Fluorescent probe and Raman spectroscopic investigation of the effects of pH, heating, and k-carrageenan on whey protein structure

by Pasdar, Nooshin Alizadeh

Surface hydrophobicity (S,) is an important structural parameter descnbing food proteins due to its correlation with functionality. A new method to measure S, was established based on the neutral fluorescent probe, 6-propionyl-2-(N,Ndimethy1amino)naphthalene (PRODAN). S, was determined for whey protein isolate (WPi), bovine serum albumin (BSA) and P-lactoglobulin (BLG) at various pH (3.0, 5.0, 7.0 and 9.0) under heated (80°C for 30 min) or unheated conditions. S, values measured by PRODAN and cis-parinaric acid (CPA) were lower at pH 3.0 than at other pH, while values measured by 1- anilinonaphthalene-8-sulfonic acid (ANS) were highest at pH 3.0. The anionic probes ANS and CPA yielded opposing results on the effects of pH and heating on protein hydrophobicity. Heating, pH and addition OF K-carrageenan (KCG) at low (1.0:62.5), medium (1.0:6.0) or high (1.O: 1.2) ratios of KCG:protein, as weII as the interactions between these factors, signiticantly (Pc0.05) affected S, measured by PRODAN. Generally, proteins had higher S, and were more sensitive to heating and KCG addition at pH 9.0, than at other pH. Heating increased S, of high ratio KCG:protein mixtures at pH 3.0, but generally decreased S, of mixtures at higher pH, especially pH 9.0. Raman spectroscopy was used to analyze structural changes at higher protein concentration (15% wfv) than those (0.002-0.01%) used in spectrofluorornetry. Decreased SS and Trp band intensities, and lower helical and higher p-sheet content, were generaIIy observed after heating at pH 9.0 or KCG addition at pH 5.0. Decreased helical and increased fi-sheet contents obtained by heating BLG at pH 7.0 or 9.0 were not observed after heati & KCG:BLG mixtures at these pH. Addition of KCG to BSA at pH 7.0 or 9.0 resulted in increased helical content. Heating KCG:WPI mixtures at pH 5.0 and 9.0 resulted in large increases in SS and Trp band intensities and helical content, and decreased ratio of the tyrosine doublet, indicating a more buried or hydrophobic environment around aromatic residues. Raman and fluorescent probe spectroscopy provided information on protein structural properties as a function of pH, heating and interactions with other macrornolecuIes, which may be important in appIications of these proteins in food systems.