We propose that HMW-bLf, which is similar to ApobLf and lacks mostly the iron content, and likely contain lot of free aspartate and tyrosine residues. This confers a very open, flexible configuration to Tiratricol HMW-bLf molecule similar to Apo-bLf. Due to this, the ability of the molecule to undergo intermolecular interactions was highly increased when maintained at a high concentration and in the absence of any salt content, especially iron. These complex interactions in HMW-bLf appeared to be very strong and led to the formation of even larger aggregates upon prolonged storage. We have also UNC0224 noticed that NM-bLf, when stored at high concentration, displays trimers and dimers formation visualized on denaturing SDS-PAGE condition. In a study that investigated the effect of iron saturation on thermal aggregation of bLf in Apo state, it was shown that Apo-bLf associated into large polymers by non-covalent interactions without the participation of disulphide cross-links. The more unfolded structure of heat sensitive Apo-bLf may have increased the exposure of noncovalent sites normally buried in the core of both lobes of the protein, thereby favoring intermolecular interactions and the formation of larger aggregates. The in-depth molecular and biophysical characterization of the HMW-bLf needs to be investigated further with more powerful proteomics tools such as Mass spectroscopy and Circular Dichroism, in order to identify the chemical as well structural changes/linkages in the oligomer. To determine spontaneous association of bLf in physiologically simulated solution SAXS and LS can be employed to analyze the oligomeric states of HMW-bLf. The consumption of test drinks containing Apo-bLf and iron saturated bLf in human volunteers has shown that Apo-bLf is more susceptible to in vivo gut digestion, than the corresponding iron-saturated form. Similarly, we have reported earlier the resistant nature of Fe-bLf towards Omnizyme. In the current study, HMW-bLf was found to be more resistant to Omnizyme digestion in vitro, even after 4, 6, and 8 h incubation periods. Figure 2C shows that HMW-bLf in lanes 2, 3 and 4 showed excellent stability to digestion, with digested forms appearing as 150�C160 kDa dimers at various intervals of time while faint bands at,78, 50 and 25 kDa.