An illustration of the through-protein electron pathway (electron wire) engineered by adding a tyrosine amino acid (Tyr) to allow electrons to pass from reductants, such as vitamin C, to the heme iron - preventing toxic reactions.
Most people know that the main function of Hemoglobin (Haemoglobin) is to reversibly bind oxygen, transporting it from the lungs to the tissue. However, hemoglobin can also behave as an enzyme. The Red Blood Cell suppresses this enzymatic activity which can be toxic to the cell. However, if used outside the cell, such as in a blood substitute or as an oxygen therapeutic, this can become a danger, resulting in cytotoxicity and organ damage.
I previously discovered that engineering through protein electron pathways (essentially electron wires) can alter this enzymatic activity of hemoglobin. In turn, this allow plasma antioxidants such as ascorbate (Vitamin C) to remove the cytotoxic forms of the protein before it has had time to damage cells.
Using PEG (PolyEthylene Glycol) chains we can stop the protein from falling apart (from tetramers to dimers) which is toxic to the kidneys. PEG also expands volume, viscosity (i.e. more like blood cells) and can "mask" the protein from the patient's immune system.
At Essex we have developed (in collaboration with the University of Parma, Italy and Polytherics, UK) a way to PEGylate the protein that does not disrupt the protein's ability to bind oxygen. In addition our way generates two PEGs per hemoglobin tetramer, no more - no less.
Typically the process of PEGylating proteins is much more random, generating anything from 0 to 9 PEGs per monomer. In addition these other methods lead to significant changes in the oxygen binding ability of hemoglobin.
Polyethylene glycol (PEG) chains "bubble" the blood substitute (seen here in red), protecting the hemoglobin from damage, increasing the size and viscosity of the product and slowing down excretion.
Endothelial cells generate Nitric Oxide (NO) to relax smooth muscle cell and increase blood flow and decrease blood pressure. RBCs do not approach the endothelial cells due to fluid dynamics. Cell-free haemoglobin, however, is small enough to enter the non-turbulent flow and absorb NO, resulting in hypertension and decreased blood flow and hence oxygen flow to the tissue.
We have also worked on solving the issue of the Nitric Oxide Dioxygenase (NOD) activity of cell-free hemoglobin. This is where the hemoglobin, again acting as an enzyme, rapidly consumes Nitric Oxide (NO), a small but very important signalling molecule that regulates blood pressure (amongst other things). Giving hemoglobin-based blood substitutes (HBBSs) or hemoglobin-based oxygen carriers (HBOCs) typically cause elevated blood pressure, or hypertension. These has previously been targeted by genetic engineered (led by Prof John Olson, Rice University). Issues relating to heme retention may have caused issues, as heme released from hemoglobin is a damage associated molecular pattern (DAMP) molecule. Our product retains the heme preventing these issues and in tests do not cause hypertension.
So what now? We have completed the research and development phase of the project and are seeking funds for product manufacture and testing. Once this is complete we hope to progress to clinical trials in 2021-2022.