Topography of Mouse 2.5S Nerve Growth Factor. Reactivity of Tyrosine and Tryptophan

The chemical reactivity and degree of solvent exposure of the two tyrosine and three tryptophan residues of mouse 2.5S nerve growth factor (NGF) have been investigated. Spectrophotometric titrations and reaction with tetranitromethane indicate that both tyrosines are solvent available to a limited extent and that neither is required for activity. While the two tyrosines appear equivalent, the tryptophans exhibit a spectrum of reactivity. The reaction of these residues with N-bromosuccinimide is a kinetically ordered process. The most rapidly reacting residue is also solvent available by the criterion of N-methylnicotinamide chloride titration and is not required for activity. Reaction of the intermediate residue destroys both biological and immunological activity, while oxidation of the third residue leads to dissociation of the 2.5S dimer. Reaction of native NGF with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide leads to modification of Trp-21 and Trp-99 and leaves Trp-76 unmodified. Modification of the rapidly oxidized residue with N-bromo-succinimide followed by reaction with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide in 6 M guanidine hydrochloride reveals the modification of Trp-99 and Trp-76, identifying Trp-21 as the solvent available residue. Computer protein modeling studies show that this chemical description of the topography of the tyrosine and tryptophan residues of NGF is in extremely good agreement with topography of the corresponding residues in the three-dimensional structure of insulin. Thus the observed similarities in the function and mechanism of action of NGF and insulin may result from regions of similar primary structure dictating related three-dimensional conformation.