Exploring the heat-induced structural changes of ß-lactoglobulin -linoleic acid complex by fluorescence spectroscopy and molecular modeling techniques.

Linoleic acid (LA) is the precursor of bioactive oxidized linoleic acid metabolites and arachidonic acid, therefore is essential for human growth and plays an important role in good health in general. Because of the low water solubility and sensitivity to oxidation, new ways of LA delivery without compromising the sensory attributes of the enriched products are to be identified. The major whey protein, ß-lactoglobulin (ß-Lg), is a natural carrier for hydrophobic molecules. The thermal induced changes of the ß-Lg-LA complex were investigated in the temperature range from 25 to 85 °C using fluorescence spectroscopy techniques in combination with molecular modeling study and the results were compared with those obtained for ß-Lg. Experimental results indicated that, regardless of LA binding, the polypeptide chain rearrangements at temperatures higher than 75 °C lead to higher exposure of hydrophobic residues causing the increase of fluorescence intensity. Phase diagram indicated an all or none transition between two conformations. The LA surface involved in the interaction with ß-Lg was about 497 Çº(2), indicating a good affinity between those two components even at high temperatures. Results obtained in this study provide important details about heat-induced changes in the conformation of ß-Lg-LA complex. The thermal treatment at high temperature does not affect the LA binding and carrier functions of ß-Lg.

Monitoring the heat-induced structural changes of alkaline phosphatase by molecular modeling, fluorescence spectroscopy and inactivation kinetics investigations.

The heat induced conformational changes of calf alkaline phosphatase (ALP) were analyzed using different methods, based on fluorescence spectroscopy, molecular modeling and inactivation studies. Experimental studies were conducted in buffer solution in the temperature range between 25 and 70 °C. Molecular dynamic (MD) simulation provided details on thermally induced changes in ALP structure, highlighting that heating favored the hydrophobic exposure and important alteration of the catalytic site above 60 °C. Additional information to MD data were obtained by using different fluorescence spectroscopy methods, which revealed a complex mechanism of thermal denaturation. Therefore, the emissive properties indicated an unfolding of ALP at temperatures below 60 °C, whereas at higher temperatures, the polypeptides chains fold leading to a higher exposure of Trp residues. In order to establish a structure-function relationship, the results were correlated with inactivation studies of ALP in buffer at pH 9.0. The inactivation data were fitted using a first-order kinetic model, resulting in an activation energy value of 207.26 ± 21.68 kJ · mol(-1).