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Hydrophobic binding sites of native and defatted human serum albumin (HSA) of individual donors were tested using a lipophilic fluorescent dye, Nile red (NR), over the pH interval of 6 to 10 covering the range of the neutral to base (N yields B) transition of HSA. A single NR-binding site of high affinity is present on the protein molecule, more hydrophobic (bound dye fluorescence maximum at (lambda)max approximately 630 nm) as compared to low- affinity sites ((lambda)max approximately 640 nm). A calculation procedure has been developed for monitoring the dissociation constant (Kd) of the dye complex with protein high-affinity binding site, based on the pH-profiles of dye fluorescence registered at two protein concentrations. A pH-profile of NR decoloration rate in the presence of HSA correlates with that of Kd and reflects pH-dependence of the protein affinity to the dye. N yields B transition of HSA results in an essential increase in the protein-NR affinity (lowering Kd from 4 - 7 μM for N-form down to 1 - 3 μM for B-form). Spectral features of protein-bound NR monitor an alkaline shift of the pH-range of N yields B transition upon deliganding HSA, midpoint pH increasing from 7.6 - 8.0 to 8.2 - 8.3. Numerous changes in the parameters of protein-bound NR fluorescence were revealed over the range of pH 6 - 10. The pH-profiles of the parameters varied for HSA of different donors and were not unified upon albumin deliganding. This variability is presumably caused by individual structural differences arising from both the ligand loading and covalent modification of the protein. The value of HSA-NR affinity is considered as possessive of a diagnostic potential.
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