Environmentally Sensitive Protein Reagents*

The use of protein reagents which exhibit sensitivity to changes in molecular environment has provided a means of examining certain properties of enzymes during interaction with their substrates (1). The recently reported 2-hydroxy-5nitrobenzyl bromide provided one such reagent, the absorption spectrum of which showed sensitivity to environmental changes by virtue of the p-nitrophenolic grouping (2, 3). The properties of this reagent made the study of its homologue, 2-methoxy-5-nitrobenzyl bromide, of interest for several reasons. In the first place, although the 2-hydroxy reagent was useful as a “reporter group” (l), the spectral sensitivity to pH changes imposed by ionization of its phenolic hydroxyl group in the region of pH 7 might be undesirable for certain types of studies. In the second place, the specificity of 2-hydroxy-5-nitrobenzyl bromide as a protein reagent was intriguing. The reagent was highly selective for tryptophan at neutral or acidic pH values, had some reectivity toward cysteine, failed to produce any stable methionine derivatives, and reacted rapidly with water (halftime of hydrolysis less than 1 minute). These properties were significantly different from those of benzyl bromide which reacts with methionine as well as cysteine and tryptophan, and reacts sluggishly with water (half-time in the range of several days). The 2-methoxy derivative seemed of interest since it could (a) provide an environmentally sensitive reagent which would not be particularly affected by the pH of solution, (b) provide a protein reagent having different, but perhaps overlapping, specificity as compared to the 2-hydroxy derivative, and (c) clarify the nature of the high reactivity of the hydroxy compound. For these reasons 2-methoxy-5-nitrobenzyl bromide was prepared and its properties were studied.