Oxidative stress detection
> DNA Damage
> SOD
> GSH
> NO
> Phospholipid Peroxide
DNA Damage
- Principle of kit(click to enlarge)
Oxidative damage to DNA is a result of the interaction of DNA with reactive oxygen species (ROS), in particular, the hydroxy radical which is converted from superoxide and hydrogen peroxide by the Fenton reaction.
Hydroxy radicals produce a multiplicity of modifications in DNA. Oxidative attack by hydroxy radical on the deoxyribose moiety will lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple abasic sites (AP sites). In fact, AP sites are one of the major types of damage generated by ROS. It has been estimated that endogeneous ROS can result in about 2 x 105 base lesions per cell per day.
Aldehyde Reactive Probe (ARP) reagent (N’-aminooxymethylcarbonylhydrazino-D-biotin) reacts specifically with an aldehyde group which is the open ring form of the AP sites. This reaction makes it possible to detect DNA modifications that result in the formation of an aldehyde group.
After treating DNA containing AP sites with ARP reagent, AP sites are tagged with biotin residues. By using an excess amount of ARP, all AP sites can be converted to biotin-tagged AP sites. Therefore, AP sites can be quantified using avidin-biotin assay followed by a colorimetric detection of peroxidase or alkaline phosphatase conjugated to the avidin.
DNA Damage Quantification Kit contains all the necessary solutions, enabling the determination of 1 to 40 AP sites per 1x105 bp.
SOD
- Principle of the SOD (click to enlarge)
Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O2.-) into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes.
In order to determine the SOD activity, several direct and indirect methods have been developed. Among these methods, an indirect method using nitroblue tetrazolium (NBT) is commonly used due to its convenience and ease of use. However, there are several disadvantages to the NBT method, such as poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase.
SOD Assay Kit-WST allows very convenient SOD assaying by utilizing Dojindo’s highly water-soluble tetrazolium salt, WST-1 (2-(4-Iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon reduction with a superoxide anion.
The rate of the reduction with O2.- are linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD. Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by a colorimetric method (Patent filing).
GSH
Glutathione (GSH) is the most abundant thiol (SH) compound in animal tissues, plant tissues, bacteria and yeast. GSH plays many different roles such as protection against reactive oxygen species and maintenance of protein SH groups. During these reactions, GSH is converted into glutathione disulfide (GSSG: oxidized form of GSH).
Since GSSG is enzymatically reduced by glutathione reductase, GSH is the dominant form in organisms. DTNB (5,5’-Dithiobis(2 nitrobenzoic acid)), known as Ellman’s Reagent, was developed for the detection of thiol compounds. In 1985, Dr. Anderson suggested that the glutathione recycling system by DTNB and glutathione reductase created a highly sensitive glutathione detection method.
DTNB and glutathione (GSH) react to generate 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Since 2-nitro-5- thiobenzoic acid is a yellow colored product, GSH concentration in a sample solution can be determined by the measurement at 412 nm absorbance.
GSH is generated from GSSG by glutathione reductase, and reacts with DTNB again to produce 2-nitro-5-thiobenzoic acid. Therefore, this recycling reaction improves the sensitivity of total glutathione detection.
Total Glutathione Quantification Kit includes all the necessary reagents for total glutathione measurement except for reagents that are used for sample preparation. 5 Sulfosalicylic acid is recommended for the removal of proteins from sample solutions and for the prevention of GSH oxidation and -glutamyl transpeptidase reaction.
However, the optimum method for sample preparation differs from sample to sample, so please review the references. This kit can be used to quantify total glutathione concentrations from 1 µM to 100 µM using the standard method. For lower glutathione concentration detections, such as in blood samples, longer incubation time is required.
NO : NO2/NO3 Assay Kit-CII
- Principle of kit (click to enlarge)
Nitric oxide (NO), a gaseous paramagnetic radical, is a very important and versatile messenger in biological systems. NO is synthesized from L-arginine by NO synthase (NOS). It has been identified as an endothelial derived relaxation factor (EDRF) and antiplatelet substance. It serves as a neurotransmitter derived from a neutrophile and a cytotoxic substance from an activated macrophage. Although NO’s molecular action in the biological system is very versatile, the most important role of NO is the activation of guanylate cyclase.
The Griess assay is one of the most popular and simplest methods used to detect the NO concentration. The Griess assay mechanism is summarized as the azo coupling between diazonium species, which are produced from sulfanilamide with NO2, and naphthylethylenediamine. The NO2/NO3 Assay Kit-CII contains these dyes, nitrate reductase, enzyme co-factor, buffer solution and NO2, NO3 solutions as standards. Therefore, total NO metabolites are easily detectable using this kit. The suitable NO2 detection range is from 10 to 100 µM.
Phospholipid Peroxide
- (click to enlarge)
DPPP is a non-fluorescent triphenylphosphine compound. It reacts with hydroperoxide to generate DPPPoxide that emits fluorecent at 352 nm excitation and 380 nm emission wavelenghs. Post-column HPLC method is used to determine phospholipid peroxide in sample solutions.