Heat-induced conformational changes of bovine lactoferrin and its relationship with milk pasteurization process.
Gonçalves, R.B.; Schwarcz, W.D.; Aguiar, C.L.; #Oliveira, A.C; #Silva, J.L. and Antunes, A.J.
Centro de Pesquisa em Ciência e Tecnologia do Leite, Universidade Norte do Paraná, Fazenda Experimental da UNOPAR, 86125-000, PR, Brazil; #Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-590, RJ, Brazil.
Lactoferrin is an iron-binding protein that takes iron from the environment depriving potential pathogens of this essential nutrient. This protein is present in several secretions such as milk, tears, saliva and it has broad antimicrobial and physiological properties. Lactoferrin comprises a single polypeptide chain with 76 kDa. This monomer can be divided into two lobes (C and N), linked by an alpha helix, with two domains each. The molecule has two binding sites for ferric ion and its affinity for iron is 260 times higher than blood transferrin. During pasteurization process, milk is submitted to several temperature shifts. Since high temperature may affect protein stability and its functional properties, we decided to investigate the effect of heating on bovine lactoferrin structure and stability during milk processing. Recent studies have showed that the iron-binding lactoferrin ability is decreased at 70°C. However, the structural changes that lead to the lack of this ability are still to be understood. In this work, we studied the effects of high temperatures on lactoferrin secondary structure during pasteurization process and its relationship with protein functionality. The changes were monitored by using spectroscopic techniques such as circular dichroism (CD) and fluorescence. The CD data showed that lactoferrin secondary structure is drastically affected since 70°C when temperature is increased gradually from 25 to 105°C, being the changes an irreversible process. In order to confirm this data, those changes have been monitored by tryptophan fluorescence emission. We decided to verify the effects of simulating the pasteurization process on lactoferrin. With this aim, temperature was raised from 5 to 70ºC (incubation time 20 s) and returned to 5°C. Surprisingly, lactoferrin remains well structured during the whole process and this result is not dependent on time since heating to 70°C by 150 minutes had no effect on lactoferrin structure. Now, we are performing some experiments to understand the changes induced by heat on lactoferrin on the molecular level.
Supported by: FUNADESP/UNOPAR, CNPq, CAPES and FUJB/UFRJ.
|