Evaluating and Validating the Fluorescent Probe Methodology for Measuring the Effective Hydrophobicity of Protein,...

The fluorescent probe method with 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANSA) and 6-propionyl-2-(N,N-dimethylamino) naphthalene (PRODAN) was validated to determine the effective hydrophobicity of the whey protein isolate. The focus was on charge and hydrophobic interactions due to the complexation between the proteins and probes. Using ANSA could overestimate the effective hydrophobicity of positively charged proteins. Furthermore, the relative fluorescence intensities (RFIs) should be considered before determining the effective hydrophobicity by linear regression. This is to be confident that the obtained RFI mainly originates from the hydrophobic interaction. The validated protocol was then applied to protein hydrolyzate and amino acids to investigate the method's reliability for small molecules. Adding ANSA or PRODAN probes to solutions containing protein hydrolyzates (60-10,000 Da), or the amino acids, tryptophan, glutamic acid, and lysine (∼165.85 Da), did not affect RFI. The effective hydrophobicity of those small constituents, therefore, could not be determined by these probes.