Description
Introduction: The enzyme superoxide dismutase (SOD), catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. Several common forms of SOD exist: they are proteins cofactored with copper and zinc, or manganese, iron, or nickel. The cytosols of virtually all eukaryotic cells contain an SOD enzyme with copper and zinc (Cu-Zn-SOD). The Cu-Zn enzyme is a homodimer of molecular weight 32,500. The two subunits are joined primarily by hydrophobic and electrostatic interactions. The ligands of copper and zinc are histidine side chains. Chicken liver (and nearly all other) mitochondria, and many bacteria contain a form with manganese (Mn-SOD). The ligands of the manganese ions are 3 histidine side chains, an aspartate side chain and a water molecule or hydroxy ligand depending on the Mn oxidation state (respectively II and III). E. coli and many other bacteria also contain a form of the enzyme with iron (Fe-SOD); some bacteria contain Fe-SOD, others Mn-SOD, and some contain both. Fe-SOD can be found in the plastids of plants. The active sites of Mn and Fe superoxide dismutases contain the same type of amino acid side chains. Simply stated, SOD outcompetes damaging reactions of superoxide, thus protecting the cell from superoxide toxicity. The reaction of superoxide with non-radicals is spin forbidden. In biological systems, this means its main reactions are with itself or with another biological radical such as nitric oxide (NO). The superoxide anion radical (O2-) spontaneously dismutes to O2 and hydrogen peroxide (H2O2) quite rapidly. SOD is biologically necessary because superoxide reacts even faster with certain targets such as NO radical, which makes peroxynitrite. Similarly, the dismutation rate is second order with respect to initial superoxide concentration. Thus, the half-life of superoxide, although very short at high concentrations is actually quite long at low concentrations. In contrast, the reaction of superoxide with SOD is first order with respect to superoxide concentration. Moreover, superoxide dismutase has the fastest turnover number of any known enzyme, this reaction being only limited by the frequency of collision between itself and superoxide.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to SOD. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for SOD and Avidin conjugated to 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 SOD, 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 SOD in the samples is then determined by comparing the O.D. of the samples to the standard curve