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Date: 20-12-2015
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Alkylation
Alkyl groups have the general formula Cn H2n + 1–. Alkylation is a reaction that introduces an alkyl group into a compound. Commonly used alkylating agents are olefins, alkyl halides, and alcohols. Biologically important alkylation occurs on nitrogen and oxygen atoms in polynucleotides. Alkylating agents that cause these reactions are mutagens and carcinogens and include methyl methanesulfonate, dimethyl sulfate, N-methyl-N-nitrosourea, dimethylnitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine, etc. Some alkylating agents are used as anticancer drugs, including cyclophosphamide, nitrogen mustard and its oxide, and triethylenephosphoramide.
Alkylating agents easily alkylate nucleophiles in proteins such as those on cysteine (1), histidine (2), and methionine (3) residues, amino groups (4), and sometimes tyrosine residues and carboxyl groups (5). The most well-known alkylation is that for blocking free thiol groups of cysteine residues to prevent their oxidation, especially in the course of determining the primary structure of a protein (see Protein Sequencing).
1. Reduction and Alkylation of Proteins
The protein at a concentration of 2% (w/v) is dissolved in 0.5 M Tris buffer, pH 8.1, containing 6 M guanidinium chloride and 0.002 M EDTA and reduced with dithiothreitol (50 mol/mol of disulfide bond) at 30° to 40°C for 4 h under nitrogen (6). Then the solution is cooled to room temperature, and iodoacetamide (100 mol/mol of the original disulfide bond) is added, with the addition of NH4OH as necessary to maintain a constant pH. Other alkylating agents that can be employed instead are iodoacetic acid, 4-vinylpyridine, N-ethylmaleimide, and ethyleneimine.
References
1. J. Bridgen (1972) Biochem. J. 126, 21–25.
2. A. M. Crestfield, W. H. Stein, and S. Moore (1963) J. Biol. Chem. 238, 2413–2420.
3. J. M. Gleisner and R. L. Blaklay (1975) J. Biol. Chem. 250, 1580–1587.
4. H. J. Goren and E. A. Barnard (1970) Biochemistry 9, 974–983.
5. K. Takahashi, W. H. Stein, and S. Moore (1967) J. Biol. Chem. 242, 4682–4690.
6. M. J. Waxdal et al. (1968) Biochemistry 7, 1959–1966.
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