Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes

“…Preparation of HAMLET and GAMLET-We prepared HAMLET and GAMLET by two different methods: (i) a heat treatment method (19), in which the mixture of ␣-LA and OA was heated at pH 2.0 and 50 -60°C for 10 min, followed by removal of excess OA and lyophilization, and (ii) the original column method, in which HAMLET and GAMLET were prepared by column chromatography using an OA-preconditioned ion exchange column (2). The anti-tumor activities of the HAMLET and GAMLET complexes prepared by the two methods were identical to each other (data not shown).…”

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…These include OA-conditioned anion exchange chromatography using either a DEAE-Trisacryl M in the conventional method [3,5,6,19] or a DEAE-Sepharose column in an alternative method [53]. Direct mixing under heat denaturation [30,36,52], heat denaturation of -LA and OA-conditioned anion exchange chromatography [62], direct mixing in solution at room temperature with alternated pH conditions [45,48,49,63], direct mixing followed by anion exchange chromatography [61] and alkaline conditions [64] have also been used.…”

“…Regardless of the preparation method used in the different experiments, it has been shown that the cytotoxicity of the resulting complexes was similar to that of conventionally prepared HAMLET or BAMLET [30,36,48,49,54,[61][62][63][64][65].…”

“…The classical molten globule of -LA was defined as the acid denatured compact state of -LA at pH 2.0 with fluctuating tertiary structure [13,33,34]. However, -LA can adopt molten globule-like states during Ca 2+ -free (apo-state) conditions, during high temperatures at 90°C and in the presence of denaturants [14,17,35,36]. Similar conformational states can also be formed by reduction of the disulfide bonds in -LA [37,38].…”

Interaction of α-lactalbumin with lipids and possible implications for its emulsifying properties – A review

“…It is worthwhile to mention that the conversion of the native inactive human or bovine a-LA to a tumoricidal molecule was carried out by the decalcification of the protein by ethylenediaminetetraacetic acid (EDTA) followed by ion-exchange chromatography in a column of DEAE Trisacryl M matrix conditioned with oleic acid (Svensson et al, 2003). Similarly, the complexing of the apo-protein with oleic acid was also achieved by direct incubation of both molecules or by heat treatment of the mixture at 50 or 60 C (Brodkorb & Lisková, 2010;Kamijima et al, 2008;Knyazeva et al, 2008;Zhang et al, 2009). To better characterize the bioactive complex, the ability of the apo conformer of bovine and human a-LA to bind oleic acid has been studied by means of fluorescence spectroscopy and partition equilibrium (Barbana et al, 2006;Barbana, Pérez, Pocoví, Sánchez, & Wehbi, 2008).…”