Purification and physical characteristics of a hemoglobin solution modified by coupling to 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP).
Human stroma-free hemoglobin (Hb) was crosslinked with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP), purified over crosslinked dextran, and eluted with a linear salt gradient. The oxygen dissociation curve of this crosslinked hemoglobin appeared to be shifted to the right with a standard P50 of 49 torr (PO2 for 50% saturation with oxygen at a pH of 7.40, a PCO2 of 40 torr, and a temperature of 37 degrees C) compared with a P50 of 12 to 15 torr for the unmodified Hb. The Hill coefficient n of HbNFPLP was 2.1, versus 2.8 for Hb. The proton Bohr factor of HbNFPLP, calculated from P50 values in the pH range of 7.1 to 7.7, was found to be -0.19, versus -0.29 for unmodified Hb. The oxygen capacity of HbNFPLP was not affected by the crosslinking and was found to be 1.410 ml of O2 per g of HbNFPLP, versus 1.407 ml of O2 per g of Hb for unmodified Hb. Four derivatives of HbNFPLP, i.e., deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, and methemoglobin, were prepared, and the light adsorption spectra were recorded in the region of 480 to 680 nm. No differences were detected in comparison with the spectra of unmodified Hb. The alpha and beta chains of the tetramer were separated by reverse-phase chromatography. Comparison of the elution patterns of the chains of Hb and HbNFPLP revealed a retardation of the beta chains due to crosslinking with NFPLP. This indicates that the binding of NFPLP to Hb occurred only between the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)
van der Plas
J
J
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
de Vries-Van Rossen
A
A
Koorevaar
J J
JJ
Buursma
A
A
Zijlstra
W G
WG
Bakker
J C
JC
D001802:Blood Substitutes / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010100:Oxygen / Q000097:blood; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000097:blood; D013053:Spectrophotometry
D001802:Blood Substitutes; D003432:Cross-Linking Reagents; D003911:Dextrans; D006454:Hemoglobins; C045124:2-nor-2-formylpyridoxal-5'-phosphate; D011732:Pyridoxal Phosphate; D010100:Oxygen