Reduction dithiothreitol

In the known NMR structures of murine, hamster and human recombinant PrP, the invariant disulfide bond Cysl79-Cys214 is entirely buried in the interior of the structured, C-terminal domain and not accessible to external reductants. In accordance with the structural data is the finding that the disulfide bond of murine PrP(23-231) is extremely resistant against the strong reductant dithiothreitol (DTT). 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

The carbamate, prepared from the 4-nitrophenyl carbonate, is cleaved by reduction with dithiothreitol (DTT) and TEA to give the aniline, which triggers fragmentation, releasing the amine. ... 

There are other substrates for the E. coli Met(0) peptide reductase, one of which is Met(0)-a-l-PI. The native protein is the major serum elastase inhibitor that functions by forming a binary complex with elastase which inhibits its activity. Met(0)-a-l-PI, on the other hand, which can be formed by treatment of the protein with TV-chlorosuccinimide, cannot form a complex with elastase and therefore is not able to inhibit elastase activity117,118. Table 6 shows, however, that when Met(0)-a-l-PI is incubated in the presence of Met(0)-peptide reductase and dithiothreitol the protein regains its ability to form a complex with elastase and inhibit elastase activity119. Similar to results found with Met(0)-L12 reduced thioredoxin could replace the dithiothreitol as reductant in the enzymatic reaction. 

The reduction of ribonucleoside triphosphates by various dithiols which are capable of intramolecular cyclization on oxidation (dihydrolipoate, dithioerythritol, dithiothreitol) yields 2 -deoxyribonucleoside triphosphates. These reactions also require 5-deoxyadenosylcorrinoids. 

Rodrlguez-Garrido B, MC Arbestain, MC Monterroso, F Macfas (2004) Reductive dechlorination of a, 3,5, and y-hexachlorocyclohexane isomers by hydroxocobalamin in the presence of either dithiothreitol or titanium(III) citrate as reducing agents. Environ Sci Technol 38 5046-5052. 

FIGURE 8. The reduction of fMet(O)-Leu-Phe by a human neutrophil and purified E. coli Met(O) peptide reductase. Each assay contained in a final volume of 30 /j1 25 mM Tris-HCl (pH 7.4), lOmM MgCl, 15 mm dithiothreitol 540pmole fMet(O)-[ H]Phe-Leu and Met(0)-peptide reductase. After incubating at 37 °C for 60 min, the incubation mixture is acidified and extracted with ethyl acetate. After centrifugation, an aliquot of the organic phase is removed and assayed for radioactivity. Reproduced by permission of Academic Press from Fliss and coworkers ... 

Dithiothreitol (DTT) and dithioerythritol (DTE) are the trans and cis isomers of the compound 2,3-dihydroxy-1,4-dithiolbutane. The reducing potential of these versatile reagents was first described by Cleland in 1964. Due to their low redox potential (—0.33 V) they are able to reduce virtually all accessible biological disulfides and maintain free thiols in solution despite the presence of oxygen. The compounds are fully water-soluble with very little of the offensive odor of the 2-mercaptoethanol they were meant to replace. Since Cleland s original report, literally thousands of references have cited the use of mainly DTT for the reduction of cystine and other forms of disulfides. 

The following protocol for labeling proteins with 5-IAF is adapted from Gorman (1987). It is a bit unusual in that it involves reduction of disulfides with dithiothreitol (DTT) and immediate reaction with 5-IAF in excess without removal of excess reductant. The procedure can be changed to include a gel filtration step after disulfide reduction to remove excess DTT, but in any case, it should be optimized for each protein to be modified. An alternative to the use of DTT to produce sulfhydryls is thiolation with a compound that can generate free thiols upon reaction with a protein (Chapter 1, Section 4.1). 

Bewley, T.A., Dixon, J.S., and Li, C.H. (1968) Human pituitary growth hormone. XVI. Reduction with dithiothreitol in the absence of urea. Biochim. Biophys. Acta 154, 420-422. 

Konigsberg, W. (1972) Reduction of disulfide bonds in proteins with dithiothreitol. In Methods in Enzymology, (C.H.W. Hirs, and S.N. Timaseff, eds.), Vol. 25 p. 185. Academic Press, New York. 

