Spectral Studies on Hen Egg-white Lysozyme Modified with Diisopropylphosohorofluoridate*

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Abstract
On chemical modification of hen egg-white lysozyme [EC 3. 2. 1. 17] with diisopropylphosphorofluoridate (DFP) diisopropylphosphoryl (DIP) enzyme was obtained. The spectral characteristics of various DIP-lysozyme preparations obtained under different conditions were studied. The spectral studies included measurements of the ultraviolet difference spectra of DIP-lysozyme minus lysozyme, the change in the spectra of DIP-lysozyme at alkaline pH, spectrophotometric titration curves for ionizable phenolic hydroxyl groups, measurement of optical rotatory dispersion in the far ultraviolet region, and measurement of circular dichroism spectra in the range of 200–320 mμ. Modification of the tyrosyl residues in DIP-lysozyme was demonstrated by the changes in the absorption spectra of DIP-lysozyme at neutral and alkaline pH values and also by the changes in its circular dichroism spectra at an alkaline pH. One or. slightly more than one tyrosyl residue per molecule was found to have been modified by DFP at pH 10.2–11.0. However, when the reaction of lysozyme with DFP was carried out in the presence of 3 M or 5 M guanidine hydrochloride, the number of modified tyrosyl residues increased markedly. A DIP-lysozyme specimen in which nearly all the three tyrosyl residues were modified showed a significant decrease in ordered conformation as judged by its optical rotatory dispersion and circular dichroism spectra. Other DIP-lysozyme samples tested seemed to retain a conformation indistinguishable from that of native lysozyme.

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