Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes.

The influence of type of fluorescent probe on the surface hydrophobicity values determined for three native and heated proteins was assessed using uncharged [6-propionyl-2-(N, N-dimethylamino)naphthalene or PRODAN] versus anionic aliphatic (cis-parinaric acid or CPA) and aromatic (1-anilinonaphthalene-8-sulfonic acid or ANS) probes. Surface hydrophobicities of whey protein isolate, beta-lactoglobulin, and bovine serum albumin under heated (80 degrees C for 30 min) and unheated conditions and at varying pH values (3.0, 5.0, 7.0, and 9. 0) were measured using ANS, CPA, and PRODAN. ANS and CPA yielded opposing results for the effects of pH and heating on protein hydrophobicity. Hydrophobicity was lower at pH 3.0 than at other pH values for all proteins measured by PRODAN, whereas the values measured by ANS and CPA at pH 3.0 were quite high compared to those at other pH values, suggesting the influence of electrostatic interactions on anionic probe-protein binding. These results suggest that the presence or absence of a permanent charge as well as the aromatic and aliphatic nature of fluorescent probes can affect protein hydrophobicity values measured under various pH conditions.