Vitamin B12 catalyzed also the dechlorination of tetrachloroethene (PCE) to tri-chloroethene (TCE) and 1,2-dichloroethene (DCE) in the presence of dithiothreitol or Ti(III) citrate [137-141], but zero-valent metals have also been used as bulk electron donors [142, 143]. With vitamin B12, carbon mass recoveries were 81-84% for PCE reduction and 89% for TCE reduction cis-l,2-DCE, ethene, and ethyne were the main products [138, 139]. Using Ni(II) humic acid complexes, TCE reduction was more rapid, leading to ethane and ethene as the primary products [144, 145]. Angst, Schwarzenbach and colleagues [140, 141] have shown that the corrinoid-catalyzed dechlorinations of the DCE isomers and vinyl chloride (VC) to ethene and ethyne were pH-dependent, and showed the reactivity order 1,1-DCE>VC> trans-DCE>cis-DCE. Similar results have been obtained by Lesage and colleagues [146]. Dror and Schlautmann [147, 148] have demonstrated the importance of specific core metals and their solubility for the reactivity of a porphyrin complex. 

Screening of over 66,000 compounds from the MLSMR by scientists at the PCMD for inhibitors of Cathepsin B resulted in the identification and characterization of an alternate substrate, SID 16952359 [29]. This study also describes issues relating to the nucleophilicity of dithiothreitol (DTT) and cysteine, reductants frequently used in HTS protocols, and the potential for reactivity with electrophilic sites of probe molecules. 

In the presence of a strong reductant such as titanium citrate, dithiothreitol, or sulfide, cofactor F430, and vitamin B12 can dechlorinate CT to either less chlorinated products (CF, DCM, and CM) or to completely non-chlorinated products as CO, C02, and formic acid at relatively high rates [262,390]. 

Example The cleavage of disulfide bonds by reduction with 1,4-dithiothreitol causes the unfolding of the protein. This exposes additional basic sites to protonation, and therefore results in higher average charge states in the corresponding ESI spectrum (Fig. 11.14). [88]... 

However, near-stoichiometric Fe " ion binding to NifU-1 or NifU was observable only in experiments conducted at 2°C in anaerobic samples that had been pretreated with dithiothreitol to ensure reduction of any intrasubunit or intersubunit disulfides. At room temperature, <10% of the NifU-1 or NifU was in a Fe bound form, and colorimetric analysis indicates that the remainder of the Fe is in solution was in the form of free Fe " ion. Hence this mononuclear Fe -bound species is more likely to be an intermediate in the reduction of Fe ion by NifU or NifU-1 rather than an initial step in cluster assembly on the NifU-1 domain of NifU. In this connection, it is important to note that Fe is rapidly reduced to Fe by cysteine in aqueous solution (Schubert, 1932). The physiological significance (if any) of the apparent ferric reductase activity associated with the NifU-1 domain of NifU remains to be established. 

Dithiothreitol can be used as the source of electrons for the reaction in vitro, although it is widely held that the reduction of the GR in vivo is linked to the pool of reduced nucleotides (NADH or NADPH). A more detailed description of the characterization of each component of the GR is given below. 

Selenoprotein A is remarkably heat stable, as seen by the loss of only 20% of activity on boiling at pH 8.0 for lOmin (Thrner and Stadtman 1973). Although selenoprotein A contains one tyrosine and no tryptophan residues, it contains six phenylalanine residues and thus has an unusual absorbance spectrum (Cone et al. 1977). Upon reduction, a unique absorption peak emerges at 238 nm, presumably due to the ionized selenol of selenocysteine, which is not present in the oxidized enzyme. The activity of selenoprotein A was initially measured as its ability to complement fractions B and C for production of acetate from glycine, in the presence of reducing equivalents (e.g., dithiothreitol). Numerous purification schemes were adopted for isolation of selenoprotein A, all of which employed the use of an anion exchange column to exploit the strongly acidic character of the protein. 

Clostridium sticklandii also expresses a proline reductase that can reduc-tively cleave proline to 8-aminovalerate (Seto and Stadtman 1976). PR was first purified by Seto and Stadtman (1976) by following the decomposition of proUne in the presence of dithiothreitol or NADH. They found PR to have a denatured mass of approximately 30kDa (sodium dodecyl sulfate-polyacrylomide gel electrophoresis SDS-PAGE) and a native size of approximately 300 kDa. The addition of selenite to the growth medium of C sticklandii did increase the specific activity of PR in extracts by threefold however, no selenium was detected in the purified enzyme. It should be noted that this purified enzyme had lost the ability to couple reduction of proline to NADH and thus probably was missing one or more components of the complete enzyme complex. 

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