jamlooki.blogg.se - Quantitation of protein carbonylation by dot blot

Cigarette smoke and aldehydes also introduce carbonyls into proteins. 10Redox cycling cations bind to proteins and, in conjunction with attack by ROS, lead to the formation of amino acid derivatives containing carbonyl groups (ketones, aldehydes). The most common marker of protein oxidation is protein carbonyl content. Many additional species can damage DNA and RNA as well. and ONOO - molecules are representative ROS and RNS species that causes damage.8-Hydroxy-2’-deoxyguanosine - the deoxynucleoside from DNAĪssays that can detect multiple oxidized guanine species capture a more complete set of biologically relevant products of oxidative damage than do assays that are restricted to analysis of only one (e.g., 8-hydroxy-2'-deoxyguanosine).8-Hydroxyguanosine - the nucleoside from RNA.8-Hydroxyguanine - the ribose-free base.

The repair processes that are initiated to correct this damage release the following oxidized guanine species into the urine: Guanine is the base that is most prone to oxidation when DNA and RNA are damaged. Alternatively, in vitro NOS activity can be detected using the chemistry of the Griess reaction once the excess NADPH, added as a cofactor for NOS activity, is removed using an oxidization step that is catalyzed by lactate dehydrogenase. NOS activity is detected in tissues and cells by harnessing the NOS-driven conversion of a radiolabeled arginine to citrulline in the presence of the necessary factors. Subsequent reaction with Griess reagents or DAN, both of which only react with NO 2 −, will determine a total concentration of NO 2 −. 4,5These assays first convert NO 3 − to NO 2 −using NADPH-dependent nitrate reductase.

reactions whose total production can be detected using either Griess reagents or DAN.

NO 3 − and NO 2 − are end products of in vivo NO

itself also reacts with thiols and iron-sulfur enzymes, whereas peroxynitrite reacts with tyrosine residues to form nitrotyrosine.

− lead to the formation of peroxynitrite.

), synthesized by nitric oxide synthase (NOS), and O 2.

Reactive nitrogen species (RNS) are also produced during oxidative stress. 1The activity of this enzyme can be measured by allowing XO to degrade hypoxanthine and capturing the H 2O 2 byproduct of this reaction via a probe like ADHP coupled to an enzyme like HRP. Xanthine oxidase (XO) produces both H 2O 2 and O 2

− to form 2-hydroxyethidium (ex: 500-530, em: 590-620 nm) or by non-specific oxidation by H 2O 2 or other sources of ROS to form ethidium (ex: 480 nm, em: 576 nm).This redox-sensitive probe is oxidized by O 2 1 Dihydroethidiumĭihydroethidium (hydroethidine or DHE) can be used directly in live cells. Assay specificity is improved significantly when an H 2O 2 scavenger, such as catalase, is included as a control. H 2O 2 can be detected using sensitive probes such as ADHP coupled to an enzyme like HRP. Assays for ROS do not discern the source of ROS production (i.e., normal versus disease state), but if the experimental model is under stress, an increase in ROS and alteration to molecular components is probable. Damage to cellular macromolecules occurs when uncontrolled oxidation stresses a biological system. Oxygen is electronically reduced as part of normal metabolism, resulting in the formation of various ROS, including hydrogen peroxide (H 2O 2) and superoxide (O 2 To help you determine which is best to use in your experimental system, here is a breakdown of the assay technology used to detect the most common oxidative damage biomarkers. Although direct measurement of ROS is ideal, the indirect methods are often relied on more heavily due to the relative stability of damage markers on biomolecules compared to the transient nature of ROS. Oxidative stress can be evaluated directly by measuring reactive oxygen species (ROS) or indirectly by the associated damage to lipids, proteins, and nucleic acids that occurs upon overproduction of ROS.