Mutant hemoglobin stability depends upon location and nature of single point mutation

Abstract

The temperature dependence of the rates of heme release from the B subunits of methemoglobin A and 5 p mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the cur& interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.

Stability of the Heme-Globin Linkage in αβ Dimers and Isolated Chains of Human Hemoglobin

“…When the heme acceptor has a very high affinity for heme, like apomyoglobin, the kinetics of the heme transfer process is followed. This is governed by the rate of heme release from the donor protein as the rate of heme association is very high (2,3). When the heme donor and acceptor proteins have similar affinities for heme, the heme is partitioned between the two proteins and hence both kinetic and equilibrium features of the heme transfer reaction can be exploited (4,5).…”

“…In the case of human hemoglobin (HbA) there are relatively few quantitative estimates of the stability of the heme-globin linkage despite its relevance in the study of mutant and chemically modified hemoglobins (2)(3)(4)(5)(6). The interaction between heme and globin is known to be affected by a number of parameters.…”

A Comparison of Rates of Heme Exchange: Site-Specifically Cross-Linked Versus Polymerized Human Hemoglobins

“…Therefore, with unmodified HbA 0 and site-specifically cross-linked hemoglobin tetramers, heme loss from 1 globin likely provided the majority of the observed absorption change. Release of heme from (x subunits is more difficult to measure because of globin precipitation [9,15]. The smaller rate found in these experiments may correspond to heme reiease from ex subunits, but the overall extent of the reaction does not allow a truly definitive interpretation at th2.…”

The spontaneous hemin release from Lumbricus terrestris hemoglobin

“…1 did not give us greater confidence in results from a multiple first order fit than the simple fit of a single first order reaction, so the single rate constant is used in discussion. In addition, a slow, linear absorbance increase was observed at the end of the experiments which was presumably due to precipitation of some apoHb and made estimation of the end absorption difficult, as has been observed for human Hb (14). This slow reaction was not further studied.…”

“…Hemin is also continuously formed in the blood of apparently healthy individuals as a product of chloride assisted oxidation of hemoglobin at a rate of l-2% daily (19). Using apoMb as the hemin sink, we have shown that the spontaneous release of hemin from the p chains of human Hb and single point mutants is a continuous process with a half-life of only a few hours (14). This system measures only the hemin release and is independent of hemin apoMb combination (4).…”