Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82 beta 1 and 82 beta 2 lysyl residues by bis(3,5-dibromosalicyl) fumarate.
We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82 beta 1 and lysine-82 beta 2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93 beta toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.
Shibayama
N
N
Department of Physics, Jichi Medical School, Tochigi, Japan.
Imai
K
K
Hirata
H
H
Hiraiwa
H
H
Morimoto
H
H
Saigo
S
S
D000328:Adult; D000595:Amino Acid Sequence; D001241:Aspirin / Q000031:analogs & derivatives*; D003432:Cross-Linking Reagents; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008239:Lysine / Q000097:blood / Q000737:chemistry*; D008969:Molecular Sequence Data; D010108:Oxyhemoglobins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D010108:Oxyhemoglobins; C021630:bis(3,5-dibromosalicyl)fumarate; D008239:Lysine; D001241:Aspirin