A complex of alpha-lactalbumin with oleic acid, also known as HAMLET/BAMLET (human/bovine alpha-lactalbumin made lethal to tumor cells), causes apoptosis-like death in tumor cells but has little effect on healthy differentiated cells. The aim of this study was to examine whether irreversible denaturation of alpha-lactalbumin is detrimental to the formation and cytotoxicity of BAMLET. Commercial bovine holo alpha-lactalbumin (1-4% w/v) was heated at 80 degrees C for up to 100 min. With an increasing concentration of protein, the denaturation of alpha-lactalbumin proceeded faster, and aggregation became more extensive. Native and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a high proportion of the aggregates was linked by disulfide bonds. BAMLET was prepared from native and heat-treated alpha-lactalbumin according to a previously described chromatographic method. Despite the high content of denatured and aggregated alpha-lactalbumin in the heat-treated samples, their conversion into BAMLET was not negatively affected, resulting in BAMLET complexes partly composed of covalently linked aggregates of alpha-lactalbumin. The cytotoxicity of all prepared BAMLET samples was comparable to that of the control sample prepared from native alpha-lactalbumin (LD(50) = 34.6 +/- 2.7 mumol L(-1)). It was concluded that alpha-lactalbumin is not required to be in its native conformation for the conversion into its biologically active BAMLET complex.