Dithiothreitol reduction with

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. ... 

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. 

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]... 

Figure 1. Reversed-phase HPLC chromatograms of human relaxin side fraction before and after reduction with dithiothreitol. The chromatography was performed on a Vydac C4 column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient from 18 to 50%.

The preparation of nine 75Se derivatives of steroids has been described these labelled steroids have proved useful in assaying the comparative binding of proteins.77 Reaction of 75Se02 with dehydroepiandrosterone yields a selenium derivative (183) which upon reduction with dithiothreitol or HSCH2CH2OH and then methylation furnishes the corresponding methylseleno-steroid. 

Figure 4.26. Disulfide-Bond Reduction. Polypeptides linked by disulfide bonds can be separated by reduction with dithiothreitol followed by alkylation to prevent reformation.

Reduction with dithiothreitol (DTT). The use of DTT, in place of mercaptoethanol, has gained wide acceptance. The reduction proceeds essentially to completion at low levels of DTT, because of the thermodynamically favored formation of a 6-membered ring. The equilibrium constant at pH 7 and 25°C for the reduction of cystine by DTT is 10", as compared to approximately 1 for mercaptoethanol (Konigsberg 1972). 

Both cross-linking between the P- and y-subunits as well as inactivation of ATPase (hydrolysis) are completely reversed by reduction with excess dithiothreitol (DTT), as shown in the third lane. As shown in lane4, the double mutant EcF,[P Ser-383->Cys/y Cys-87- Ser], in which the original cysteine in the y-subunit was replaced by serine, apparently underwent no cross-linking to form anintersubunit p-y bond nor suffered inactivation of ATPase hydrolysis. However, a minor band at 101 -kDa is present in the SDS-PAGE for the double-mutant membrane this band has been attributed to a dimer of P-subunits, whose predicted molecular mass is 100.6 kDa. 

An extensive study revealed that the A-dithiasuccinyl-protected azide 224 offers a major advantage in the synthesis of A-glycans. Efficient reduction of the A -dithiasuccinyl- and azido-functionality in 224 could be achieved, either in solution by utilizing simultaneous in situ reduction with Zn in THE AcOH AC2O, or on solid phase upon treatment with ethyldiisopropylamine and an excess of dithiothreitol, propane-1,3-dithiol, or 2-mercapto-A-methylacetamide leading to the known 1 or 225. 

Other time-resolved synchrotron radiation scattering studies have been performed to study the dissociation of aspartate transcarbamylase by mercurials. These showed that a time resolution of the order of 100 msec is possible [142aj. The aggregation of bovine serum albumin after cleavage of its disulphide bonds by reduction with dithiothreitol could be traced in time intervals of 50 sec and also showed that experiments with a time interval of 100 msec are possible [142b],... 

Reduction with dithiothreitol or alkylation with iodoacetamide has been shown to abolish the antigenicity of species-specific antigenic determinants of proteoglycan preparations from chick epiphyseal cartilage and rat chondrosarcoma, as detected by immunodiffusion and radioimmunoassay. Some, but not all, of the species-common antigenic determinants were also sensitive to the treatment. 

Example 9.1 The molecular mass of a protein is 10,275 Da. Upon reduction with dithiothreitol and alkylation with iodoacetic acid, its mass increased to 10,865 Da. In a separate experiment, the protein was treated only with iodoacetic acid. The molecular mass of the protein was found to be 10,391 Da. Calculate the number of disulfide bonds in this protein. 

The partly purified reductase from the Novikoflf tumor is specific for ribonucleoside diphosphates, and a single enzyme reduces CDP, UDP, ADP, and GDP. In analogy with the E. coli enzyme, the tumor reductase appears to require nonheme iron, as indicated by stimulation of reductase activity with iron salts and inhibition with iron-chelating agents. The purified reductase requires either a reductant such as dihydrolipoate or dithiothreitol, or an NADP-linked hydrogen transport system the E. coli thioredoxin-thioredoxin reductase couple will link the tumor reductase with NADPH. There appears to be a thioredoxin-like hydrogen transport system in the tumor and in rat liver 32) that links nucleotide reduction with NADPH. 

Ascorbic acid and dehydroascorbic acid have been determined by reversed-phase h.p.l.c., post-column reduction of dehydroascorbic acid to ascorbic acid with dithiothreitol, reaction of excess reagent with JV-ethylmaleimide, and electrochemical detection. Ascorbic acid and its 2-phosphate were determined by h.p.l.c. on an aminopropyl bonded-phase silica column. Dehydroascorbic acid could also be determined by the increase in the ascorbic acid content after reduction with dithiothreitol The method was applied to raw apple and potato to which these compounds are added to prevent browning. ... 

Hen egg-white lysozyme has been esterified specifically at L-aspartic acid-52 with the affinity label 2,3-epoxypropyl j8-di-iV-acetylchitobioside. The disulphide and L-aspartic acid ester bonds of the derivatized enzyme were then reduced with 1,4-dithiothreitol and sodium borohydride, respectively, resulting in the removal of the affinity label and the formation of homoserine residues. The enzyme obtained on re-oxidation with glutathione was inactive, whereas reduction (with 1,4-dithio-threitol) and re-oxidation (with glutathione) of the original enzyme did not abolish its activity. It was concluded that L-aspartic acid-52 is essential for enzymic activity. 

Uridine or 2 -deoxyuridine give the 5-thiocyanato-derivatives by substitu-ticm with thiocyanogen chloride, " opening a route to biologically active 5-mercaptopyrimidines through dithiothreitol reduction of the thiocyanate. 

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,). 

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. 

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