Preparation And Properties Of Modified Porcine Hemoglobins And Reconstructed Relaxin Receptors

Chemical modification and genetic recombination technique were used to reconstruct two functional proteins, porcine hemoglobin and relaxin receptor protein, to optimize them to be more suitable for scientific research and clinical application.Blood shortage and blood-borne contamination have promoted a constant search for blood substitutes, among them hemoglobin-based blood substitutes is the most attractive and promising one. Animal blood is the preferred raw material for blood substitutes due to its abundance in resources, safety, convenience of storage and dispensation.For many years research on hemoglobin based oxygen carrier at home and abroad has focused on bovine hemoglobin-based blood substitutes. However, porcine hemoglobin emerged as an ideal substitute for bovine one in China based on its richness and cheapness, especially its close similarity to human hemoglobin in structure, oxygenation/dissociation modification mechanism and ow immunogenecityIn the first part of this paper, stroma-free porcine hemoglobin was prepared, modified using a variety of physical or chemical methods and the structure and oxygen-carrying property of the product were further evaluated.Firstly, stroma-free porcine hemoglobin was separated and purified from fresh porcine blood by hemolyzation, centrifugation and filtration with micropore films. SDS-PAGE analysis of purified porcine hemoglobin showed just one band indicating that stroma-free porcine hemoglobin is dissociated easily from tetramer to monomer.Small molecular compounds, such as oxidized raffinose and bis (3, 5-dibromosalicyl) fumarate (DBBF), were used to modify stroma-free porcine hemoglobin to prevent the dissociation of porcine hemoglobin and its high oxygen affinity when out of red blood cells.Hemoglobins cross-linked with these small molecular modifiers turn out to be more stable. The cross-linking between hemoglobin subunits was proven by the SDS-PAGE analysis of raffinose-pHb and DBBF-pHb.Oxidized raffinose is utilized to modify porcine oxyhemoglobin and deoxyhemoglobin, respectively. The oxygen dissociation curve of pHb modified with oxidized raffinose was observed shift to right remarkably, favorable to modulate the product’s oxygen-carrying capacity, the P50 and Hill coefficient of modify porcine deoxyhemoglobin were 2.93 kPa and.2.1, respectively. Similar experiments were repeated to explore the optimal reaction conditons to modify porcine deoxyhemoglobin with bis (3,5-dibromosalicyl) fumarate (DBBF). when 5.0 mmol/L IHP was added in the reaction system, the oxygen dissociation curve of the modified hemoglobin shifted towards right. The optimal reaction time was 2 hours. P50 and Hill Coefficient of the product were close to normal physiological range when the molar concentration of DBBF to pHb was 2:1.Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions.In the present study, four derivatives of PEG with different activation groups, and several PEGs with different molecular weights were covalently bound to porcine hemoglobin (pHb). PEG-pHbs exhibited a variety of differences in their properties depending on the molecular weights of the used PEGs, the amounts of bound PEGs and the presence or absence of allosteric cofactors.Furthermore, both PEG and DBBF were used simultaneously to modify pHb. The results indicate that the pHbs modified with PEG and DBBF have more stable tetrameric conformations. Their oxygen half-saturation pressure (P50) is around 3.33 kPa which approximates the physiological P50 of human red blood cells.To evaluate the safety of the products, both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by ELISA. At the same time, the function of kidney, liver, heart etc after intravenous infusion were also evaluated. The results showed that the modified products are pretty safe without obvious damage to kidney, liver, heart functions.Relaxin is not only an important reproductive hormone involving all the processes of fertilization, implantation, pregnancy maintainance, delivery and lactation It also plays an important role in the maintenance of the functions of heart, vessels, kidney and brain, and it is also related to the occurrence of certain tumors. Fully understanding the function and its role in the biological mechanism is extremely important to treat the relevant diseases and develop new drugs.Expression of these receptors by genetic cloning to is a new strategy to study the receptor-ligand mechanism and to develp and screen new drug candidates.Genetic recombination was used to reconstruct the relaxin receptor proteins. Stably transfected HEK293T cell lines expressing the fusion proteins were selected.Radioligand binding assays showed that hybrid proteins accurately anchored on the cell surface retained high-affinity ligand binding:7BP transfected cell surface presented with high affinity binding sites with H2 and H3 relaxin with features similar to those of reported RXFP1; 8BP transfected cells presented high affinity with insulin-like peptide-3, relative high affinity to H2 relaxin, but no affinity to H3 relaxin, in accordance with the characteristics of RXFP2.Cells stably expressing the 7BP only receptor were cultured and 7BP protein was cleaved from the cell surface using thrombin and purified using the hexa-HIS tag and flag tag.33P-RLX Cross-linking analyses confirmed the formation of high molecular weight complexes between the receptor ecto-domains and its specific ligand.The ability of 7BP to block cAMP accumulation in cells expressing the LGR7 or LGR8 receptors was measured and results showed that the purified 7BP could interfer with RLX binding to its receptor and blocked cAMP production stimulated by RLX, Indicating that 7BP can act as a functional relaxin antagonist.The 7BP were further immobilized on SELDI Protein Chips. These could be used for SELDI MS-based examination of the nature of binding of the relaxin ligand.