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Effect of chelating agents on alcohol dehydrogenase, a zinc containing enzyme
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Resumo(s)
O zinco é um metal globalmente presente na comida, na água e no ar e também é o segundo oligoelemento mais abundante nos organismos vivos. Está envolvido em muitos processos fisiológicos e o corpo humano possui mecanismos eficientes, tanto a nível sistémico quanto celular, para manter a sua homeostasia numa ampla faixa de exposição. Consequentemente, o zinco tem uma toxicidade bastante baixa e a intoxicação severa com impacto na saúde humana é um evento relativamente raro. No entanto, a nível celular, o zinco pode ser um regulador crucial da apoptose, bem como a morte neuronal após lesão cerebral. Entre outros, o zinco também está presente na estrutura da Álcool desidrogenase (ADH) e é necessário para o seu papel catalítico, que reside na oxidação reversível do etanol em acetaldeído com a redução concomitante de NAD + a NADH. Como muitos agentes quelantes de metais, mesmo usados clinicamente, não são seletivos, desta forma, também podem quelar o zinco. O objetivo deste estudo foi, nesse sentido, testar se diferentes quelantes podem bloquear a função enzimática da ADH. Resumidamente: diferentes quelantes dissolvidos em DMSO foram misturados com ADH de Saccharomyces cerevisiae (0,04 UI / mL). Etanol (3 V / V%) e NAD + (concentração: 6.8 mM) em tampão de pH 8,8 foram adicionados para iniciar o processo enzimático a 25 ºC. A reação foi monitorizada durante 6 minutos, medindo a absorvância a 340 nm, que corresponde ao NADH formado. Os resultados preliminares com 12 quelantes mostraram que a maioria deles, em particular os quelantes hidrofílicos, apresentaram um efeito muito baixo ou quase nulo na atividade da ADH. Os quelantes mais ativos foram os derivados nitro ou halogenados das 8-hidroxiquinolinas, embora a sua atividade para concentrações inferiores a 100 µM mostrou ser muito baixa. Em resumo, estes resultados revelam que os agentes quelantes avaliados neste estudo, podem interferir na enzima ADH apenas para concentrações mais elevadas.
Zinc is an ubiquitous metal present in food, water and air and it is also the second most abundant trace element in living organisms. It is involved in many physiological processes and the human body has efficient mechanisms, both on systemic and cellular levels, to maintain its homeostasis over a broad exposure range. Consequently, zinc has rather a low toxicity, and severe intoxication with impact on human health is a relatively rare event. Nevertheless, on the cellular level zinc may be a crucial regulator of apoptosis, as well as neuronal death following brain injury. Among others, zinc is contained as well in alcohol dehydrogenase (ADH) and is needed for its catalytic role which lies in reversible oxidation of ethanol to acetaldehyde with the concomitant reduction of NAD+ to NADH. Since many, even clinically used, metal chelators are non-selective, they can chelate zinc as well. The aim of this study was hence to test if different chelators can block enzymatic function of ADH. Shortly: different chelators dissolved in DMSO were mixed with ADH from Saccharomyces cerevisiae (0,04 IU/mL). Ethanol (3 V/V%) and NAD+ (final concentration: 6,8 mM) in a buffer of pH 8.8 were added to start the enzymatic process at 25 ºC. The reaction was monitored for 6 minutes measuring the absorbance at 340 nm, which corresponds to the formed NADH. Our preliminary results with 12 chelators have shown that most of them, in particular hydrophilic chelators, had very low or almost no effect on ADH activity. The most active chelators were nitro or halogen derivatives of 8-hydroxyquinolinoles but their activity at concentrations below 100 µM was quite low. In conclusion, it seems that metal chelators can interfere with ADH only at higher concentrations.
Zinc is an ubiquitous metal present in food, water and air and it is also the second most abundant trace element in living organisms. It is involved in many physiological processes and the human body has efficient mechanisms, both on systemic and cellular levels, to maintain its homeostasis over a broad exposure range. Consequently, zinc has rather a low toxicity, and severe intoxication with impact on human health is a relatively rare event. Nevertheless, on the cellular level zinc may be a crucial regulator of apoptosis, as well as neuronal death following brain injury. Among others, zinc is contained as well in alcohol dehydrogenase (ADH) and is needed for its catalytic role which lies in reversible oxidation of ethanol to acetaldehyde with the concomitant reduction of NAD+ to NADH. Since many, even clinically used, metal chelators are non-selective, they can chelate zinc as well. The aim of this study was hence to test if different chelators can block enzymatic function of ADH. Shortly: different chelators dissolved in DMSO were mixed with ADH from Saccharomyces cerevisiae (0,04 IU/mL). Ethanol (3 V/V%) and NAD+ (final concentration: 6,8 mM) in a buffer of pH 8.8 were added to start the enzymatic process at 25 ºC. The reaction was monitored for 6 minutes measuring the absorbance at 340 nm, which corresponds to the formed NADH. Our preliminary results with 12 chelators have shown that most of them, in particular hydrophilic chelators, had very low or almost no effect on ADH activity. The most active chelators were nitro or halogen derivatives of 8-hydroxyquinolinoles but their activity at concentrations below 100 µM was quite low. In conclusion, it seems that metal chelators can interfere with ADH only at higher concentrations.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019
Palavras-chave
Zinco Quelantes Álcool Desidrogenase NAD+ NADH Mestrado Integrado - 2019