Description
Introduction: Alcohol dehydrogenase (ADH) is an enzyme discovered in the mid-1960s in Drosophila melanogaster. Since then, there has been extensive research on the enzyme. Alcohol dehydrogenase is a dimer, weighing 80 kDa. Alcohol dehydrogenases are a group of seven dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD+ to NADH. In humans and many other animals, they serve to break down alcohols which could otherwise be toxic; in yeast and many bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation. In humans, alcohol is metabolized by a rate of 1 ounce or 7 to 10 g/hour. Alcohol dehydrogenase is responsible for catalyzing oxidation of primary and secondary alcohols to aldehydes and ketones, respectively, and also can effect the reverse reaction. It does not work well with primary alcohols. Instead, it works the best with secondary and cyclic alcohols. In humans, it exists in multiple forms as a dimer and is encoded by at least seven different genes. There are five classes (I-V) of alcohol dehydrogenase, but the hepatic form that is primarily used in 3 humans is class 1. Class 1 consists of A,B, and C subunits that are encoded by the genes ADH1A, ADH1B, and ADH1C. The enzyme is contained in the lining of the stomach and in the liver. It catalyzes the oxidation of ethanol to acetaldehyde. This allows the consumption of alcoholic beverages, but its evolutionary purpose is probably the breakdown of alcohols naturally contained in foods or produced by bacteria in the digestive tract. Alcohol dehydrogenase is also involved in the toxicity of other types of alcohol: for instance, it oxidizes methanol to produce formaldehyde and ethylene glycol to ultimately yield glycolic and oxalic acids. Humans have at least six slightly different alcohol dehydrogenases. All of them are dimers (consist of two polypeptides), with each dimer containing two zinc ions Zn2+. One of those ions is crucial for the operation of the enzyme: it is located at the catalytic site and holds the hydroxyl group of the alcohol in place.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to ADH. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for ADH and Avidin conjugated to 4 Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain ADH, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of ADH in the samples is then determined by comparing the O.D. of the samples to the standard curve