Disulfide bridges reduction

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

CH2SH + 1/2 O2 -CH2-S-S-CH2 + H2O This reaction requires an oxidative environment, and such disulfide bridges are usually not found in intracellular proteins, which spend their lifetime in an essentially reductive environment. Disulfide bridges do, however, occur quite frequently among extracellular proteins that are secreted from cells, and in eucaryotes, formation of these bridges occurs within the lumen of the endoplasmic reticulum, the first compartment of the secretory pathway. 

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,). 

This thiol-disulfide interconversion is a key part of numerous biological processes. WeTJ see in Chapter 26, for instance, that disulfide formation is involved in defining the structure and three-dimensional conformations of proteins, where disulfide "bridges" often form cross-links between q steine amino acid units in the protein chains. Disulfide formation is also involved in the process by which cells protect themselves from oxidative degradation. A cellular component called glutathione removes potentially harmful oxidants and is itself oxidized to glutathione disulfide in the process. Reduction back to the thiol requires the coenzyme flavin adenine dinucleotide (reduced), abbreviated FADH2. 

As attractive as the transannular bridging of bis(thiolactones) to bicyclic bis(oxepane) frameworks is, our inability to convert the disulfide bridging product (see 25, Scheme 5) to a mmv-fused bre-vetoxin-type bis(oxepane) (see 28) necessitated the development of a modified, stepwise strategy. This new stepwise approach actually comprises two very effective methods for the construction of cyclic ethers the first of these is the intramolecular photo-induced coupling of dithioesters, and the second is the reductive cyclization of hydroxy ketones. We will first address the important features of both cyclization strategies, and then show how the combination of the two can provide an effective solution to the problem posed by trans-fused bis(oxepanes). 

Fungal cutinases show no free SH groups but have 4 Cys residues, indicating that they are in disulfide linkage [119]. The reaction of the native enzyme with DTE was extremely slow but in the presence of SDS at its CMC rapid reduction could be observed [102]. Reduction of the disulfide bridge resulted in irreversible inactivation of the enzyme and the protein tended to become insoluble. CD spectra of cutinase in the 205-230 nm region, before and after DTE reduc-... 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

Figure 28.8 The heterobifunctional crosslinkers sulfo-SAND, SANPAH, and sulfo-SANPAH contain an amine-reactive (sulfo)NHS ester on one end and a photoreactive phenyl azide group on the other end. Sulfo-SAND allows release of conjugates by reduction of its internal disulfide bridge.

The A and B peptide chains in insulin are linked through disulfide bridges. Their presence was suspected from the change in molecular weight which followed the reduction of insulin. For quantitative analyses the S-S bridges had to be broken. Sanger, following the approach used by Toennies and Homiller (1942), oxidized the protein with performic acid, so that the half-cystines were converted to cysteic acid. After oxidation, insulin could be separated into its A and B chains, the A peptide with 20 amino acid residues and the B with 30. 

The kinetics of disappearance from the circulation of intravenously administered human insulin (Fig. 6.32) is nonlinear [145]. Within a few minutes after injection, it becomes localized in the liver, heart, and kidneys, where it is rapidly metabolized. Indeed, the hepatic extraction could be as high as 70% on a single passage, whereas kidneys could account for 10-40% degradation. Enzymatic reduction of the disulfide bridges appears to be the first step in the in vivo metabolism of insulin, although this reaction appears of limited significance under in vitro conditions. 

Oxido-reduction processes may come into play, for example, by closing or opening a ring when disulfide bridges are present ... 

Typically, the incorporation of carbohydrazide obtained with antibody A5B7 was about 0.5 molecules of carbohydrazide per F(ab )2 as determined by incorporation of 55Fe-labeled aldehyde 201 ESI-MS after reduction of disulfide bridges by dialysis in 10 mM DTT confirmed that the light chain was not modified by this procedure. On the other hand, the heavy-chain mass was 26 489 Da in the F(ab )2-carbohydrazide and only 26 416 in the F(ab )2. The 73 Da difference between the two heavy chains is in agreement with the 72 Da increment expected by the incorporation of one carbohydrazide group. 

Alternatively, both peptide chains could be protected at one cysteine residue as a 5-Acm derivative and at the second cysteine residue by an acid-labile [Trt, Mob, Xan, or Bzl(4-Me)], base-labile (Fm), or reduction-labile (5-tBu) group. Both peptide chains may then be separately converted into the free thiol/Acm-protected form for selective activation of one chain as S-SPy or. S -Npys derivatives by reaction with di(2-pyridyl)disulfide or di[5-nitro(2-pyridyl)]disulfide, or as a sulfenohydrazide derivative by reaction with azodicarbocylic acid derivatives for formation of the first interchain disulfide bridge. 

Cys(StBu) with phosphines only a slight excess of tributylphosphine is required for this purpose. Under these conditions reduction of the diselenide does not occur at all, and subsequent air oxidation of the two cysteine residues at high dilution leads in a highly selective manner to the diselenide- and disulfide-bridged peptides. The selectivity of the disulfide bridging is assured by the complete absence of thiol/diselenide exchange reactions even at alkaline pH values due to the very low reactivity of the diselenide toward mono-thiols as a result of their highly differentiated redox potentials. ... 

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