Fig. 2B shows that Hp almost fully inhibits the reaction. Bityrosines that form may be either intramolecular or intermolecular. As only one protein molecule exists per LDL particle, intermolecular cross-linking results in aggregation. An SDS-PAGE was run in order to analyze the aggregation state of the protein. Lanes 1 and 2 in Figure 2C indicate that within 3.5 hours, little protein remained as the original 500 kDa monomer. Most of the protein had become cross-linked in a covalent intermolecular form. In contrast, in the reaction mixtures containing Hp, most of the protein remained as an apoB monomer, indicating that the presence of haptoglobin was able to prevent the formation of covalent aggregates of LDL. There are certain in vivo conditions in which LDL is more vulnerable to oxidation. One condition is a lack of vitamin E within the particle, a vitamin E-depleted form, rendering it more sensitive to oxidation. Another condition is a type of haptoglobin which has a weaker AZD3293 protective effect against hemin release, Hp 2-2. To evaluate the degree of protection from cell-free Hb provided by Hp to the vasculature, we tested whether Hp 2-2 could protect dLDL. Ferric-Hb was incubated with dLDL, with and without Hp 2-2. As a double control, dLDL and Hp were each incubated alone. As dLDL is highly negatively charged, an agarose column was used to separate the components of the reaction mixtures according to charge. The column was developed using a salt gradient of 0�C1 M NaCl, buffered with tris-HCl at pH 7.4, and protein fractions were eluted. The study shows that in the absence of free oxygen and at low peroxide levels, ferrous-Hb undergoes oxidation to ferric-Hb. Under such conditions, hemin disintegrates rapidly, judging by the partial loss in absorbance of the ferric Soret band. In corroboration with a previous study, while the association of Hb with Hp did not inhibit the oxidation of ferrous to ferric iron, hemin was protected from disintegration. The current study further demonstrates that when a solution of cell-free Hb contains THZ1 circulatory components of plasma, like hydrophobic LDL, hemin readily transfers from globin to these components.