Spectral Detection of the Reaction of Formaldehyde with the Histidine Residues of α-Chymotrypsin

The absorption spectra of both imidazole and acetylhistidine undergo bathochromic shifts in the presence of formaldehyde. When the spectra in formaldehyde are compared to those in water, the difference maximum is at 220 and 229 mµ for imidazole and acetylhistidine, respectively. In high concentrations of formaldehyde (about 7 m), the limiting value for the molar difference extinction coefficient of acetylhistidine is about 1600. In conjunction with other evidence discussed, the spectral data permit the conclusion that the difference peak generated by the imidazole group in formaldehyde is due to the formation of a hydroxymethyl derivative. When the spectrum of chymotrypsin in formaldehyde is compared to that in water, a difference peak is observed at 230 mµ. Although the major contribution to this low wave length peak is the result of solvent-induced red spectral shifts of the absorption envelopes of the aromatic chromophores (primarily tryptophyl groups), part of the total difference absorbance is due to the reaction between formaldehyde and the imidazole groups of the histidine residues. The contribution of the latter effect to the observed difference absorbance has been assessed from the results of spectral measurements of the enzyme and appropriate model mixtures in formaldehyde solution. The limiting molar difference extinction coefficient for the reaction between the histidine residues of chymotrypsin and formaldehyde, corrected for solvent effects, is equal to twice that observed for acetylhistidine. The change in the corrected molar difference extinction of chymotrypsin, as a function of formaldehyde concentration, agrees with the theory for the reaction of 1 mole of the enzyme with 2 moles of formaldehyde.