Studies with Site-Specific Labeled HAMLET Figure 7. Measurement of tumoricidal activity during conversion of recombinant protein to tumoricidal complex. The effect on the cell viability of SK-BR-3 cells was studied after treatment with mg-hLA, mg-hLA/OA, and mg-hLA-ext and mg-hLA-ext/OA. The cells were incubated with molten globule proteins alone or complexed with OA, and their viability was investigated using Trypan blue (A) and by FACS analysis with Annexin V (B). When cells were incubated with the apo-form of hLA (mg-hLA and mg-hLA-ext), viability was comparable to the control (media alone) in the cell lines studied. When molten globule protein derived from hLA or hLA-ext was complexed with OA in a 1:10 ratio, viability of the cell lines SK- BR-3 was reduced. Results are shown as mean 6 SEM (*p,0.05). doi:10.1371/journal.pone.0026093.g007 various protease digested fragments of native a-LA can also form a tumoricidal complex with OA [32,36]. Earlier protease digestion and spectroscopic studies on the molten globule state of a-LA indicated that the b-domain of the protein may be more disordered than the a-domain [37]. Furthermore, the deletion of the b-domain from a-LA has been shown to have little impact on the tertiary structure of the a-domain [29]. Therefore, we have chosen the a-domain of the hLA protein for further investigation in the present study. Like the full-length protein, the a-domain, after refolding also remained a monomer and formed a tumoricidal complex with OA. In all studies, the molten globule state of the a-LA was found to be essential for forming a tumoricidal complex with OA. It has been shown that, in the molten globule state a-LA is more hydrophobic compared to its holo-form [38]. Thus, this state may facilitate the binding of the fatty acids, such as OA. A simple addition of OA to the molten globule a-LA solution may not be enough to make the complex, as most OA molecules exist as micelles in water. Simple heating of such a solution may disrupt the micelles enough to cause a few OA molecules to dissociate and bind to a molten globule a-LA molecule, thus generating a HAMLET molecule. Although, it may be hard to predict how many OA molecules may be bound to the hydrophobic surface of a a-LA molecule, the number we have determined in our complex is comparable to the ones reported by others. We have earlier developed a site-specific labeling technique of proteins. In this technique, the target protein is made with a C- terminal 17–amino acid fusion peptide. This peptide contains a single Thr residue to which a C2-keto-galactose sugar is transferred from its UDP-derivative by the ppGalNAc-T2 enzyme [26]. Since the glycosylated protein carries a unique chemical handle at its sugar moiety, it can be used for site-specific PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26093