| Abstract
| - The influence of ionic interactions on quantitation of protein surface hydrophobicity was assessedby comparing the protein binding of an uncharged fluorescent probe, 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN), with that of an anionic probe, 1-(anilino)naphthalene-8-sulfonate(ANS-). Binding constants for the protein−probe complexes involving bovine serum albumin (BSA)and ovalbumin (OVA) in phosphate buffer (pH 7.0, I = 0.01 M) at 30 °C were fluorometricallydetermined to be KP-BSA = (1.00 ± 0.01) × 106 M-1 and KP-OVA = (4.2 ± 0.1) × 103 M-1, respectively,for PRODAN, compared to KA-BSA = (6.21 ± 0.04) × 106 M-1 and KA-OVA = (1.97 ± 0.09) × 103 M-1,respectively, for ANS-. A procedure was established using PRODAN to determine protein surfacehydrophobicity (S0) values from the initial slope of relative fluorescence intensity versus proteinconcentration plots, and the results were compared to S0 values measured using ANS-. Increasingionic strength up to 1.0 M decreased the S0 values of BSA measured by ANS-, increased S0 of BSAmeasured by PRODAN and of OVA measured by ANS-, and had no significant effect on the S0 ofOVA measured by PRODAN. These results demonstrate the importance of considering charge effectswhen determining protein surface hydrophobicity. Keywords: Protein hydrophobicity; fluorescent probe; electrostatic interactions; PRODAN; ANS
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