Original Article
Clinical Nanomedicine
Interaction of hemoglobin and copper nanoparticles: implications in hemoglobinopathy

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Abstract

Here we study the interaction of copper nanoparticles (CuNPs) with different variants of hemoglobin (Hb). The study reports analysis with HbA0 (the major component of human Hb) and HbA2 (a variant that is associated with β-thalassemia). In the case of HbA0, the major fraction of human Hb, the CuNPs trigger protein aggregation, and this is followed by the precipitation of the protein. The aggregative response is largely attenuated in the case of HbA2. The difference between the two variants is thus amenable to detection by simple optical methods. We verified that CuNPs co-precipitated with specific Hb variants using atomic absorption spectroscopy (AAS) and high-pressure liquid chromatography (HPLC). An associated observation was the reversal of zeta potential of HbA0 induced by the CuNPs (from –11 mV to +13 mV). Dynamic light-scattering (DLS) studies indicated that in the case of HbA0, protein initially broke the nanoclusters into smaller sizes (4 nm), and this was followed by a gradual increase in cluster size. Assays of heme peroxidase activity indicated that the protein unfolded during the process. It is suggested that interaction between the CuNPs and HbA0 stimulates the molten-globule state of the protein, leading to the onset of such an aggregative pathway. When studied for other variants, HbE, a common mutant of Hb, showed similar aggregative behavior, and on the other hand, rare variants such as HbC tended to remain in solution. A suitable scaling up of the approach may have important implications in screening hemoglobinopathies such as β-thalassemia.

Section snippets

Hemolysate preparations

Blood samples were collected in EDTA vials from normal persons. The whole blood was centrifuged at 3000g for 10 minutes and washed three times in normal saline, forming packed red blood cells (RBCs). Hemolysis was performed by mixing 100 μL of packed RBCs in 500 μL of lysis buffer (2.5 M NaCl, 100 mM EDTA–Na salt, 1% Triton X-100, and 10% DMSO). The solution was applied to a Sephadex G-50 column equilibrated with 0.1 M phosphate buffer, pH 7.0. Elution was carried out with 0.1 M phosphate

Results and discussion

Dynamic light scattering (DLS) studies indicate that the clusters of the CuNPs [9] assume a size of 160 nm. Notably, the clusters are broken when one adds protein (HbA0) to the CuNP suspension. Figure 1 shows initial declustering of the NPs at different intervals of time, followed by formation of an extended structure (see differently colored DLS size graphs in Figure 1). Longer incubation with CuNPs leads to protein aggregation and precipitation. The initial increase in absorbance that is

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