Synthesis of bioactive hemoglobin-based oxygen carrier nanoparticles via metal-phenolic complexation.

The transfusion of donor red blood cells (RBCs) is seriously hampered by important drawbacks that include limited availability and portability, the requirement of being stored in refrigerated conditions, a short shelf life or the need for RBC group typing and crossmatching. Thus, hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) which make use of the main component of RBCs and the responsible protein for O2 transport, hold a lot of promise in modern transfusion and emergency medicine. Despite the great progress achieved, it is still difficult to create HBOCs with a high Hb content to attain the high O2 demands of our body. Herein a metal-phenolic self-assembly approach that can be conducted in water and in one step to prepare nanoparticles (NPs) fully made of Hb (Hb-NPs) is presented. In particular, by combining Hb with polyethylene glycol, tannic acid (TA) and manganese ions, spherical Hb-NPs with a uniform size around 350-525 nm are obtained. The functionality of the Hb-NPs is preserved as shown by their ability to bind and release O2 over multiple rounds. The binding mechanism of TA and Hb is thoroughly investigated by UV-vis absorption and fluorescence spectroscopy. The binding site number, apparent binding constant at two different temperatures and the corresponding thermodynamic parameters are identified. The results demonstrate that the TA-Hb interaction takes place through a static mechanism in a spontaneous process as shown by the decrease in Gibbs free energy. The associated increase in entropy suggests that the TA-Hb binding is dominated by hydrophobic interactions.

Structural and functional alterations of polydopamine-coated hemoglobin: New insights for the development of successful oxygen carriers.

One of the major factors that is currently hindering the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) is the autoxidation of Hb into nonfunctional methemoglobin. Modification with polydopamine (PDA), which is a biocompatible free radical scavenger has shown the ability to protect Hb against oxidation. Due to its tremendous potential in the development of successful HBOCs, herein, we conduct a thorough evaluation of the effect of PDA on the stability, aggregation, structure and function of the underlying Hb. By UV-vis spectrometry we show that PDA can prevent Hb's aggregation while thermal denaturation studies indicate that, although PDA coating resulted in a lower midpoint transition temperature, it was also able to protect the protein from full denaturation. These results are further corroborated by differential scanning calorimetry. Circular dichroism reveals that PDA can promote changes in Hb's secondary structure while, by UV-vis spectroscopy, we show that PDA also interacts with the porphyrin complex located in Hb's hydrophobic pocket. Last but not least, affinity studies show that PDA-coated Hb has a higher capability for oxygen release. Such an effect is further enhanced at lower pH. Importantly, through molecular docking simulations we provide a plausible explanation for the observed experimental results.

The Effect of pH on Globular State of Lipase-3646; an Appropriate Model for Molten Globule Investigations.

Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.