<title>Monitoring structural and ligand-loading status of serum albumin with an uncharged hydrophobic fluorescent probe, Nile red</title>

Abstract

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-*B) transition ofHSA. A single NR-binding site of high affinity is present on the protein molecule, more hydrophobic (bound dye fluorescence maximum at 630 nm) as compared to low-affinity sites (? 640 mn). A calculation procedure has been developed for monitoring the dissociation constant (Kd) '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 decolouration rate in the presence of HSA correlates with that of Kd and reflects pH-dependence of the protein affinity to the dye. N-B transition of HSA results in an essential increase in the protein-NR affinity (lowering Kd from 4-7 xM for N-form down to 1-3 piM for Bform). Spectral features of protein-bound NR monitor an alkaline shift of the pH-range of N-*B transition upon deliganding HSA, midpoint pH increasing from 7.6-8.0 to 8.2-83. Numerous changes in the parameters of protein-bound NR fluorescence were revealed over the range ofpH 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 ofthe protein. The value ofHSA-NR affinity is considered as possessive of a diagnostic potential.

A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

“…Nile Red can interact with many proteins, is photostable and can detect the exposure or formation of hydrophobic surfaces (20,21). The Nile Red fluorescence was shown in several studies to be unaffected at a variety of pH ranges: 4.5-8.5 (20), 6.0-10.0 (22). We measured a significant increase in Nile Red fluorescence emission in three salmon calcitonin formulations at pH 4.0 (sodium citrate, sodium citrate phosphate and potassium phthalate) and up to pH 10.5 compared to the control solutions.…”