Dithiothreitol reduction reaction

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,). 

Grollmann U, Schnabel W (1980) On the kinetics of polymer degradation in solution, 9. Pulse radiolysis of polyethylene oxide). Makromol Chem 181 1215-1226 Hamer DH (1986) Metallothionein. In Richardson CC, Boyer PD, Dawid IB, Meister A (eds) Annual review of biochemistry. Annual Reviews, Palo Alto, pp 913-951 Held KD, Harrop HA, Michael BD (1985) Pulse radiolysis studies of the interactions of the sulfhydryl compound dithiothreitol and sugars. Radiat Res 103 171-185 Hilborn JW, PincockJA (1991) Rates of decarboxylation of acyloxy radicals formed in the photocleavage of substituted 1-naphthylmethyl alkanoates. J Am Chem Soc 113 2683-2686 Hiller K-O, Asmus K-D (1983) Formation and reduction reactions of a-amino radicals derived from methionine and its derivatives in aqueous solutions. J Phys Chem 87 3682-3688 Hiller K-O, Masloch B, Gobi M, Asmus K-D (1981) Mechanism of the OH radical induced oxidation of methionine in aqueous solution. J Am Chem Soc 103 2734-2743 Hoffman MZ, Hayon E (1972) One-electron reduction of the disulfide linkage in aqueous solution. Formation, protonation and decay kinetics of the RSSR radical. J Am Chem Soc 94 7950-7957... 

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. 

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). 

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. 

A third enzyme is required to reduce the epoxide to vitamin K (Eq. 15-57). The biological reductant is uncertain but dithiols such as dithiothreitol serve in the laboratory.518 See also Eq. 18-47. Protonation of an intermediate enolate anion would give 3-hydroxy-2, 3-dihydrovitamin K, an observed side reaction product. 

Carboxy terminal amino acid or peptide thiols are prepared from various p-amino alcohols by conversion into a thioacetate (R2NHCHR1CH2SAc) via a tosylate followed by saponification.Several methods have been used to prepare N-terminal peptide thiols, the most common procedure is the coupling of (acetylsulfanyl)- or (benzoylsulfanyl)alkanoic acids or add chlorides with a-amino esters or peptide esters, followed by deprotection of the sulfanyl and carboxy groups. 8 16 Other synthetic methods include deprotection of (trit-ylsulfanyl)alkanoyl peptides, 1718 alkaline treatment of the thiolactones from protected a-sulfanyl acids, 19 and preparation of P-sulfanylamides (HSCH2CHR1NHCOR2, retro-thior-phan derivatives) from N-protected amino acids by reaction of P-amine disulfides with carboxylic acid derivatives, followed by reduction. 20,21 In many cases, the amino acid or peptide thiols are synthesized as the disulfides and reduced to the corresponding thiols by the addition of dithiothreitol prior to use. 

Neta P, Eluie RE, Mosseri S, Shastri LV, Mittal JP, Maruthamuthu P, Steenken S (1989) Rate constants for reduction of substituted methylperoxyl radicals by ascorbate ions and N,N,N, Al -tetrameth-yl-p-phenylenediamine. J Phys Chem 93 4099-4104 Netto LES, Stadtman ER (1996) The iron-catalyzed oxidation of dithiothreitol is a biphasic process hyudrogen peroxide is involved in the initiation of a free-radical chain of reactions. Arch Biochem Biophys 333 233-242... 

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. 

The incubation mixture for assay of y-glutamylcysteine synthetase contained in a final volume of 0.3 mL 0.1 M Tris-HCl (pH 8.2), 6 mM ATP, 50 mM KC1, 6 mW dithiothreitol, 20 mM MgCl2, 3 mW L-cysteine, and 15 mM L-glutamic acid. The mixture was incubated at 37°C for 15 minutes to ensure the complete reduction of cysteine. The reaction was initiated by addition of hemolysate. Samples of 20 /xL were withdrawn at various times between 10 and 30 minutes and added to 50 /xL of 50 mM N-ethylmorpholine (pH 8.4) and 10 /xL of monobromobimane in acetonitrile. After incubation for 15 minutes in the dark at room temperature, the reaction was stopped by addition of 80 /xL of 10% sulfosalicylic acid. The volume was made up to 500/xL with water before centrifugation and analysis of the resulting supemate by HPLC. 

Since azides are smoothly reduced on solid supports with SnCybenzenethiol/TEA (5 mmol/ 25 mmol/25 mmol for 1 mmol resin-bound azide rt, 2 this alternative procedure has been exploited for the synthesis of oligoureas on resin using a-azido acids.Similarly, azides are efficiently reduced to amines with dithiothreitol (DTT),P 1 a procedure that was successfully transferred to the reduction of a-azidoacyl peptides on resin for the SPPS with a-azido acids.In the case of sterically hindered azides, addition of small amounts of 2-sulfanylethanol to the DTT/DIPEA mixture or the use of DBU as a base enhances the reaction rates (Scheme Other reductions of azides include the use of H2S/pyridine/... 

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