Who Is Mr. HAMLET? Interaction of Human α-Lactalbumin with Monomeric Oleic Acid

Abstract

A specific state of the human milk Ca(2+) binding protein alpha-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 degrees C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 degrees C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca(2+)-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca(2+) association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca(2+)-free hLA were estimated. The far-UV CD spectra of Ca(2+)-free hLA show that all OA-bound forms of the protein are characterized by elevated content of alpha-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties.

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

“…The sodium elaidate and sodium stearate were insoluble at room temperature making any meaningful dynamic light scattering measurements impossible; however, the addition of the protein greatly increased the solubility of these fatty acids. 14 The results outlined here show that the previously used single-phase system used for complex preparation was successfully employed to form complexes with a variety of fatty acids. While ratios of 2, 5, and 10 fatty acids per α-LA were used when preparing the complexes, not all the fatty acid bound to α-LA.…”

“…Several variations of this approach had been employed previously to make BAMLET-like complexes [12][13][14][15]. Excess fatty acid was removed by extensive dialysis of the complex.…”

“…In addition it was found that it is possible to make BAMLET-like complexes without the need for a pretreated anion-exchange column; Knyazeva et al formed complexes of α-LA and OA by titrating the protein solution with OA under alkali conditions [14]. Furthermore, complexes of peptide fragments of α-LA have been formed by mixing the protein with OA in ethanol dispersed in a buffer [15] and BAMLET has also been made by mixing α-LA with the more soluble sodium salt of OA, sodium oleate [16].…”

“…The structure of the H/BAMLET has been a subject of multiple studies, often with conflicting results. The number of OA molecules bound to α-LA has reported values between 1 and 13 [2,9,14,20].…”

Mleko kobiece jako naturalny produkt leczniczy

“…Kompleks a-LA z kwasem oleinowym może być utworzony w roztworze wodnym, co sprawia, że proces przygotowania aktywnego związku w warunkach laboratoryjnych staje się bardziej przejrzysty i kontrolowany, a to otwiera dodatkowe możliwości dostosowania jego właściwości dla szczególnych potrzeb. Jednakże dokładny mechanizm działania kompleksu HAMLET wymaga dalszych wnikliwych badań [14].…”