Tryptophan residues of the gamma subunit of 7S nerve growth factor: intrinsic fluorescence, solute quenching, and N-bromosuccinimide oxidation.
The environment of tryptophan residues of the gamma subunit derived from the 7S nerve growth factor has been studied by intrinsic fluorescence, solute quenching, and oxidation with N-bromosuccinimide (NBS). The native protein has a fluorescence emission maximum of 345 nm with a tryptophan quantum yield of 0.10. A red shift in the emission maximum occurs between 3 and 6 M urea; a 20% increase in quantum yield occurred at 7-8 M urea. NBS (20-24 mol of NBS/mol of protein) completely oxidized one tryptophan of the gamma subunit, causing a 70% quenching of the intrinsic fluorescence, a complete loss of esterolytic activity toward synthetic substrates, an inability to combine with alpha and beta subunits to re-form the 7S complex, and a shift of about 12% of the structure from beta strands to random coil as determined by circular dichroism. About 80% of the fluorescing tryptophans are accessible to quenching by acrylamide but not to potassium iodide. These results suggest the presence of an exposed tryptophan contributing greater than or equal to 70% of the native fluorescence, located near a negative charge, critical to the esterase activity and possibly to interaction with the beta subunit to re-form 7S NGF.
D001974:Bromosuccinimide / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D046911:Macromolecular Substances; D008433:Mathematics; D009414:Nerve Growth Factors / Q000032:analysis*; D013388:Succinimides / Q000378:metabolism*; D014364:Tryptophan / Q000032:analysis*
D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D013388:Succinimides; D014364:Tryptophan; D001974:Bromosuccinimide