Excited-state intramolecular proton transfer as a fluorescence probe for protein binding-site static polarity.
A fluorescence probe is introduced for protein conformation and binding-site monitoring as the proton-transfer (PT) tautomer fluorescence by using 4-hydroxy-5-azaphenanthrene (HAP) as a prototype. A typical grossly-wavelength-shifted PT fluorescence for HAP is observed in the 600-nm spectral region for this UV-absorbing molecule (absorption onset, 400 nm), for which case PT occurs even in protic solvents. It is shown that PT fluorescence of HAP can serve as a protein-binding-site static-polarity calibrator, shifting from a lambda max of 612 nm in cyclohexane to 585 nm in ethanol at 298 K, contrary to the usual dispersion red shift. A small mechanical solvent-cage effect is noted in ethanol at 77 K, but solvent dielectric relaxation is not apparent from the fluorescence spectrum. Thus, HAP serves to distinguish static solvent-cage polarity from dynamical solvent dielectric relaxation and other solvent-cage effects (mechanical restriction of molecular conformation). HAP as a PT-fluorescence probe is applied to human serum albumin (HSA) and beaver apomyoglobin.
Sytnik
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A
Institute of Molecular Biophysics, Florida State University, Tallahassee 32306-3015.
Kasha
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M
D001059:Apoproteins / Q000737:chemistry*; D001665:Binding Sites; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D009211:Myoglobin / Q000737:chemistry*; D009284:Naphthols / Q000737:chemistry*; D010616:Phenanthrenes / Q000737:chemistry; D011485:Protein Binding; D011522:Protons; D011725:Pyridines / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*; D012997:Solvents; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature
D001059:Apoproteins; D007202:Indicators and Reagents; D009211:Myoglobin; D009284:Naphthols; D010616:Phenanthrenes; D011522:Protons; D011725:Pyridines; D012709:Serum Albumin; D012997:Solvents; C010857:apomyoglobin; C089213:4-hydroxy-5-azaphenanthrene