I found that NBT can be used to specifically stain proteins that have been modified covalently by oxidized polyphenols (after electrophoretic transfer onto a nitrocellulose or PVDF membrane). All literature I have found so far states that staining of such modified proteins is INDICATIVE for covalent bonds between protein and polyphenols. I was wondering how useful/reliable such data really is? All literature is basically just referencing back to the original paper by Paz et al , 1991. Any comments are welcomed.
Thanks
Nat
Polyphenols exhibit a propensity to bind proteins by both non-covalent and covalent interactions. In beverages and fruits, a combination of hydrogen bonds and hydrophobic interactions characterizes predominant non-covalent interactions between proanthocyanidins and proteins. Covalent interactions of dietary polyphenols such as caffeic acids with milk proteins (casein) are also known. Auto-oxidation of polyphenols results in the formation of irreversible, covalent bonds between quinones and proteins. Phenoxyl radicals generated during such autoxidation can readily react with nucleophilic centers of amino acids and proteins (such as the epsilon amino group of L-lysine or the sulfhydryl group of cysteine) and form covalent bonds. Such quinone complexation of proteins can be found in melanins and lignins. The quinone cofactor of certain bacterial enzymes, PQQ (pyrroloquinoline quinone) is dissociable. Certain mammalian quinoproteins contain covalently bound dopa quinone. At alkaline pH, quinones oxidize the amino acid, glycine. Superoxide, released in the cycling reaction of quinones with sodium glycinate, reduces NBT to its formazan (redox cycling staining), which facilitates detection of qunione containing proteins in polyacrylamide gels. I do not know the usefulness or application of such information on oxidized polyphenols.
Dear Nathalie,
Dear Nathalie,
http://europepmc.org/abstract/MED/25956828 Dopamine o-quinone, formed by dopamine oxidation, covalently modifies cysteinyl proteins forming a quinoprotein adduct. Employing two-dimensional gel electrophoresis and NBT/glycinate redox-cycling staining, quinoprotein adducts have been compared in the substantia nigra of rats of different age. Certain key enzymes in energy metabolism and mitochondrial function were readily conjugated by dopamine o-quinone.
NBT, combined with another agent, has been used to stain beta-galactosidase activity in histological sections.
Serum glycated proteins have been separated by agarose gel electrophoresis and NBT staining. NBT staining has been employed to detect enzymes of starch synthesis and alkaline phosphatse in Western blots. SOD is detected in polyacrylamide gels utilizing NBT dye reduction; this is followed by ferricyanide precipitation for negative staining of catalase on the same gel.
Dear Chithan,
Thanks so much for your feedback!
So when I do see that proteins are stained by NBT (well, the visible formazan), that really DOES mean that I do have quinoproteins on my blot (proteins that have COVALENTLY been modified by oxidized polyphenols, quinones)?
In my research I am complexing food proteins with plant polyphenols...if that helps
Dear Nathalie,
It does not mean that you have (covalently modified) quinoproteins on your blot. NBT/glycinate appears to detect a broad spectrum of quinonoid cofactors. Staining apparently is applied for the identification of PQQ-like molecules and quinonoid substances in quinoproteins, quinopeptides and biological samples. Only certain mammalian quinoproteins seemed to have tested positive with the NBT/glycinate staining.
Dear Chithan,
please see the attached paper, this is just one of several that I found, showing the use of NBT redox cycling to detect proteins that have covalently been modified by oxidized polyphenols (quinones). Dr. Ann Hagerman (she works a lot on polyphenol-protein interactions as well) also has a few papers on this matter for example...
Best
Nat
Dear Nathalie,
Thanks very much. I presume that the NBT redox cycling can also detect quinone containing amine oxidases besides proteins with noncovalently bound quinones. Dr Anne Hagerman's NBT procedure is modified from Paz et al (1991). Hagerman and Butler described the specificity of (sorghum) proanthcyanidin-protein interactions in 1981.
Best...
I see! Thanks so much for taking the time in giving me feedback. I appreciate it!
All the best,
Nat
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