Determination of myoglobin concentration in blood-perfused tissue. Eur J Appl Physiol

Faculty of Human Sciences, Institute of Human and Social Science, Kanazawa University, Kanazawa, Japan.
Arbeitsphysiologie (Impact Factor: 2.19). 10/2008; 104(1):41-8. DOI: 10.1007/s00421-008-0775-x
Source: PubMed


The standard method for determining the myoglobin (Mb) concentration in blood-perfused tissue often relies on a simple but clever differencing algorithm of the optical spectra, as proposed by Reynafarje. However, the underlying assumptions of the differencing algorithm do not always lead to an accurate assessment of Mb concentration in blood-perfused tissue. Consequently, the erroneous data becloud the understanding of Mb function and oxygen transport in the cell. The present study has examined the Mb concentration in buffer and blood-perfused mouse heart. In buffer-perfused heart containing no hemoglobin (Hb), the optical differencing method yields a tissue Mb concentration of 0.26 mM. In blood-perfused tissue, the method leads to an overestimation of Mb. However, using the distinct (1)H NMR signals of MbCO and HbCO yields a Mb concentration of 0.26 mM in both buffer- and blood-perfused myocardium. Given the NMR and optical data, a computer simulation analysis has identified some error sources in the optical differencing algorithm and has suggested a simple modification that can improve the Mb determination. Even though the present study has determined a higher Mb concentration than previously reported, it does not alter significantly the equipoise PO(2), the PO(2) where Mb and O(2) contribute equally to the O(2) flux. It also suggests that any Mb increase with exercise training does not necessarily enhance the intracellular O(2) delivery.

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    • "These orthologous globins share much of their overall structure (characteristic ''globin fold''), and many key regions are highly conserved [10]. With similar structure and heme binding properties, the optical characteristics of these proteins are similar [11] [12] (Figs. 2 and 3) and these hemoproteins cannot be distinguished by spectroscopy when in solution together [11]. At wavelengths of 500–700 nm, the absorption spectra of HbO 2 and MbO 2 both show twin absorption peaks: 544 and 582 nm for MbO 2 and 542 and 578 nm for HbO 2 . "
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    ABSTRACT: An accurate determination of myoglobin (Mb) oxygen affinity (P50) can be difficult due to hemoglobin (Hb) contamination and autoxidation of Mb to metMb which is incapable of binding oxygen. To reduce Mb autoxidation, P50 is often measured at refrigerated temperatures. However, the temperature dependent shift in Mb oxygen affinity results in a greater oxygen affinity (lower P50) at colder temperatures than occurs at physiological temperature (ca. 37-39 °C) for birds and mammals. Utilizing the temperature dependent pH shift of Tris buffer, we developed novel methods to extract Mb from vertebrate muscle samples and remove Hb contamination while minimizing globin autoxidation. Cow (Bos taurus) muscle tissue (n = 5) was homogenized in buffer to form a Mb solution, and Hb contamination was removed using anion exchange chromatography. A TCS Hemox Blood Analyzer was then used to quickly generate an oxygen dissociation curve for the extracted Mb. The oxygen affinity of extracted bovine Mb was compared to commercially available horse heart Mb. The oxygen affinity of extracted cow Mb (P50 = 3.72 ± 0.16 mmHg) was not statistically different from commercially prepared horse heart Mb (P50 = 3.71 ± 0.10 mmHg). With high yield Mb extraction and fast generation of an oxygen dissociation curve, it was possible to consistently determine Mb P50 under physiologically relevant conditions for endothermic vertebrates.
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    • "At the basal myocyte P O2 of ca. 10mmHg, the concentration of Mb-bound O 2 (260moll –1 ) in rat heart stands about 20 times higher than the level of free O 2 (13moll –1 ) (Masuda et al., 2008). Even though free O 2 diffuses more rapidly than Mb, the concentration difference between free O 2 and Mb-bound O 2 can still confer a significant role for Mb in contributing to the overall O 2 flux if Mb has sufficient diffusive mobility in the cell (Gros et al., 2010; Johnson et al., 1996). "
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    ABSTRACT: Despite a century of research, the cellular function of myoglobin (Mb), the mechanism regulating oxygen (O(2)) transport in the cell and the structure-function relationship of Mb remain incompletely understood. In particular, the presence and function of pores within Mb have attracted much recent attention. These pores can bind to Xe as well as to other ligands. Indeed, recent cryogenic X-ray crystallographic studies using novel techniques have captured snapshots of carbon monoxide (CO) migrating through these pores. The observed movement of the CO molecule from the heme iron site to the internal cavities and the associated structural changes of the amino acid residues around the cavities confirm the integral role of the pores in forming a ligand migration pathway from the protein surface to the heme. These observations resolve a long-standing controversy - but how these pores affect the physiological function of Mb poses a striking question at the frontier of biology.
    Preview · Article · Aug 2010 · Journal of Experimental Biology
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    • "The HbO 2 and metHb signals do not coresonate (Ho and Russu, 1981). In tissue samples, the cytochrome concentration appears too low to produce any significant spectral interference (Feng et al., 1990; Masuda et al., 2008). "
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    ABSTRACT: Myoglobin, a mobile carrier of oxygen, is without a doubt an important player central to the physiological function of heart and skeletal muscle. Recently, researchers have surmounted technical challenges to measure Mb diffusion in the living cell. Their observations have stimulated a discussion about the relative contribution made by Mb-facilitated diffusion to the total oxygen flux. The calculation of the relative contribution, however, depends upon assumptions, the cell model and cell architecture, cell bioenergetics, oxygen supply and demand. The analysis suggests that important differences can be observed whether steady-state or transient conditions are considered. This article reviews the current evidence underlying the evaluation of the biophysical parameters of myoglobin-facilitated oxygen diffusion in cells, specifically the intracellular concentration of myoglobin, the intracellular diffusion coefficient of myoglobin and the intracellular myoglobin oxygen saturation. The review considers the role of myoglobin in oxygen transport in vertebrate heart and skeletal muscle, in the diving seal during apnea as well as the role of the analogous leghemoglobin of plants. The possible role of myoglobin in intracellular fatty acid transport is addressed. Finally, the recent measurements of myoglobin diffusion inside muscle cells are discussed in terms of their implications for cytoarchitecture and microviscosity in these cells and the identification of intracellular impediments to the diffusion of proteins inside cells. The recent experimental data then help to refine our understanding of Mb function and establish a basis for future investigation.
    Full-text · Article · Aug 2010 · Journal of Experimental Biology
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