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.
Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.
The specific complexes of human alpha-lactalbumin (alpha-LA) with oleic acid (OA), HAMLET and LA-OA-17 (OA-complexes), possess cytotoxic activity against tumor cells but the mechanism of their cell penetration remains unclear. To explore the molecular mechanisms underlying interaction of the OA-complexes with the cell membrane, their interactions with small unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles and electroexcitable plasma membrane of internodal native and perfused cells of the green alga Chara corallina have been studied. The fractionation (Sephadex G-200) of mixtures of the OA-complexes with the vesicles shows that OA-binding increases the affinity of alpha-LA to DPPC vesicles. Calcium association decreases protein affinity to the vesicles; the effect being less pronounced for LA-OA-17. The voltage clamp technique studies show that LA-OA-17, HAMLET, and their constituents produce different modifying effects on the plasmalemmal ionic channels of the Chara corallina cells. The irreversible binding of OA-complexes to the plasmalemma is accompanied by changes in the activation-inactivation kinetics of developing integral transmembrane currents, suppression of the Ca(2+) current and Ca(2+)-activated Cl(-) current, and by increase in the nonspecific K(+) leakage currents. The latter reflects development of nonselective permeability of the plasma membrane. The HAMLET-induced effects on the plasmalemmal currents are less pronounced and potentiated by LA-OA-17. The control experiments with OA and intact alpha-LA show their qualitatively different and much less pronounced effects on the transmembrane ionic currents. Thus, the modification of alpha-LA by OA results in an increase in the protein association with the model lipid bilayer and in drastic irreversible changes in permeability of several types of the plasmalemmal ionic channels.
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