Copper react with proteins

Lowry method uses a combination of the Biuret copper-based reagent and the Folin-Ciocalteau reagent, which contains phosphomolybdic-phosphotungstic acid. Reagents react with protein, yielding a blue colour that displays an absorbance maximum at 750 nm... 

Skin sensitization is believed to occur as a result of nickel binding to proteins (particularly on the cell surface) and hapten formation. Essentially, the body perceives the nickel-protein complex as foreign and mounts an immune reaction to it. For example, sweat may react with the nickel in plated jewelry that comes in direct contact with skin dissolved metal may penetrate and react with proteins in the skin and lead to immune sensitization. Nickel may substitute for certain other metals (especially zinc) in metal-dependent enzymes, leading to altered protein function. High nickel content in serum and tissue may interfere with both copper and zinc metabolism. It also readily crosses the cell membrane via calcium channels and competes with calcium for specific receptors. 

In alkaline solution biuret, HN(CONH2)2 reacts with copper(II) sulfate to give a characteristic violet colour due to the formation of the complexes [Cu2(/l-OH)2(NHCONHCONH)4] (Fig. 28.6a) and [Cu(NHCONHCONH)2] . This is the basis of the biuret test in which an excess of NaOH solution is added to the unknown material together with a little CUSO4 soln a violet colour indicates the presence of a protein or other compound containing a peptide linkage. 

Bradford reagent contains the dye Coomassie blue G-250 in an acidic solution. The dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm Copper-containing reagent that, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid. The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein - very sensitive method... 

Protein determination procedures using bicinchonic acid were developed by Pierce Chemicals, who hold a patent on the product. The procedure entails the use of a copper-based reagent containing bicinchonic acid. Upon incubation with a protein sample, the copper is reduced. In the reduced state it reacts with bicinchonic acid, yielding a purple colour that absorbs maximally at 562 nm. 

The NO/NO+ and NO/NO- self-exchange rates are quite slow (42). Therefore, the kinetics of nitric oxide electron transfer reactions are strongly affected by transition metal complexes, particularly by those that are labile and redox active which can serve to promote these reactions. Although iron is the most important metal target for nitric oxide in mammalian biology, other metal centers might also react with NO. For example, both cobalt (in the form of cobalamin) (43,44) and copper (in the form of different types of copper proteins) (45) have been identified as potential NO targets. In addition, a substantial fraction of the bacterial nitrite reductases (which catalyze reduction of NO2 to NO) are copper enzymes (46). The interactions of NO with such metal centers continue to be rich for further exploration. 

Figure 18.16 Hypothetical model for the metallobiology of AP in Alzheimer s disease. (From Bush, 2003. Copyright 2003, with permission from Elsevier.) The proposed sequence of events (1) concentration of iron and copper increase in the cortex with aging. There is an overproduction of APP and AP in an attempt to suppress cellular metal-ion levels. (2) Hyper-metallation of AP occurs which may facilitate H202 production. (3) Hyper-metallated AP reacts with H202 to generate oxidized and cross-linked forms, which are liberated from the membrane. (4) Soluble AP is released from the membrane and is precipitated by zinc which is released from the synaptic vesicles. Oxidized AP is the major component of the plaque deposits. (5) Oxidized AP initiates microglia activation. (6) H202 crosses cellular membranes to react with Cu and Fe, and generate hydroxyl radicals which oxidize a variety of proteins and lipids.

Mixture of D-penicillamine (pen, Fig. 22) and copper(II) chloride forms a complex of the formula [Cu(I)8Cu(II)6pen12]5-. The Ri value of this complex at 300 MHz and 23°C is 0.586 mM 1 s-1 [178]. The carboxylate groups on the cluster have been shown to react with aziridines such as XAMA-7 (Fig. 23) which in turn can be reacted with biotin [179]. The copper clusters can then be attached to antibodies or other biotinylated proteins via an avidin linker molecule. 

Cu has a bifunctional role in copper-quinoprotein amine oxidases, catalyzing the formation of TPQ from the specific tyrosy l residue in the precursor protein and playing a role in the catalytic mechanism, most probably with 02 reacting with Cu(I) in the oxidative part of the cycle. The recent discovery of LTQ suggests that other combinations of quinone cofactors may be found in the future. 

The biuret test. This is one of the most general tests for proteins. When a protein reacts with copper(II) sulfate, a positive test is the formation of a copper complex which has a violet color. 

The Folin-Ciocalteau Assay of Protein Concentration. The Folin-Ciocalteau assay is one of the most sensitive and most commonly used assays to determine protein concentration (sensitive to about 10 /rg/m I protein). This procedure employs two color-forming reactions to assay protein concentration photometrically. In the first reaction (a biuret reaction), compounds with two or more peptide bonds form a dark blue-purple color in the presence of alkaline copper salts. In the second reaction, tryptophan and tyrosine side chains react with the Folin solution to produce cuprous ions. This reaction is most efficient under basic condi-... 

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