Chemical Modification

Chemical modification is one of the most useful methods of identifying the functional groups of a protein. Chemical modification is also used for labeling proteins with reporter groups to monitor their conformations for radiolabeling and to increase their stability. If proteins are modified chemically under mild conditions, the modifications occur to the native conformation, and the modified proteins usually retain their native conformations. However, conformational changes in the modified proteins sometimes occur, and one must be careful about checking the conformation of the modified proteins, especially when they lose their functions. One must also be careful about the side reactions that often accompany otherwise specific chemical modifications.

It is possible to modify chemically the residues of aspartic acid, glutamic acid, histidine, lysine, arginine, methionine, tryptophan, tyrosine, and cysteine, and less easily serine, threonine, asparagine, and glutamine, but it is not possible to modify specifically glycine, alanine, valine, leucine, isoleucine, phenylalanine, and proline residues.

Residues that participate in the function are usually accessible to the solvent and consequently susceptible to reaction with a chemical reagent. The residues that show different reactivity in the presence and absence of a ligand are important for the function. A good method for discriminating the functional residues after modification involves analyzing the responsible residues within the proteins retained by an appropriate affinity column. Affinity labeling is a sophisticated way of modifying active site residues selectively. Modification with a suicide substrate is a very good method for modifying catalytic residues specifically.

 The stability of a protein can be altered by chemical modification. Proteins are stabilized by introducing additional stabilizing forces such as hydrophobic forces, electrostatic interactions, and hydrogen bonds. Intra- or intermolecular cross-links are also effective for stabilizing proteins, as is attaching polymers to their surfaces.

Amino groups are reactive nucleophiles that can be modified by many chemical reactions, including acylation, amidination, and guanidination. Carboxyl groups can be modified after activation with carbodiimides or by esterification. Tyrosine residues are modified by nitration with tetranitromethane, iodination, or acetylation with acetylimidazole. The thiol group is the strongest nucleophile among all of the functional groups of amino acids, so there are many reagents that react specifically with it. Among these reactions are metal binding with p-mercurylbenzoate (PCMB); mixed-disulfide formation with disulfide reagents, such as 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, or Ellman's reagent) or dithiodipyridine; alkylation with iodoacetate, methyl iodide, ethyleneimine or N-ethylmaleimide, cyanylation with 2-nitro-5-thiocyanobenzoic acid (NTCB), and oxidation with many oxidants. Reduction of the disulfide bonds of proteins and alkylation of the resulting thiol groups is an important step in protein chemistry. The imidazole group of histidine residues can be modified with diethylpyrocarbonate, or they can be photooxidized in the presence of photosensitizing dyes. The sulfur of methionine residues can be oxidized to the sulfoxide by air or by oxidants, or alkylated with agents like methyl iodide under acidic conditions. The latter reaction can be reversed by thiols, so an isotopic label can be introduced in 50% of the terminal methyl group of methionine residues using labeled methyl iodide. The guanido group of arginine residues forms heterocyclic condensation products with 1,2- and 1,3-dicarbonyl compounds, such as phenylglyoxal,               , 2,3-butanedione, and 1,2-cyclohexanedione. The indole ring of the tryptophan residue can be modified with various oxidants, such as N-bromosuccinimide, iodine, or ozone, or by electrophilic reagents, such as 2-hydroxy-5-nitrobenzylbromide or 2-nitrophenylsulfenylchloride.