Dithiothreitol , reactions with

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 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). 

The reaction of OH with thiolate ions, taken as an overall reaction, is an ET reaction [reaction (36)]. One must, however, again take into account that a three-electron bonded intermediate is formed in the first step (Chap. 7). In semi-de-protonated dithiothreitol reaction (32) dominates over the H-abstraction reaction (33) (Akhlaq and von Sonntag 1987), although the rate constant for the reaction of OH with a thiol and a thiolate ion are both diffusion controlled (k = 1.5 x 1010 dm3 mol-1 s 1). This is another example of the potentially high regio-selectivity of OH reactions. 

It is usual to effect cleavage of disulphide bonds by reduction or oxidation. Addition of a large excess of a thiol such as 2-mercaptoethanol or 1,4-dithiothreitol to a polypeptide reduces cystine residues to cysteine (Scheme 5.1). In order to prevent reoxidation in air, the generated thiol groups are blocked, usually by reaction with iodoacetic acid. The product yields S -carboxymethylcysteine (5.9) on hydrolysis for amino-acid analysis. Alternatively, oxidative cleavage of disulphide bonds can be achieved with performic acid each half of the cysteine residue is converted into a residue of cysteic acid (5.10). 

For optimal reaction with SDS, samples must be boiled for 3-5 min in the presence of reagents such as 3-mercaptoethanol or dithiothreitol to disrupt disulphide bonds. DTT may be preferable in the presence of urea as S-mercaptoethanol can result in anomalous molecular weight values under these conditions (30). 

To determine the number of disulfide bonds, the protein is reduced and alkylated as described in Section 8.5.1 by reacting it with either dithiothreitol or 2-mercaptoethanol, followed by reaction with iodoacetic acid. The molecular masses of the native protein (Mnat) and reduced and alkylated protein (Mr+a) are determined with MALDl-MS or ESl-MS. S-Carboxymethylation increases the mass of each cysteine residue by 59 Da. From the change in the molecular mass, the number of cysteine residues (Ncys) and hence the number of disulfide bonds (Ns-s) can be estimated using... 

Other Properties and Problems. The low redox potential of dithiothreitol interferes with its use in the presence of certain reagents. While it gives full color yield in reactions with aromatic disulfides such as 5,5 -dithiobis(2-nitrobenzoate) or 2,2 - or 4,4 -dipyridyl disulfide, gives only 4% the expected color in the nitroprusside assay for thiols, presumably because it reduces the iron. It reduces Ci or Co , complexes of nucleotides... 

Complete reduction of the disulfide at the acetylcholine binding site is obtained with the receptors of Electrophorus and Torpedo electric tissues by reaction with 0.2 mAf dithiothreitol at pH 8.0 for 20 min at 25°. At the conclusion of the reduction, the pH is brought to 7.0 with NaHa-PO4 solution. Significant spontaneous reoxidation of reduced receptor does not occur within 30 min. 

In general, three types of sulfhydryls are available for reaction with MBTA the sulfhydryls formed by reduction at the acetylcholine binding site (RSH) sulfhydryls either preexisting or formed by reduction elsewhere on the receptor or on other proteins (PSH), and the sulfhydryls of dithiothreitol (QSH). The rate constant, k, for reaction with RSH is 6 X 10 M-i sec" the average rate constant, fcp, for reaction with PSH is roughly 600 M sec and that, /cq, for reaction with QSH is 5 X 10 sec h The kinetic equations may be written... 

It is possible to avoid the removal of the dithiothreitol before the addition of [ H]MBTA even though the concentration of dithiothreitol sulfhydryls (go) is much greater than mo. As shown above, when 95% of RSH is alkylated, 2.5% of QSH is alkylated hence, if the concentration of H-MBTA (mo) is about 2.5% of go, the reaction with receptor will be 95% complete by the time the bulk of the [ H]MBTA has reacted with dithiothreitol. This approach is suitable for the assay of small quantities (1 /ig) of receptor - as well as for the labeling of larger quantities. 

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. ... 

These methods are unique because, even though a stable covalent bond is formed between matrix and enzyme, it can be broken by reaction with a suitable agent such as dithiothreitol under mild conditions. Additionally, because the reactivity of the thiol groups can be modulated via pH alteration, the activity yield of the methods involving disulfide bond formation is usually high provided that an appropriate thiol-reactive adsorbent with high specificity is used. 

An analogous situation occurs in membrane bound CFj. Illumination of thylakoids in the presence of dithiothreitol causes the exposure of an additional SH group(s) in the y subunit to reaction with N-ethyImaleimide (Table 2). Moreover, the treatment of light- and dithiothreitol-treated thylakoids with N-ethy Imaleimide in the dark stabilizes the ability of the thylakoids to catalyze ATP hydrolysis after a light trigger (Table 3) (S. Ketcham, K. Warncke, unpublished). 

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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