Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged (PRODAN) and anionic (ANS-) fluorescent probes

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 degrees C were fluorometrically determined to be KP-BSA = (1.00 +/- 0 01) x 10(6) M-1 and KP-OVA = (4.2 +/- 0.1) x 10(3) M-1, respectively, for PRODAN, compared to KA-BSA = (6.21 +/- 0.04) x 10(6) M-1 and KA-OVA = (1.97 +/- 0.09) x 10(3) M-1, respectively, for ANS(-). A procedure was established using PRODAN to determine protein surface hydrophobicity (So) 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 So 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.