-related metabolism or through other processes are involved in the oxidation of such vital biomolecules as DNA, proteins and lipids. The types of oxidation represented here are known to cause many different diseases and disorders in human beings, such as cancer, Alzheimer's disease and ageing. In addition, oxidation by ROS causes deterioration of food and is sometimes involved in the deactivation of enzymes. A particularly important source of ROS is believed to be the reaction of hydrogen peroxide with transition metal ions, mostly iron and copper, resulting in the formation of the highly deleterious hydroxyl radical, which is able to basically oxidize any organic molecule present. Several antioxidative defence mechanisms have evolved, serving to keep the biomolecules intact and prevent them from being damaged by ROS. The action of small-molecule antioxidants having the function of reacting with free radicals so as to prevent more vital molecules from being oxidized are an important part of this defence.



The thesis is based on papers dealing with research on naturally occurring small-molecule antioxidants and the oxidation of DNA and proteins. In the case of DNA, a study was performed of the mechanisms involved, in which reaction intermediates and the formation of adducts were examined, using the iron-mediated Fenton reaction to induce oxidation of the nucleoside dG. Several oxidation products were identified and quantified by use of various reaction conditions. It was found that 8-oxodG, frequently used as a marker for oxidative DNA-damage, was highly susceptible to secondary oxidation. The measured level of this adduct being highly dependent upon both the reaction time and Fenton reagent concentrations involved. Two antioxidants, the iron-chelating antioxidant catechin and the strong radical-scavenger ascorbic acid, were evaluated by use of the same reaction system. It was concluded that catechin was more effective. Again, problems regarding 8-oxodG were observed, its being clearly shown that more reliable results could be obtained when a marker of more than one type was used to evaluate the effects of the antioxidants. In another study the effects of antioxidants were evaluated using a cellular test developed based on the Ames-tester strain Salmonella typhimurium TA102. Oxidation was induced by use of either hydrogen peroxide or the organic peroxide tert-butyl hydroperoxide (tBHP). Similar to the study just referred to, chelating antioxidants, in this case phenolic quercetin and caffeic acid, were the antioxidants found to be most effective. In the protein oxidation study carried out, use was made of the commercially interesting enzyme chloroperoxidase (CPO) from Caldariomyces fumago. This enzyme is able to catalyze various stereoselective oxygen-insertion reactions useful in the synthesis of drugs, for example, a clean source of hydrogen peroxide being used as the external oxidant. The main limitation of CPO is its poor stability, caused by its oxidative inactivation by hydrogen peroxide during the catalytic cycle. It was found that the inactivation was correlated with the oxidation of a key amino acid, a cysteine residue acting as the axial ligand to the prosthetic heme group of the enzyme, along with the disappearance of the heme. Furthermore, the addition of antioxidants significantly improved its operational stability, leading in the best case to 10 times as many turnovers of CPO being observed prior to its inactivation. Again it was found that a chelating antioxidant, caffeic acid in this case, was the antioxidant inhibiting unwanted oxidation the most.



One thing these rather differing oxidation studies had in common was that the chelating antioxidants were clearly superior to the antioxidants that only possessed a radical scavenging mechanism. The results indicate two things in particular, first that the Fenton reaction is highly relevant in vivo and is thus a good way of producing radicals in in vitro assays, and secondly that the chelating mechanism is an important antioxidative mechanism, one which involves the action of small-molecule antioxidants both in vitro and in vivo. (Less)
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author
supervisor
opponent
  • Dr. van Rantwijk, Fred, Delft University of Technology
organization
publishing date
type
Thesis
publication status
published
subject
keywords
dG, antioxidants, mass spectrometry, Analytical chemistry, Analytisk kemi, Food and drink technology, Livsmedelsteknik, Bioteknik, Biotechnology, DNA, Oxidation, Protein, Fenton reaction
pages
160 pages
publisher
Department of Biotechnology, Lund University
defense location
Lecture hall B, Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund
defense date
2006-12-20 13:15
external identifiers
  • other:ISRN: LUTKDH/TKBT--06/1103--SE
ISBN
978-91-89627-50-5
language
English
LU publication?
