Oxidation of tryptophan-21 alters the biological activity and receptor binding characteristics of mouse nerve growth factor.

The tryptophan residues of P nerve growth factor (J3NGF) were modified by reaction with varying concentrations of N-bromosuccinimide. Analysis of the oxidized derivatives by electrophoresis under denaturing conditions, before and after reduction or oxidation of disulfide bonds, revealed partial hydrolysis of specific peptide bonds. Analysis of the newly created NH2 termini by the 5-dimethylaminonaphthalene-1-sulfonyl procedure showed that the peptide bonds contained the carbonyl group of the reacted tryptophan residues and that the oxidation reaction proceeded in the order Trp21, Trp-99, and Trp-76. To obtain PNGF modified only at Trp-21, 0.9 tryptophan residues/NGF chain were oxidized. The dose-response curves for the PNGF-induced neurite outgrowth on sensory and PC12 cells showed that 20to 30-fold higher concentrations for the 0.9 Trp-OX-NGF YNGF oxidized with N-bromosuccinimide) derivative and about 1000-fold higher concentrations for a 2.0 Trp-OX-NGF derivative were required to elicit half-maximum biological response when compared with native PNGF. This decrease in biological activity was paralleled by a decreased binding to NGF receptors on both sensory and PC12 cells. On sensory cells, the affinity of 0.9 Trp-OX-NGF was 2.5% and the affinity of 2.0 Trp-OX-NGF was 0.05% of that of native PNGF. Tested on PC12 cells, 0.9 Trp-OX-NGF bound with 1.2% of the affinity of native PNGF. At the highest concentration tested, 2.0 Trp-OX-NGF did not compete with the binding of PNGF to PC12 cells. That the residual binding affinity and biological activity of 0.9 TrpOX-NGF were not due to unmodified PNGF in the preparation was ascertained by the PNGF-dependent cytotoxicity assay, which showed that less than 0.2% of unmodified PNGF remained. In contrast to the significant decrease in biological and binding activity, the derivative modified at Trp-21 retained 25% of the antigenicity of the native protein.