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The influence of ionic interactions on quantitation of protein surface hydrophobicity was assessed by 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 fluorometrically determined 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 surface hydrophobicity (S0) values from the initial slope of relative fluorescence intensity versus protein concentration plots, and the results were compared to S0 values measured using ANS-. Increasing ionic strength up to 1.0 M decreased the S0 values of BSA measured by ANS-, increased S0 of BSA measured by PRODAN and of OVA measured by ANS-, and had no significant effect on the S0 of OVA measured by PRODAN. These results demonstrate the importance of considering charge effects when determining protein surface hydrophobicity. Keywords: Protein hydrophobicity; fluorescent probe; electrostatic interactions; PRODAN; ANS
Haskard et al. (Fri,) studied this question.