Studies with Site-Specific Labeled HAMLET Figure 5. Site-specific labeling of proteins with aminooxy-biotin. Glycosylated proteins of hLA-ext (A) and aD-hLA-ext (B) were coupled with aminooxy-biotin. Proteins were separated on SDS- PAGE and transferred to the nitrocellulose membrane. Biotinylated proteins were detected with streptavidin-HRP and chemiluminescent substrate. Biotinylated HAMLET retained cytotoxic activity (data not shown). doi:10.1371/journal.pone.0026093.g005 found by others. Thus the presence of the C-terminal extension with a sugar or sugar conjugated with Alexa fluoro probe does not significantly affect the protein to OA ratio. However, when the complex is made with higher amount of OA (1:100) the protein- OA complex could not be precipitated with 1M sodium chloride and purified easily. Interestingly when the protein-OA complex is prepared with 1:25 ratio, the purified complex had a ratio of 1:35 as determined by the LC-MS. This higher OA value suggests that at higher OA concentrations the protein-OA complex associates with OA and could not be easily purified. Tumoricidal activity by the protein–OA complex The tumoricidal activity of mg-hLA, mg-hLA–OA complex, mg-hLA-ext, and mg-hLA-ext–OA complex was tested on several tumor cell lines and on immortalized, non-transformed mammary epithelial cell line MCF-10A (Table 2). Detailed studied were carried out with SK-BR-3 and MDA-MB-468 cell lines. Viability was investigated using Trypan blue and Annexin V methods. SK-BR-3 cells (Figure 7) and MDA-MB-468 cells (data not shown) were incubated for 3 h with 30 mM molten globule proteins (mg-hLA or mg-hLA-ext) alone or in complex with OA (1:10 ratio). Control cells were incubated with media only, and their viability was considered to be as 100%. Control cells were incubated with 300 mM OA alone. Three independent experi- ments were performed, with results shown as mean 6 SEM (*p,0.05) (Figure 7). When cells were incubated with the mg-hLA, viability in the cell lines studied was comparable to the control (media alone). The same results were obtained with the molten globule protein of the modified hLA with a C-terminal extension (mg-hLA-ext). When mg-hLA was complexed with OA in a 1:10 ratio, viability of the SK-BR-3 cell lines was reduced by 70% (Figure 7A). The same effect was observed with the complexes Figure 6. Liquid chromatography–mass spectrometry (LC-MS)/Single Ion Monitoring (SIM) chromatogram. Representative Profile for the Oleic Acid-Protein Complex and its 18 C13 isotopic Oleic Acid Internal Standard (ISTD). The original Complex was diluted 60 times with ISTD at 0.5 mg/mL. 25 mL sample solution was injected on to the column. doi:10.1371/journal.pone.0026093.g006 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26093