In Vitro Inhibition Studies of Human Class II Alcohol Dehydrogenase
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چکیده
Alcohol dehydrogenase (ADH) is an enzyme in the human body that processes the alcohol concentration in the blood. Alcohol dehydrogenase is a dimer which means it’s a molecule that consists of two parts which are called monomers. There are seven different dehydrogenases. ADH can be found in many organisms and it facilitates the oxidisation of alcohols to aldehydes or ketones and at the same time the reduction of the co-enzymes NAD to NADH. This enzyme is important to many organisms in order to break down alcohol compounds since these can be toxic at high concentrations. In humans ADH2 is involved in many different metabolic pathways such as retinoid metabolism and reduction of 4-hydroxynonenal. In this project inhibition studies with a spectrophotometer were used to determine inhibition type and inhibition constants. Three different inhibitors were tested, 2,2,2-Tribromoethanol, 2,2,2-Trichloroethanol and 2,2,2-Triflouroethanol. From these studies the type of inhibition mechanism was uncompetitive inhibition for all the tested inhibitors. The conclusions were based on Standard Deviation, STD, and R values as well as goodness of fitting on graph. In vitro-inhibitonsstudier av humant klass II alkoholdehydrogenas. Sammanfattning Alkoholdehydrogenas (ADH) är ett enzym i den mänskliga kroppen som bearbetar alkoholen i blodet. ADH är en dimer villket innebär att det är en molekyl som består av två delar som kallas monomerer. Det finns sju olika dehydrogenaser. ADH finns i många organsimer och det tar hand om oxidationen av alkoholer till aldehyder samt ketoner, samtidigt som oxidationen reduceras ko-factorn NAD till NADH. Detta enzym är viktigt för många organismer eftersom det bryter ner alkohol i blodet som kan vara gifitigt för dem. I bakterier och jäst katalyserar detta enzym den motsatta reaktionen, så att säga fermentering. ADH2 är inblandat i många olika metaboliska processeser till exempel retinoid metabolism och reduction av 4-hydroxynonenal. I detta projekt har inhibitions studier gjorts på ADH2 med hjälp av spektrofotometer för att fastställa inhibitonstyp och kinetiska konstanter. Tre olika inhibitorer har använts, 2,2,2Tribromoetanol, 2,2,2-Trichloroetanol och 2,2,2-Triflouroetanol. Dessa studier har resulterat i bestämning av en inhibitonsmekanism som är icke-kompetitiv för alla testade inhibitorer. Slutsatserna baserades på Standard Deviation, STD, och R-värden samt punktanpassningen av graferna. To mother, father, brother and sister.... Thank you for believing in me and your support during my journey. Many thanks to those who helped me making this project a success. I wouldn’t have done it without you all. Many thanks to my supervisors; Mikko Hellgren, Andreas Cederlund and Erik Fransén, and to everyone at Hejlab1 / MBB Karolinska Institutet.
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