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The Airway and Vasoactive Intestinal Peptide Autoantibodies and Receptors
Published in Devendra K. Agrawal, Robert G. Townley, Inflammatory Cells and Mediators in Bronchial Asthma, 2020
Pretreatment of solubilized lung extract with increasing concentrations of dithiothreitol progressively reduced the 125I-VIP binding from 43.2 ± 3.1 to 10.3 ± 1.4 fmol/mg protein.39 This suggests that disulfide bonds are important in maintaining the receptors in an active conformation.
Pathogenesis: Molecular mechanisms of osteoporosis
Published in Peter V. Giannoudis, Thomas A. Einhorn, Surgical and Medical Treatment of Osteoporosis, 2020
Anastasia E. Markatseli, Theodora E. Markatseli, Alexandros A. Drosos
Wnt proteins: Experiments conducted on mice have highlighted the role performed by Wnt proteins in osteoblastogenesis (273). Wnt are secretory glycoproteins that contain a high proportion of the amino acid cysteine. These amino acids of cysteine are linked to each other with disulfide bonds (219). The structure of Wnt proteins is insoluble (274). Since Wnt glycoproteins are secretory and are found in the extracellular matrix, their production site remains unknown.
The Modification of Cystine — Cleavage of Disulfide Bonds
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
There are several approaches to the cleavage of disulfide bonds in proteins. The majority of studies involve the cleavage of the disulfide bond of cystine to the free thiol group of cysteine by reduction. Reduction has been generally accomplished with a mild reducing agent such as β-mercaptoethanol or cysteine. Gorin and co-workers1 have examined the rate of reaction of lysozyme with various thiols. At pH 10.0 (0.025 M borate), the relative rates of reaction were β-mercaptoethanol (2-mercaptoethanol), 0.2; dithiothreitol, 1.0; 3-mercaptopropionate, 0.4; and 2-aminoethanol, 0.01. The results with aminoethanethiol were somewhat surprising since the reaction (disulfide exchange) involves the thiolate anion and 2-aminoethanethiol would be more extensively ionized than the other mercaptans. Dithiothreitol has been a useful reagent in the reduction of disulfide bonds in proteins2 as introduced by Cleland. Dithiothreitol and the isomeric form, dithioerythritol, are each capable of the quantitative reduction of disulfide bonds in proteins. Furthermore, the oxidized form of dithiothreitol has an absorbance maximum at 283 nm (Δϵ = 273) which can be used to determine the extent of disulfide bond cleavage.2 The UV spectra of dithiothreitol and oxidized dithiothreitol are shown in Figure 1. Insolubilized dihydrolipoic acid has also been proposed for use in the quantitative reduction of disulfide bonds.4
Molecular effects of ozone on amino acids and proteins, especially human hemoglobin and albumin, and the need to personalize ozone concentration in major ozone autohemotherapy
Published in Critical Reviews in Clinical Laboratory Sciences, 2023
Fouad Mehraban, Arefeh Seyedarabi
During oxidation with different oxidants such as H2O2, the free thiol group of Cys in HSA can form sulfenic acid as a key intermediate in thiol oxidation processes, which can play a role in the formation of mixed disulfides, for example, the reaction of a protein sulfenic acid (HSA-SOH) with GSH and the formation of glutathionylated proteins (HSA-SSG) [118,127]. The formation of disulfide bonds can also occur in other molecules in the blood that contain the sulfhydryl group. Disulfides are generally formed as a result of the oxidation of thiol groups of Cys and also through thiol-disulfide reactions caused by oxidants, which lead to the generation of intermolecular protein-protein crosslinks such as the crosslink formed between Cys36 from the Cys36-Cys97 disulfide of C-reactive protein with the thiol of Cys34 from HSA [128].
Self-assembled non-covalent protein-drug nanoparticles: an emerging delivery platform for anti-cancer drugs
Published in Expert Opinion on Drug Delivery, 2020
Islam A. Hassanin, Ahmed O. Elzoghby
Protein–protein interactions are required for the aggregation process and the subsequent formation of protein nano-assemblies. These interactions take place secondary to conformational changes induced by thermal denaturation or disulfide bond reduction, which expose the hydrophobic residues of a protein molecule. Therefore, it is important to assess the surface hydrophobicity and the changes following the formation of protein nano-assemblies [15]. 1-anilino-8-naphthalenesulfonate (ANS), a fluorescent probe, is used for studying protein–ligand interactions and changes in the hydrophobic regions of a protein, which may be attributed to a change in the protein environment [54]. Disulfide bond reduction increases the hydrophobic residues accessible to protein–protein interactions and protein–drug interactions. It was demonstrated, using ANS, that the change in fluorescence intensity due to hydrophobicity was 5% higher in β-ME-HSA than in the native HSA, at HSA concentration of 0.2 mg/mL, and it was 10% higher at HSA concentration of 0.34 mg/mL [15]. Conversely, a decrease in the fluorescence intensity of ANS, which may be induced due to the transfer of ANS from a hydrophobic to a polar environment, may indicate an interaction between a hydrophobic drug molecule and the hydrophobic binding site of a protein, or a protein-protein interaction [54]. Thus, changes in the protein hydrophobicity, as a result of nanoparticle formation, were found to result in a decrease in the fluorescence values, due to the presence of hydrophobic intermolecular interactions involved in the process of protein self-assembly.
Identification and quantitation of hinge cysteinylation on endogenous IgG2 antibodies from human serum
Published in mAbs, 2020
Luladey Ayalew, Alicia D. D’Souza, Yan Chen, Michael T. Kim
Cysteinylation involves the addition of a terminal cysteine onto a cysteine residue in a protein via a disulfide bond. In therapeutic mAbs, this PTM may arise from free cysteine molecules present in cell culture media forming covalent adducts via nucleophilic free thiols.12,15 Previous studies have shown that cysteinylation can occur in the hinge regions of recombinant IgG2 antibodies.15,16 The hinge regions of IgG2 antibodies equilibrate between three structural isoforms IgG2-A, IgG2-B, and IgG2-A/B17,18 (Figure 1) with estimated in vivo relative abundances of 7.2%, 21.8%, and 70.8%, respectively.19 Notably, Kita et al. reported that cysteinylation was only found in the hinge region of IgG2-A isoform in recombinant IgG2 mAbs.15 In this case, two terminal cysteines are incorporated into a reduced hinge disulfide bond of the IgG2-A isoform as shown in Figure 2; mono-cysteinylation, where only one terminal cysteine is incorporated, was not found in the hinge region of IgG2-A isoform.