Conformational Studies on Modified Proteins and Peptides IV. CONFORMATION OF LYSOZYME DERIVATIVES MODIFIED AT TYROSINE OR AT TRYPTOPHAN

Conformational investigations have been carried out on derivatives of lysozyme in which tyrosines 20 and 23 were nitrated (NTa-lysozyme), or in which the nitrotyrosine residues had been reduced to aminotyrosine (ATn-lysozyme). Also, a derivative modified at the 6 tryptophan residues by reaction with 2-nitrophenyl sulfenyl chloride (NPSe-lysozyme) was studied. In optical rotatory dispersion measurements, NTJysozyme and ATJysozyme showed equal rotations at the negative 233-nm minima and at the positive 199-nm maxima. Also, they had identical bo values. These two derivatives were more rotatory than lysozyme. On the other hand, NPSJysozyme showed an appreciable degree of unfolding evidenced by a decrease of all its optical rotatory dispersion parameters. Measurements of the reduced molar ellipticities showed that the present three derivatives, like lysozyme, give a negative circular dichroism band at 208 nm and a shoulder around 220 nm. Results were in agreement with optical rotation measurements. The rotatory behavior of lysozyme and the derivatives showed no change in the pH range 7 to 3. The conformational changes revealed by optical rotatory dispersion and circular dichroism measurements were also shown by increase in disulfide reducibility. Both NTJysozyme and ATz-lysozyme exhibited appreciable disulfide reducibility (one bond) relative to lysozyme (0.03 bond), and the great unfolding in NPS6lysozyme was also confirmed by the large increase in accessibility of its disulfide bonds (23 bonds). Changes in conformation obtained on binding of each of these proteins with sodium dodecyl sulfate were monitored by disulfide accessibility. It was shown that NTy-lysozyme and ATZlysozyme assume “relaxed’ conformation with identical disulfide accessibility (two bonds). The conformation of lysozyme also became ‘relaxed’, although to a lesser extent than the tyrosyl derivatives, on binding with sodium dodecyl sulfate. In contrast to these, NPSB-lysozyme, assumed a