Characteristics of bovine hemoglobin as a potential source of hemoglobin-vesicles for an artificial oxygen carrier.
Hemoglobin-vesicles (HbV) have been developed for use as artificial O(2) carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie reagent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87 degrees C, respectively, which are higher than those of HHb (80 and 78 degrees C, respectively), and resistant to pasteurization (60 degrees C, 10 h). The purified BHb was concentrated to over 40 g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280 nm. The O(2) affinity (P(50)) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl(-) (as NaCl), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P(50) = 28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5'-phosphate as a replacement for 2,3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5 cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of O(2) affinity in comparison with HHb.
Sakai
Hiromi
H
Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
Masada
Yohei
Y
Takeoka
Shinji
S
Tsuchida
Eishun
E
D019794:2,3-Diphosphoglycerate / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D002417:Cattle; D002712:Chlorides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000737:chemistry*; D007477:Ions; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D002712:Chlorides; D006454:Hemoglobins; D007477:Ions; D008051:Lipid Bilayers; D008081:Liposomes; D019794:2,3-Diphosphoglycerate; D010100:Oxygen