Disulfide bonds in peptides

Complete reduction of all the disulfide bonds in peptides and proteins is generally achieved by treatment with excesses of dithiothreitol (DTT) under denaturing conditions. Due to the highly differentiated redox potentials of the disulfides, selective reduction of single disulfide bonds can also be achieved. Subsequent alkylation of the thiol groups generated by this partial reduction leads to stable derivatives, which are useful in the analysis of disulfide connectivities. 

Cyanogen Iodide (ICN) has been used extensively for the cyanation of alkenes and aromatic compounds [12], iodination of aromatic compounds [13], formation of disulfide bonds in peptides [14], conversion of dithioacetals to cyanothioacetals [15], formation of trans-olefins from dialkylvinylboranes [16], lactonization of alkene esters [17], formation of guanidines [18], lactamization [19], formation of a-thioethter nitriles [20], iodocyanation of alkenes [21], conversion of alkynes to alkyl-iodo alkenes [22], cyanation/iodination of P-diketones [23], and formation of alkynyl iodides [24]. The products obtained from the reaction of ICN with MFA in refluxing chloroform were rrans-16-iodo-17-cyanomarcfortine A (14)... 

MeSiCl3 has been found useful as a deprotecting agent in peptide chemistry. It also promotes, in conjunction with diphenyl sulfoxide, formation of disulfide bonds in peptides (eq 12). ... 

Maruyama K, Nagasawa H, Suzuki A (1999) 2,2 -Bispyridyl disulfide rapidly induces intramolecular disulfide bonds in peptides. Peptides 20 881-884... 

A second kind of covalent bonding in peptides occurs when a disulfide linkage, RS-SR, is formed between two cysteine residues. As we saiv in Section 18.8, a disulfide is formed by mild oxidation of a thiol, RSH, and is cleaved by mild reduction. 

The three-dimensional structural architecture of plant defensins is exemplified by the structure of Rs-AFP, ° which comprises an N-terminal /3-strand followed by an ct-helix and two /3-strands (/3a/3/3 configuration). The /3-strands form a triple-stranded antiparallel /3-sheet. The three-dimensional structure is stabilized by three disulfide bonds. In general, in plant defensins two disulfide bonds form between the ct-helix and the central /3-strand. A third disulfide bond stabilizes the structure by linking the /3-strand after the helix to the coiled part after the ct-helix. This motif is called the cysteine-stabilized a/3-motif (CSa/3)" and also occurs in toxins isolated from insects, spiders, and scorpions.The fourth disulfide bond links the C-terminal end of the peptide with the N-terminal /3-strand. Two plant defensins, PhDl and PhD2, feature a fifth disulfide bond and have been proposed to be the prototypes of a new subclass within plant defensins." As a result of these structural features the global structure of plant defensins is notably different from o //3-thionins, which is one of the reasons for the different nomenclature. The structures of plant defensins Rs-AFP ° and NaDf are shown in Figure 6, where they are compared to the thionin /3-purothionin and the structurally more related drosomycin and charybdotoxin. ... 

The protected protein was deprotected with HF and then with mercury(II) acetate in 50% AcOH to remove the Acm groups. Oxidative folding gave the desired peptide. A tryptic digest was separated on HPLC and the seven identified peaks confirmed the correct disulfide bonds in the product. 

Scheme 1 General Approaches for the Production of Disulfide Bonds in Cysteine Peptides...

Many scorpion toxins, insect defensins, and enzyme inhibitors are cystine-rich polypeptides containing three to four disulfide bonds. In a large number of these toxins, two cystines are involved in the consensus Cys-(Xaa)1-Cys/Cys-(Xaa)3-Cys framework which is responsible for the common characteristic fold consisting of an a-helix and a two- or three-stranded antiparallel (3-sheet (a 3 3-fold or 3a 3 3-fold). For a review see ref[69]. The overall compact globular structures of these cystine-rich peptides contain the cystine stabilized a-helix motif (Section 6.1.5.1.2) which is further stabilized by a third disulfide bond between the N-terminus and the (3-strand adjacent to the helix and in some cases by an additional fourth disulfide bridge. Due to the presence of the cystine stabilized a-helix motif, a preferred initial formation of this motif followed by its stabilization via the additional disulfides was expected. However, in contrast to what was observed for the cystine peptides containing only the cystine stabilized a-helix motif, simple air oxidation is not successful. 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

Absorbance of peptide bonds at 2170 nm is usually used as a key band for the calibration of protein content measurement. However, the intensity of the absorption depends on the structure and conformation of the protein. Yamashita et al [8] investigated the change in absorption at 2170 nm caused by the conformational change of protein using bovine serum albumin (BSA) as the model protein. A mixture of dithiothreitol (a reducing agent) and BSA (5%) was taken in a quartz cell attached to the NIR instrument, and then spectra were recorded at 10-minute intervals. Dithiothreitol reduced the disulfide bond in BSA. The... 

Vasopressin is administered by intravenous, intramuscular, or intranasal routes oral absorption is slight. The half-life of circulating ADH is approximately 20 minutes, with renal and hepatic catabolism via reduction of the disulfide bond and peptide cleavage. A small amount of vasopressin is excreted as such in the urine. 

The native three-dimensional conformation of a protein is maintained by a range of noncovalent interactions (electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulfide bonds), in addition to the peptide bonds between individual amino acids. 

Limitations of the TR-FRET approach in terms of peptide sequence flexibility must be considered as well. The prerequisites and limitations for successful peptide labeling, for example, for the LANCE Ultra approach include (1) need for a cysteine in position zero, i.e., the peptide s N terminus, (2) no other free cysteine allowed in the peptide sequence, (3) overall charge different from zero, (4) no disulfide bonds in the peptide, (5) no tryptophan or histidine at the C terminal position, and (6) maximum length of 30 amino acids (personal communication). 

In previous papers, we have (a) reviewed elimination reactions of disulfide bonds in amino acids, peptides, and proteins under the influence of alkali (5) (b) analyzed factors that may operate during alkali-induced amino acid crosslinking and its prevention (6) (c) demonstrated inhibitory effects of certain amino acids and inorganic anions on lysinoalanine formation during alkali treatment of casein, soy protein, wheat gluten, and wool and on lanthionine formation in wool ( 7, 9) (d) demonstrated that... 

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