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

Abstract

Abstract Conformational investigations have been carried out on derivatives of lysozyme in which tyrosines 20 and 23 were nitrated (NT2-lysozyme), or in which the nitrotyrosine residues had been reduced to aminotyrosine (AT2-lysozyme). Also, a derivative modified at the 6 tryptophan residues by reaction with 2-nitrophenyl sulfenyl chloride (NPS6-lysozyme) was studied. In optical rotatory dispersion measurements, NT2-lysozyme and AT2-lysozyme showed equal rotations at the negative 233-nm minima and at the positive 199-nm maxima. Also, they had identical b0 values. These two derivatives were more rotatory than lysozyme. On the other hand, NPS6-lysozyme 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 NT2-lysozyme and AT2-lysozyme exhibited appreciable disulfide reducibility (one bond) relative to lysozyme (0.03 bond), and the great unfolding in NPS6-lysozyme was also confirmed by the large increase in accessibility of its disulfide bonds (2½ 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 NT2-lysozyme and AT2lysozyme 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, NPS6-lysozyme, assumed a ‘constrained’ conformation on binding with sodium dodecyl sulfate. It was concluded that conformational changes take place upon modification of the tyrosine or tryptophan residues and that NT2-lysozyme and AT2-lysozyme had identical conformations. From these results and the previously reported immunochemical behaviors of these derivatives, it may be concluded that one (or both) of tyrosines 20 and 23 is located in an antigenic reactive site in lysozyme. The results also indicated that, although the antigenic reactivity of native proteins is highly influenced by changes in conformation, not every conformational change will exert an effect on the antigenic reactivity. This should be dependent on the protein and the nature of the conformational change.