Hydrophobicity of Bovine Serum Albumin and Ovalbumin Determined Using Uncharged (PRODAN) and Anionic (ANS-) Fluorescent Probes

Abstract

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 K P - BSA = (1.00 ± 0.01) × 10 6 M - 1 and K P - OVA = (4.2 ± 0.1) × 10 3 M - 1, respectively, for PRODAN, compared to K A - BSA = (6.21 ± 0.04) × 10 6 M - 1 and K A - OVA = (1.97 ± 0.09) × 10 3 M - 1, respectively, for ANS - . A procedure was established using PRODAN to determine protein surface hydrophobicity ( S 0 ) values from the initial slope of relative fluorescence intensity versus protein concentration plots, and the results were compared to S 0 values measured using ANS - . Increasing ionic strength up to 1.0 M decreased the S 0 values of BSA measured by ANS -, increased S 0 of BSA measured by PRODAN and of OVA measured by ANS -, and had no significant effect on the S 0 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