Stability of the heme-globin linkage in alphabeta dimers and isolated chains of human hemoglobin. A study of the heme transfer reaction from the immobilized proteins to albumin.
The stability of the heme-globin linkage in alphabeta dimers and in the isolated chains of human hemoglobin has been probed by studying the transfer of heme from the proteins immobilized onto CNBr-activated Sepharose 4B to human albumin. The kinetic and equilibrium features of the reaction have been measured spectrophotometrically given the stability of the heme donors and the ease with which heme donor and acceptor can be separated. Isolated alpha and beta chains transfer heme to albumin at similar rates (1 6 x 10(-2) s-1 at pH 9.0 and 20 degrees C) in the ferrous CO-bound and in the ferric state. In alpha beta dimers the heme-globin linkage is strengthened considerably, albeit to a different extent in the ferrous CO-bound and ferric met-aquo derivatives. Only in the latter heme is lost at a measurable rate, 0.065 +/- 0.011 x 10(-2) s-1 for alpha heme and 2.8 +/- 0.6 x 10(-2) s-1 for beta heme at pH 9.0 and 20 degrees C, which is very close to the rate measured with soluble met-aquo-hemoglobin at micromolar concentrations. These results indicate that in human hemoglobin the heme-globin linkage in the alpha chains is stabilized by interactions between unlike chains at the alpha1 beta1 interface, whereas heme binding to the beta chains is stabilized by interactions at the alpha1beta2 interface. These long range factors have to be taken into account in addition to the local factors at the heme pocket when evaluating the effect of point mutation and chemical modification.
Gattoni
M
M
CNR Center of Molecular Biology, Department of Biochemical Sciences "A. Rossi Fanelli," University La Sapienza, 00185 Rome, Italy.
Boffi
A
A
Sarti
P
P
Chiancone
E
E
D000418:Albumins / Q000737:chemistry; D002263:Carboxyhemoglobin / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D005914:Globins / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008706:Methemoglobin / Q000737:chemistry
D000418:Albumins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006454:Hemoglobins; D006418:Heme; D006427:Hemin; D005914:Globins; D008706:Methemoglobin; D002263:Carboxyhemoglobin