Effect of temperature on the structure and cytotoxicity effect of α-lactalbumin-oleic acid complexes against the L1210 cell line

• Temperature effects on protein structure in α-La-OA samples were studied.
• FTIR spectroscopy showed small changes in the protein secondary structures.
• Preparation temperatures influenced protein tertiary unfolding and OA concentration.
• Preparation temperature effects on protein structure influenced cytotoxic function.
• Protein tertiary structure unfolded by two distinct transitions below and above 60 °C.

α-Lactalbumin-oleic acid (α-La-OA) complexes with cytotoxicity against selected tumor cells are potential functional food ingredients. This research employed Fourier-transform infrared (FTIR), circular dichroism (CD), and intrinsic fluorescence spectroscopic techniques to investigate the protein and lipid structures in such complexes and their cytotoxic function against the L1210 cell line as mediated by the preparation temperature under conditions amenable to commercial production. Food-grade α-La-OA samples were prepared from 3.53 mM α-La and 0.354 mM OA at pH 6.5 by heating at 25, 50, 60, 70, or 80 °C for 20 min. FTIR spectroscopy revealed that OA, the cytotoxic component in the α-La-OA samples, was protonated. OA concentration increased with temperature up to a ‘saturation limit’ of 50:1 lipid:protein molar ratio attained at 60 °C, 70 °C, 80 °C. Cytotoxicity against tumor cells increased with temperature up to 60 °C and then declined, such that α-La-OA samples prepared at 70 and 80 °C were not cytotoxic. Near-UV CD spectroscopy demonstrated positive correlation in magnitude of unfolding in protein tertiary structure with temperature (r2 = 0.99), and with OA concentration (r2 = 0.99) in the 25–60 °C range. Distinct transitions of unfolding in tertiary structure below and above 60 °C were detected by intrinsic fluorescence spectroscopy. This research demonstrated that temperature-dependent effects on protein structure affected α-La-OA complex tumor cell cytotoxic function. Minimizing loss in cytotoxic function in α-La-OA complexes prepared above 60 °C could generate interest in their potential use in the functional food industry.

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