A Triply Modified Hemoglobin-based Oxygen Carrier And The Study On Its Oxygen Delivery And Unloading Activity

Blood transfusion is a major clinical therapy of multiple diseases.However,natural blood has many disadvantages,including insufficient sources and matching before application to prevent cross infection.Thus,many international researchers turned their interest on red cell substitutes.The article aims is regulating oxygen affinity of human adult hemoglobin by chemical modification to acquire triply-modified HbA with low oxygen affinity,high auto-oxidation rate and high tetramer stability.In the thesis,HbA as the research object,was modified by intramolecular cross-linking,reductive alkylation and succinimide chemistry.The functional and structural properties of the triply modified HbA were investigated.The project on chemical modification of HbA can be divided into two sections.At first,HbA was intramolecular cross-linked by using bis(3,5-dibromosalicylate)fumarate,which can improve tetramer stability and oxygen affinity similar with HbA in red blood cells Subsequently,sodium glyoxylate monohydrate was used to modify N-terminus of crosslinked HbA by reductive alkylation.This method can maintain lower oxygen affinity state(T state)for the static repulsion.Three HbA samples(??-Hb,Glx-Hb and Glx-??-Hb)were acquired by these methods.Furthermore,the structural and functional properties of the modified HbA were investigated.Subsequently,four HbA samples were covalently modified using polyethylene glycol with eight succinimide motifs(Eight-arm PEG-NHS 10 kDa)and acquired PEGylated HbA samples(PEG-Hb?PEG-??-Hb?PEG-Glx-Hb and PEG-Glx-??-Hb)to further improve the tetramer stability and increase the hydrodynamic volume for reduction of renal toxicity caused by vascular overflow and decrease immunogenicityResults show that P50 value of Glx-??-Hb is 34.6 mmHg,about three times than HbA.The dual modification significantly increased oxygen transfer efficient of HbA from 9.1%to 33.1%.Eight-arm PEG-based polymerization slightly decreased the P50 of the Hb derivatives to 27.8 mmHg and OTE to 30.5%,followed by increase in the auto-oxidation rate and the metHb concentrate.It is worth noting that the higher the degree of chemical modification,the greater auto-oxidation rate and the greater content of metHb.Glx-??-Hb has the highest auto-oxidation rate of 0.036 h-1 and metHb concentration of 12%(after four hours),while PEG-Glx-??-Hb was 0.044 h-1 and 15%.The far-UV region of circular spectrum(260?190 nm)results showed that chemical modification did not change the secondary structure of HbA.However,the near-UV region(480?260 nm)showed that dual modification and triple modification have some impacts on quaternary structure of hemoglobin and microenvironment of heme.SDS-PAGE analysis shows that the intramolecular cross-linking hemoglobin has a band at 32 kDa,due to that the fumarimide bridge is constructed at Lys-99(?)between two a subunits.The mass of PEGylated HbA is mainly in the region of 90 kDa.It can be seen that PEGylation can significantly increase the molecular mass of HbA.Size exclusion chromatography analysis showed that the peak position of PEGylated HbA move to the left compared with its corresponding original protein and hydrodynamic volume of PEGylated hemoglobin increased significantly,which was related to enhanced stability.The Raman spectrum shows that Raman intensity of dual modification significantly increases in the region of 1340?1390 cm-1,which has relation with the change of quaternary structure.The result of tryptic peptide mapping and reserved-phase high performance liquid chromatography shows that dual modified sites are Val-1(?)and Lys-99(?).According to the kinetics of UV spectrophotometry,the number of thiol number in eight chemical modified samples including the original protein has not changed,while the different chemical modified methods have the different effect on the reaction activity of thiol.The main reason is that chemical modification disturbs the ?1 ?2 interfaceThe triple modified hemoglobin-based oxygen carriers based on intramolecular cross-linking,reductive alkylation and succinimide chemical reaction has low oxygen affinity and can effectively release oxygen to hypoxic tissues along with an increase in auto-oxidation rate and the content of metHb.Due to the formation of fumarimide bridges and the modification of eight-arm PEG-NHS,the interaction between subunits is enhanced and the stability of tetramer is improved.