Immobilized apo‐myoglobin, a new stable reagent for measuring rates of heme dissociation from hemoglobin

Abstract

Apo-myoglobin covalently linked on CNBr-activated Sepharose 4B is proposed as a new heme acceptor for investigating the heme transfer reaction from hemoproteins. Immobilized apo-myoglobin has the desirable properties of an ideal heme acceptor in that it is characterized by a high affinity for ferric heme, a high stability towards denaturation even at physiological temperatures and can be lyophilized for long-term storage. The study of heme release from myoglobin at pH 5.0 and 37³C indicates that heme affinity is increased at least 10-fold relative to the soluble protein. Experiments with human hemoglobin allowed the estimation of the heme release rates from both K K and L L chains and brought out the greater temperature sensitivity of the K K chain heme-globin linkage. z 1998 Federation of European Biochemical Societies.

Heme proteins and oxidation in fresh and processed meats

“…Longissimus dorsi from beef contained 15±42% Hb while in the semimembranosus, Hb comprised 32±34% of the total heme pigment (Rhee and Ziprin, 1987). Hb concentrations can be underestimated relative to Mb due to the greater tendency of Hb to release its porphyrin moiety during extraction (Gattoni et al, 1998). Optical density of Hb and Mb between 700 and 400 nm is often used to quantify the heme protein concentration.…”

“…Human Hb is known to release its ferric hemin moiety much more rapidly compared to human Mb (Bunn and Jandl, 1968;Gattoni et al, 1998). In fact apoMb can completely extract hemin from Hb (Banerjee, 1962).This is relevant because released hemin is capable of stimulating extensive lipid oxidation through decomposition of pre-formed lipid hydroperoxides (Tappel, 1955, Van der Zee et al, 1996 (reactions 20 and 21).…”

Detection of coexisting protein conformations in capillary zone electrophoresis subsequent to transient contact with sodium dodecyl sulfate solutions

“…At 2.0 mmol?L 21 SDS 77% of Hb occurred in the "SDSform", which indicates a slightly higher stability than encountered for Mb at identical detergent concentration (Table 1). Allosteric effects due to the quaternary structure of Hb, which has to become disintegrated to monomers before a release of prosthetic groups is initiated, can provide an explanation [79,80]. Probably the more rigid T-structure of Hb contributes further to the stabilization [70].…”

Assessing the Relative Stabilities of Engineered Hemoglobins Using Electrospray Mass Spectrometry

“…Heme loss has not been observed for hemoglobins in the monomeric ␣-globin frame, suggesting that gas-phase heme dissociation from the native tetramer of hemoglobins is secondary to the dissociation into subunits. A loss of heme has also been observed in the solution phase, primarily from the biologically inactive metHb(Fe III ) form of hemoglobins (37,38). The increase in the relative stability observed in the described experiments is consistent with the increase of T m , measured by differential scanning calorimetery (DSC) and/or other thermal denaturation experiments (A. Mathews and T. Fattor, personal communication) for hemoglobins expressed in di-␣ frame (pseudo-tetramers) vs expressed in mono-␣ frame (tetramers).…”