Abstract
The apparent dielectric constant, ε, of the hydrophobic pyrene binding sites in erythroid spectrin and human serum albumin (HSA) were estimated using the linear relationship [Turro, N.J., Kuo, P.L., Somasundaran, P. and Wong, K. (1986). J. Phys. Chem. 90, 288–291] between the ratio of the first (373 nm) and the third (384 nm) vibronic peak intensities (l1/l3) and the dielectric constant of the bulk medium. Binding of the hydrophobic fluorescent probe, pyrene, to erythroid spectrin and HSA was determined from concentration dependent change in the ratio l1/l3 from the emission spectra. Pyrene binds to spectrin (Kapp = 6.2 × 106 M−1) with a higher affinity than that of HSA (Kapp = 3.7 × 104 M−1) and the binding in both cases are saturable. The ε for spectrin and HSA was estimated to be 7 ± 2.1 and 5.4 ± 1.6 respectively. A case study with spectrin, covalently labeled with pyrene maleimide, have been presented for aging of pyrene-labelled spectrin showing the potential of the use of vibrational peak ratios (l1/l3) in the study of polarity of microenvironments in the neighborhood of cysteine residues of a protein. Large changes in the pyrene spectral components indicated conformational changes in the cysteine microenvironment of the protein upon storage at 4°C.
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Haque, M.E., Ray, S. & Chakrabarti, A. Polarity Estimate of the Hydrophobic Binding Sites in Erythroid Spectrin: A Study by Pyrene Fluorescence. Journal of Fluorescence 10, 1–6 (2000). https://doi.org/10.1023/A:1009402126863
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DOI: https://doi.org/10.1023/A:1009402126863