Evaluating and\nValidating the Fluorescent Probe Methodology\nfor Measuring the Effective Hydrophobicity of Protein, Protein Hydrolyzate,\nand Amino Acid

The fluorescent probe method with 8-anilino-1-naphthalenesulfonic\nacid ammonium salt (ANSA) and 6-propionyl-2-(<i>N</i>,<i>N</i>-dimethylamino) naphthalene (PRODAN) was validated to determine\nthe effective hydrophobicity of the whey protein isolate. The focus\nwas on charge and hydrophobic interactions due to the complexation\nbetween the proteins and probes. Using ANSA could overestimate the\neffective hydrophobicity of positively charged proteins. Furthermore,\nthe relative fluorescence intensities (RFIs) should be considered\nbefore determining the effective hydrophobicity by linear regression.\nThis is to be confident that the obtained RFI mainly originates from\nthe hydrophobic interaction. The validated protocol was then applied\nto protein hydrolyzate and amino acids to investigate the method’s\nreliability for small molecules. Adding ANSA or PRODAN probes to solutions\ncontaining protein hydrolyzates (60–10,000 Da), or the amino\nacids, tryptophan, glutamic acid, and lysine (∼165.85 Da),\ndid not affect RFI. The effective hydrophobicity of those small constituents,\ntherefore, could not be determined by these probes.