title: 6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin.
abstract: The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.
authors: Department of Chemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
authors: R S
authors: RS
authors: Phillips
authors: R Q
authors: RQ
authors: Marmorstein
mesh_terms: D000465:Algorithms; D001059:Apoproteins / Q000032:analysis*; D001426:Bacterial Proteins; D002942:Circular Dichroism; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D012097:Repressor Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis*; D012997:Solvents; D014157:Transcription Factors / Q000032:analysis*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism; D014367:Tryptophan Synthase / Q000378:metabolism; D014368:Tryptophanase / Q000378:metabolism
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D012709:Serum Albumin; D012997:Solvents; C027190:TRPR protein, E coli; D014157:Transcription Factors; C055424:6-nitrotryptophan; D014364:Tryptophan; D014368:Tryptophanase; D014367:Tryptophan Synthase