yes
id
88a122d8-eea6-43fc-aa91-79e2671c2566 (old id 547741)
date added to LUP
2007-10-13 10:49:21
date last changed
2016-09-19 08:45:02
@phdthesis{88a122d8-eea6-43fc-aa91-79e2671c2566,
  abstract     = {Reactive oxygen species (ROS) consisting of various oxygen-based free radicals as well as other reactive non-radical species produced in O2-related metabolism or through other processes are involved in the oxidation of such vital biomolecules as DNA, proteins and lipids. The types of oxidation represented here are known to cause many different diseases and disorders in human beings, such as cancer, Alzheimer's disease and ageing. In addition, oxidation by ROS causes deterioration of food and is sometimes involved in the deactivation of enzymes. A particularly important source of ROS is believed to be the reaction of hydrogen peroxide with transition metal ions, mostly iron and copper, resulting in the formation of the highly deleterious hydroxyl radical, which is able to basically oxidize any organic molecule present. Several antioxidative defence mechanisms have evolved, serving to keep the biomolecules intact and prevent them from being damaged by ROS. The action of small-molecule antioxidants having the function of reacting with free radicals so as to prevent more vital molecules from being oxidized are an important part of this defence.<br/><br>
<br/><br>
The thesis is based on papers dealing with research on naturally occurring small-molecule antioxidants and the oxidation of DNA and proteins. In the case of DNA, a study was performed of the mechanisms involved, in which reaction intermediates and the formation of adducts were examined, using the iron-mediated Fenton reaction to induce oxidation of the nucleoside dG. Several oxidation products were identified and quantified by use of various reaction conditions. It was found that 8-oxodG, frequently used as a marker for oxidative DNA-damage, was highly susceptible to secondary oxidation. The measured level of this adduct being highly dependent upon both the reaction time and Fenton reagent concentrations involved. Two antioxidants, the iron-chelating antioxidant catechin and the strong radical-scavenger ascorbic acid, were evaluated by use of the same reaction system. It was concluded that catechin was more effective. Again, problems regarding 8-oxodG were observed, its being clearly shown that more reliable results could be obtained when a marker of more than one type was used to evaluate the effects of the antioxidants. In another study the effects of antioxidants were evaluated using a cellular test developed based on the Ames-tester strain Salmonella typhimurium TA102. Oxidation was induced by use of either hydrogen peroxide or the organic peroxide tert-butyl hydroperoxide (tBHP). Similar to the study just referred to, chelating antioxidants, in this case phenolic quercetin and caffeic acid, were the antioxidants found to be most effective. In the protein oxidation study carried out, use was made of the commercially interesting enzyme chloroperoxidase (CPO) from Caldariomyces fumago. This enzyme is able to catalyze various stereoselective oxygen-insertion reactions useful in the synthesis of drugs, for example, a clean source of hydrogen peroxide being used as the external oxidant. The main limitation of CPO is its poor stability, caused by its oxidative inactivation by hydrogen peroxide during the catalytic cycle. It was found that the inactivation was correlated with the oxidation of a key amino acid, a cysteine residue acting as the axial ligand to the prosthetic heme group of the enzyme, along with the disappearance of the heme. Furthermore, the addition of antioxidants significantly improved its operational stability, leading in the best case to 10 times as many turnovers of CPO being observed prior to its inactivation. Again it was found that a chelating antioxidant, caffeic acid in this case, was the antioxidant inhibiting unwanted oxidation the most.<br/><br>
<br/><br>
One thing these rather differing oxidation studies had in common was that the chelating antioxidants were clearly superior to the antioxidants that only possessed a radical scavenging mechanism. The results indicate two things in particular, first that the Fenton reaction is highly relevant in vivo and is thus a good way of producing radicals in in vitro assays, and secondly that the chelating mechanism is an important antioxidative mechanism, one which involves the action of small-molecule antioxidants both in vitro and in vivo.},
  author       = {Grey, Carl},
  isbn         = {978-91-89627-50-5},
  keyword      = {dG,antioxidants,mass spectrometry,Analytical chemistry,Analytisk kemi,Food and drink technology,Livsmedelsteknik,Bioteknik,Biotechnology,DNA,Oxidation,Protein,Fenton reaction},
  language     = {eng},
  pages        = {160},
  publisher    = {Department of Biotechnology, Lund University},
  school       = {Lund University},
  title        = {Protection of Biomolecules by Antioxidants - Mechanisms and Applications},
  year         = {2006},
}