Sulfhydryl groups modification

Intracellular and extracellular ROS activate tyrosine and serine-threonine kinases (i.e., the MAPK family members). Following TNF-a, TGF-f5 or EGF stimulation, intracellular ROS are generated which stimulate various signaling pathways [73], Tyrosine kinase receptors (e.g., EGF, PDGF and TGF-a) may be activated by ROS directly via protein sulfhydryl group modifications, or inhibition of phosphotyrosine phosphatases (PTPases) and subsequent receptor activation. The latter is possible as PTPases contain a redox-sensitive cysteine at their active site [78], and oxidation of protein sulfhydryl groups results in the inactivation of PTPases. 

Akhand, A.A., Pu, M., Senga, T., Kato, M., Suzuki, H., Miyata, T., Hamaguchi, M., and Nakashima, I. (1999). Nitric oxide controls src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. J. Biol. Qiem. 274, 25821-25826. 

After It has been determined which chain In the variant Is aberrant, specific structural studies are required. Several procedures are available which differ from one laboratory to the other and Include chain separation by column chromatography, modification of the sulfhydryl groups through reaction with... 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Figure 1.60 Methyl 4-mercaptobutyrimidate forms 2-iminothiolane, which can react with a primary amine to create a sulfhydryl group. The modification preserves the positive charge of the original amine.

Dissolve the sulfhydryl-containing protein or macromolecule to be modified at a concentration of l-10mg/ml in 50mM Tris, 0.15M NaCl, 5mM EDTA, pH 8.5. EDTA is present to prevent metal-catalyzed oxidation of sulfhydryl groups. The presence of Tris, an amine-containing buffer, should not affect the efficiency of sulfhydryl modification. Not only do amines generally react slower than sulfhydryls, the amine in Tris buffer is of particularly low reactivity. If Tris does pose a problem, however, use 0.1M sodium phosphate, 0.15M NaCl, 5mM EDTA, pH 8.0. 

Thus, iodoacetamide has the highest reactivity toward cysteine sulfhydryl residues and may be directed specifically for —SH blocking. If iodoacetamide is present in limiting quantities (relative to the number of sulfhydryl groups present) and at slightly alkaline pH, cysteine modification will be the exclusive reaction. For additional information on a-haloacetate reactivities and a protocol for blocking, see Section 4.2 (this chapter). 

Methyl methanethiosulfonate (MMTS) is a small reversible blocking agent for sulfhydryl groups (Thermo Fisher, Toronto Research). It reacts with free thiols to form a dithiomethane modification with release of sulfinic acid (Figure 1.122). The sulfinic acid component decomposes into volatile products, which don t affect the disulfide formed from the MMTS reaction Alkylthiosulfonates react rapidly with thiols under mild conditions at physiological pH. The MMTS compound is a liquid at 10.6 M concentration and is conveniently added to a reaction medium by pipette. Complete thiol modifications of available cysteine residues in proteins can... 

Sulfhydryl groups also can be added to 5 -phosphate end of DNA probes (Chapter 27, Section 2.2). Biotinylation at these sites avoids disruption of base pairing with complementary DNA targets, since the point of modification is restricted to a single end position on the oligonucleotide. 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

Purify the thiolated toxin from unreacted Traut s reagent by gel filtration using 0.1M sodium phosphate, 0.15M NaCl, pH 7.5, containing lOmM EDTA. The presence of EDTA in this buffer helps to prevent oxidation of the sulfhydryl groups with resultant disulfide formation. The degree of —SH modification in the purified protein may be determined using the Ellman s assay (Chapter 1, Section 4.1). 

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

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