Proteins SH groups

Another chemical variant of PRODAN is ACRYLODAN [6-acryloyl-2-(dimethyl-amino)naphthalene], which covalently binds to protein-SH groups. 

In their natural habitat, proteins are usually surrounded by other proteins and organic factors. When these are removed or diluted as during purification, the protein becomes surrounded by water on all sides. Proteins react differently to a pure aqueous environment many are destabilized and rapidly denatured. A common remedial measure is to add 5%-20% glycerol to the purification buffer. The organic surface of the glycerol is believed to simulate the environment of the protein in the intact cell. Two other ingredients that are most frequently added to purification buffers are mercaptoethanol and ethylenedia-minetetraacetate (EDTA). The mercaptoethanol inhibits the oxidation of protein —SH groups, and the EDTA chelates... 

In addition to the isotopically labeled Mannich bases, it is worth mentioning the paramagnetic Mannich base 492, which represents a very good spin-label for the protein SH group,as well as the cyclic Mannich base deriving from 1,-DOPA (493), which exhibits much enhanced optical rotatory power with respect to the open chain precursor allowing it to be more precisely estimated. --... 

Three other metal ions that have been implicated in damage to vessel walls are mercury, chromium, and arsenic. Mercury, which interferes with protein -SH groups, may cause vasoconstriction of pre-glomerular vessels in the kidney. 

Fig. 8. Modification of a protein SH group with carbohydrate derivatives.

The administration of TNT to laboratory animals leads to the excretion of 4-NHOH-DNT, 2-NH2-DNT, and 4-NH2-DNT in the urine [59], and to the formation of covalent adducts with microsomal liver and kidney proteins, hemoglobin, and other blood proteins [60], The acid hydrolysis of adducts yielded mainly 2-NH2-DNT (2-ADNT) and 4-NH2-DNT (4-ADNT). Incubation of rat liver microsomes with TNT and NADPH under aerobic conditions resulted in the formation of NH2-DNTs and the transient metabolite 4-NHOH-DNT [57], The formation of covalent protein adducts with TNT metabolites was enhanced by the presence of 02 and decreased by GSH. This is consistent with the scheme of the TNT adduct formation with the central role of the nitroso metabolite (NO-DNT) reaction with protein or nonprotein thiols (RSH Equation 9.11) [57], The acid hydrolysis of the sulfinamide adduct (RS(0)-NH-DNT) formed after the rearrangement of the semimercaptal (RS-N(OH)-DNT Equation 9.12) will yield NH2-DNT. The mixture of NHOH-DNTs inhibits bacterial glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase more efficiently than TNT [61]. This was attributed to the covalent modification of protein -SH groups. 

Resultant dehydroascorbic acid is reduced to ascorbic acid by DHA reductase. Reduction is coupled to oxidation of flour protein SH groups... 

Gonadotropin inhibition Byakangelicin (30) Interacted with sulfhydiyi group in pregnant mare s serum gon otropin, possibly through addition of protein -SH group to the 3,4-double bond of the coumarin [215,216]... 

The ability of LOX to catalyze cooxidation reactions has long been recognized (e.g., its carotene oxidase activity see Section I) and has been used as the basis for some LOX assays (see Section IV) and in commercial applications e.g., soybean or Vida faba flours (both rich in LOX activity) are added to bleach wheat flour pigments in white bread production. Cooxidation is clearly manifested in the bleaching of pigments (chlorophyll, carotenoids, etc.) but also results in the oxidation of protein-SH groups and of unsaturated fatty acids, including substrates for LOX. 

According to Lii and Hendrich 94), two pathways exist for the in vivo S-glutathiolation of protein SH groups (a) glutathione disulfide (GSSG)-protein mixed disulfide exchange (equation 4) and (b) direct combination of thiol free radicals to mixed disulfides, possibly as shown in equations 5-7. 

The formation of protein-glutathione mixed disulfide prevents oxidation of protein SH groups to, for example, a sulfonic acid derivative (PSO3). The GS- and PS radicals derived from the action of r-butyl peroxide (r-BuOOH) on GSH and PSH (equations 5,6) can dimerize to form PS-SG (equation 7). The net effect is protection by glutathione of sensitive protein SH groups against oxidative damage. 

As already mentioned, dehydroalanine is the postulated reactive precursor for lysinoalanine. Direct evidence for dehydroalanine reactivity was obtained by Friedman et al. (1977). They showed that dehydroalanine derivatives convert lysine side chains in casein, bovine serum albumin, lysozyme, wool, or polylysine to lysinoalanine residues at pH 9 to 10. Related studies showed that protein SH groups generated by reduction of disulfide bonds are completely alkylated at pH 7.6 to lanthionine side chains. These studies demonstrate that lysinoalanine and lanthionine residues can be introduced into a protein under relatively mild conditions, without strong alkaline treatment. They also imply that it should be possible to explore nutritional and toxicological consequences of lysinoalanine and lanthionine consumption in the absence of racemiza-tion (see below). 

Iodine in molar potassium iodide appears to be a useful agent for the oxidation of protein SH groups since it reacts fairly rapidly in dilute solution it can be estimated precisely, the conditions of the reaction are those of maximum stability of most proteins, and thus far it has not failed to oxidize all the SH groups in the native protein... 

Ferricyanide has the advantage of reacting quite rapidly with protein SH groups, under biological conditions. It is stable, and readily available. The ferrocyanide formed can be easily and precisely determined as Prussian blue in extremely low concentrations. However, this reagent appears to be non-specific in some instances and completely ineffective in others. 

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