Reducing cysteine

Insulin, a small protein of molecular mass 6000 daltons, is composed of two chains designated A and B. There are no reduced cysteine residues in insulin, but it contains three essential disulfide bonds two that crosslink the A and B chains, and one internal to the A chain to stabilize the overall tertiary stmcture. These disulfide bonds are cleaved in the presence of excess AuX4, leaving A and B chains that have cysteine residues that have become oxidized to sulfonic adds [119]. With smaller amounts of AuX4, a single disulfide bond will be attacked to form sulfinic acid [119]. The reaction is second order for AuCU while AuBr4 reacts too quickly for accurate monitoring. 

Among the commercially available fluorescein derivatives that have been widely used are fluorescein isothiocyanate (FITC) 49, carboxyfluorescein (FAM) succini-midyl ester 50, and fluorescein dichlorotriazine (DTAF) 51. FITCs are the most commonly used fluorescein derivatives. They have been used to react with sulfhy-dryl [127], targeting reduced cysteine chains and especially amino groups in peptides or proteins [128]. 

At the two extremes, lysine is observed as the amino acid most accessible on the surface of proteins while cysteine is the least exposed amino acid. The inaccessibility of cysteine probably stems from the fact that disulfides are typically buried within the polypeptide structure of proteins, whether they are intrachain or interchain in nature, and proteins rarely contain many reduced cysteine thiols. 

However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme. 

The sequences of synthetic peptides used to form two-stranded a-helical coiled coils with an interchain disulfide bond are shown above. The positions of the cysteine residues are indicated by the solid circles above the sequences. The peptide with a cysteine at position 2 (position a) is designated as C2a. b Line plots (a) A plot of [Gdn HCl]1/2, the transition midpoint of the denaturation profiles in the presence of Gdn HC1 (guanidinium chloride), versus the position of an interhelical disulfide bond at positions 2, 5, 9,12, 16,19,23,26,30, or 33. Open triangles denote the coiled coils with a terminal disulfide bond at either position 2 or position 33. Open squares denote the coiled coils with nonterminal disulfide bonds at position d (5,12,19, or 26). Open circles denote the coiled coils with nonterminal disulfide bonds at position a (9, 16, 23, or 30). Closed symbols denote the coiled coils without a disulfide bridge (reduced cysteine residues). The broken line indicates the [GdnHCl]1/2 of the native coiled coil. 

To gain quantitative data without the inherent problems associated with gel electrophoresis would seem to be an added advantage. ICAT, (Fig. 17.10), also based on LC, labels reduced cysteine residues from two sample states with a multipart tag. This tag consists of an affinity tag to bind to AC media along with an isotopically coded linker, generally heavy and light forms... 

Biomass may also sorb As(III) from water. Teixeira and Ciminelli (2005) removed considerable As(III) with ground chicken feathers treated with ammonium thioglycolate. X-ray absorption near edge structure (XANES) spectra indicate that the adsorbed arsenic is still in the +3 valence state and that each atom is bound to three sulfur atoms associated with reduced cysteine amino acids (HC>2CCH(NH2)CH2SH) in the feathers. At pH 5 and biomass dosages of 2.0gL 1, the sorption capacity of the material was as high as 0.265 mmol As(III) g-1 biomass (19.9 mg As(III) g-1 biomass Table 7.2). The presence of 0.01 mol L-1 of phosphate had only minor effects on the sorption capacity, which was 0.260 mmol As(III) g 1 biomass (19.5 mg As(III) g-1 biomass) (Teixeira and Ciminelli, 2005, 898). 

While the affinity of transferrin see Iron Proteins for Storage Transport their Synthetic Analogs) for vanadyl is 10-fold greater than that of albumin, the latter can bind up to 20 vanadyl ions including a specific interaction with cys-34, the only reduced cysteine residue in the protein. 

Figure 19-10. The pyruvate formation with the N-terminal cysteine. The C-2 carbonyl in pyruvic acid initially forms a ketimine intermediate (A). The sulfhydryl (SH) group of Cys-1 generated from the reduced cysteine 1-98 disulfide bond in Iso tends to favor the formation of the more thermodynamically stable cyclic thiazoUdiae pyruvate intermediate (B).

