Stopped-flow chemical modification with N-bromosuccinimide: a good probe for changes in the microenvironment of the Trp 62 residue of chicken egg white lysozyme

Abstract

The stopped-flow chemical modification with N-bromosuccinimide (NBS) of Trp 62 of hen (chicken) egg white lysozyme (EC 3.2.1.17) was found to depend greatly on pH: it was not observed at pH's above 7, but it was observed at pH's lower than 6. In addition, at pH's between 6 and 7 the NBS modification showed a delta epsilon pH profile similar to a "titration curve," giving a pK (congruent to 6.5) nearly equal to the pK (congruent to 6.2) of a catalytic residue, Glu 35. The stopped-flow chemical (NBS) modification of N-acetyl-L-tryptophan ethyl ester, a model compound of Trp 62, does not depend on pH at the pH's examined, approximately 3.5-8.5. These experimental results suggest that a change in the state of Trp 62 at Subsite C is induced by protonation-deprotonation of an ionizable residue, which could be Glu 35 (catalytic site), indicating that stopped-flow NBS modification is a good probe for detection of changes in the micorenvironment around the tryptophan residue(s) of enzymes.