Chemical modifications sulfhydryl groups

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.

Oligonucleotides containing amine groups introduced by enzymatic or chemical means may be modified with SATA (Chapter 1, Section 4.1) to produce protected sulfhydryl derivatives. The NHS ester end of SATA reacts with a primary amine to form a stable amide bond. After modification, the acetyl protecting group can be removed as needed by treatment with hydroxylamine under mildly alkaline conditions (Fig. 401). The result is terminal sulfhydryl groups that can be used for subsequent labeling with thiol-reactive probes or activated-enzyme derivatives (Kumar and Malhotra, 1992). 

Chemical modification of soybean agglutinin by acetylation of its amino groups resulted in little loss of agglutinating activity, whereas the protein was quite sensitive to modification of its tyrosyl residues.547 Failure of the protein to react with 2-iodoacetamide or p-(chloro-mercuri)benzoate in 6 M urea confirmed that it was devoid of sulfhydryl groups. A metalloprotein containing151 Ca2+ and Mn2+, the soybean lectin is inactivated by Al3+, Fe3+, and Pb2+, whereas Mn2+, Ba2+, Mg2+, Ag+, Li+, and K+ are without effect.532... 

Chemical modification of the single free sulfhydryl group of gastric lipases, with either 4,4 -dithiopyridine or 5,5 -dithiobis(2-nitrobenzoic acid) [83, 84], induced a complete loss of activity, when water-soluble p-NPA or emulsified tributy-roylglycerol (tributyrin) were used as substrates [85]. Both catalytic activities were... 

Most of the enzyme modification reactions, and hence of the coupling reactions, are nucleophilic reactions, in particular bimolecular nucleophilic substitution reactions following an 5 2-type mechanism. Therefore, the chemical reactivity is basically a function of nucleophilicity of the amino acid side chain. Following the overall nucleophilic order of Edwards and Pearson [22], the sulfhydryl group of cysteine is the most potent nucleophile in the protein, especially in its thiolate form. 

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