These differences are correlated with differing functions. The intracellular reduced thioredoxins are thermodynamically the best reductants of disulfide linkages in proteins and they help keep intracellular proteins reduced. Glutaredoxin can be reduced efficiently by reduced glutathione or by NADPH and glutathione reductase and can, in turn, reduce cysteine and the oxidized form of vitamin C, dehydroascorbic acid (Box The peri-... 

Sometimes the two joined cysteines are called cystine. Disulfide bond formation is an oxidative process the sulfurs each lose a hydrogen atom, becoming more oxidized. Proteins found outside the cell are more likely to have disulfides than are proteins found inside the cell. This is due to the more reducing environment in the cell. Thus, for example, digestive enzymes found in the small intestine have disulfides, while many enzymes involved in cell metabolism have free (reduced) cysteine -SH groups. 

Using accurate mass spectrometry, how is it possible to determine if a small protein contains a pair of disulfide-bonded cysteine residues (i.e., cystine) or two reduced cysteine residues ... 

Figure 7 Steps in ArsC arsenate reductase function coupled to glutathione and glutaredoxin. Step 1 binding of arsenate to Cysl2 thiol. Step 2 binding of reduced glutathione (GSH) to As(V) with reduction to As(IV) level. Step 3 binding of reduced glutaredoxin (GrxSH) and reduction to As(III) level. Step 4 release of arsenite and reforming of reduced cysteine Cl2.

Disulfides can be reduced chemically, electrochemically or enzymatically. Chemical reduction is often used prior to derivatisation of thiols to fluorophores, but it can also be used with HPLC-ED. Common reducing agents are mercapto-ethanol, DTT, dithioerythritol, cyanide and sodium borohydride, but their efficiency varies towards different disulfides. Cyanide or borohydride rapidly reduce cystine at room temperature, but require over 24 h to reduce cysteine-D-penicillamine disulfide completely, whilst the sterically hindered D-penicillamine disulfide is not reduced at all. ... 

In general, the digestion process has to be optimized to achieve maximum efficiency based on a number of parameters affecting the enzymatic reaction that include (i) solubilization and denaturation of proteins, (ii) reduction of disulfide bonds, (iii) alkylation of reduced cysteines, and (iv) digestion conditions. 

Reduction of Disulfide Bonds and Alkylation of Reduced Cysteines... 

Recently, 2,4-dihydroxy-2,5-dimethyl-3(2ff)-thiophenone (DHDMT) was identified in soy sauce as a low-molecular-weight pigment formed by the MaiUard reaction (11BBB1240). Murata et al. (13FF1076) discovered that DHDMT contributed color, aroma, and functional properties to food, and was formed more from cystine than cysteine. It was detected in various brown foods (roasted brown rice) and beverages (beer, but not coffee).The authors su ested that the various kinds of reductants formed during the MaiUand reaction (98FSTIT258) reduced cysteine to cystine, which subsequently leads to DHDMT formation. 

Prima facie, the O2 would be quickly scavenged by one or both reactions, but at what cost to adhesion The cost for reactions in eqn (9.3) and (9.5) is still unclear however, there is experimental evidence that the reaction in eqn (9.3) effectively undermines adhesion in vitro by reducing the contribution of Dopa to adhesion in an SFA (Figure 9.7). To prevent this from happening, eqn (9.4) is again recruited to restore Dopa and adhesion with mfp-6 providing reducing cysteines ... 

Fig. 2. Structure of mercuric reductase (modeled after the crystal structure of ScHiERiNG et al. 1991). is indicated bound to four cysteine sulfurs (see text). NADPH and FAD are perpendicular to the plane of the page. Amino (N) and carboxyl (C) termini of the amino acid backbone are indicated, as is the dithiol (5-5, although probably reduced cysteines in vivo) near the amino terminus. The central dot represents the twofold rotational axis...

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