Gattoni M, Piro M C, Boffi A, Brinigar W S, Fronticelli C, Chiancone E
CNR Center of Molecular Biology, Department of Biochemical Sciences, University La Sapienza, Rome, Italy.
Arch Biochem Biophys. 2001 Feb 15;386(2):172-8. doi: 10.1006/abbi.2000.2185.
The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of beta chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.
已测定了在血红素口袋、α1β1或α1β2界面处具有位点特异性β链突变的铁重组人血红蛋白的血红素-珠蛋白和二聚体-四聚体平衡。血红素口袋突变V67T导致β链血红素显著稳定，且不影响二聚体-四聚体缔合常数K2,4。在C112突变体中，相对于重组HbA（HbA-wt），β链血红素丢失的内在速率仅在C112G中显著增加，同时也有一些血红素从α链释放。凝胶过滤实验表明，K2,4值在C112G和C112L中基本未改变，但在C112V中增加，在C112N中降低。用A或M取代半胱氨酸93会导致β链血红素释放速率略有降低，而观察到的K2,4值与HbA-wt相似。除V67T、C93A和C112G外，在所有突变血红蛋白中均观察到氧亲和力的改变。数据表明，四聚体稳定性、β链血红素亲和力和血红蛋白功能之间没有相关性，因此表明这些特性受到独立调节。
