Cytotoxic Complexes of Sodium Oleate with β-Lactoglobulin

pre-printA complex of α-lactalbumin and oleic acid has previously been shown to induce apoptosis in cancer cells in a number of in vitro and in vivo trials. This complex is called HAMLET or BAMLET, depending on the origin of α-la (human/bovine alpha-lactalbumin made lethal to tumour cells). In the current study, it was shown that bovine β-lactoglobulin (β-lg), upon binding sodium oleate (NaOle), the salt of oleic acid, also acquires cytotoxicity towards tumour cells (human monocytic cells U937), analogously to HAMLET/BAMLET complexes. The properties of the complex were characterized using FIR spectroscopy, HPLC and SDS-PAGE. It was shown that the level of covalent oligomerization (dimers and trimers) of β-lg increased with increasing the molar ratio of sodium oleate NaOle:β-lg in the preparation procedure. At the same time, increasing the molar ratio of NaOle:β-lg increased the cytotoxicity of the complex. The increase in cytotoxicity appeared to be dependent on the amount of bound NaOle in the complex, but not on the content of multimeric forms of β-lg. The NaOle/β-lg complex also showed similarity with BAMLET in penetrating the cell membrane and co-localizing with the cell nucleus. Furthermore, DNA fragmentation studies suggested that tumour cells (U937) treated with the complex died by apoptosis, as in the case of BAMLET, and healthy cells appeared to be less affected by treatment, as shown with model rat adrenal pheochromocytoma cells PC12. In conclusion, β-lg and NaOle can form complexes with apoptosis-inducing qualities comparable to those of BAMLET.The work was funded by the Irish Dairy Research Trust and The Department of Agriculture (Food Institutional Research Measure – FIRM project 08RDTMFRC650) under the National Development Plan 2007-2013. K. Lišková was funded under the TeagascWalsh Fellowship Scheme

Novel pulse sequences for time-resolved photo-CIDNP

The properties of a new class of pulse sequences for photo-CIDNP (photochemically induced dynamic nuclear polarization) are analysed in detail, and guidelines for their optimization and applicability are derived. Sensitivity is a central problem with time-resolved photo-CIDNP experiments. By using multiple laser flashes per acquisition and storing the polarizations temporarily in the spin system, a significant improvement is achieved. An alternative application is the reduction of the absorbed light needed to attain a given sensitivity. Compared to conventional signal averaging with the same number n of flashes, a maximum additional improvement by a factor of slightly more than 0.5?n can be achieved in both cases. By an analysis of the transfer pathways, it is shown that multiplet signals and CIDNP multiplet effects can also be investigated in this way, even for strongly coupled spin systems. Experimental examples are given

Multiple subsets of side-chain packing in partially folded states of a-lactalbumins

Photochemically induced dynamic nuclear polarization NMR pulse- labeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (mol- ten globule) states of bovine and human -lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rap- idly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spec- trum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the -lactalbumin fold can be formed from interactions between a variety of different hydro- phobic residues in both native and nonnative environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains

Can misfolded proteins be beneficial? The HAMLET case.

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms

Rapid Sample-Mixing Technique for Transient NMR and Photo-CIDNP Spectroscopy: Applications to Real-Time Protein Folding

We describe the development and application of a novel rapid sample-mixing technique for real- time NMR (nuclear magnetic resonance) spectroscopy. The apparatus consists of an insert inside a conventional NMR tube coupled to a rapid injection syringe outside the NMR magnet. Efficient and homogeneous mixing of solutions in the NMR tube is achieved with a dead time of tens of milliseconds, without modification of the NMR probe or additional hardware inside the magnet. Provision is made for the inclusion of an optical fiber to allow in situ laser irradiation of samples, for example to generate photo- CIDNP (chemically induced dynamic nuclear polarization). An NMR water suppression method has been implemented to allow experiments in H2O as well as in deuterated solvents. The performance of the device has been tested and optimized by a variety of methods, including sensitive detection of residual pH gradients and the use of NMR imaging to monitor the extent of mixing in real time. The potential utility of this device, in conjunction with the sensitivity and selectivity of photo-CIDNP, is demonstrated by experiments on the protein hen lysozyme. These measurements involve the direct detection of spectra during real-time refolding, and the use of CIDNP pulse labeling to study a partially unfolded state of the protein under equilibrium conditions. Magnetization transfer from this disordered state to the well-characterized native state provides evidence for the remarkable persistence of nativelike elements of structure under conditions in which the protein is partially denatured and aggregation prone