Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate.
It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).
D004529:Egg White; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009113:Muramidase; D011487:Protein Conformation
D007461:Iodoacetates; D006639:Histidine; D009113:Muramidase