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. 2022 Apr 26;23(9):4747.
doi: 10.3390/ijms23094747.

Myoglobin Interaction with Lactate Rapidly Releases Oxygen: Studies on Binding Thermodynamics, Spectroscopy, and Oxygen Kinetics

Kiran Kumar Adepu  1   2 , Dipendra Bhandari  1 , Andriy Anishkin  3 , Sean H Adams  4   5 , Sree V Chintapalli  1   2
Affiliations

Myoglobin Interaction with Lactate Rapidly Releases Oxygen: Studies on Binding Thermodynamics, Spectroscopy, and Oxygen Kinetics

Kiran Kumar Adepu et al. Int J Mol Sci. .

Abstract

Myoglobin (Mb)-mediated oxygen (O2) delivery and dissolved O2 in the cytosol are two major sources that support oxidative phosphorylation. During intense exercise, lactate (LAC) production is elevated in skeletal muscles as a consequence of insufficient intracellular O2 supply. The latter results in diminished mitochondrial oxidative metabolism and an increased reliance on nonoxidative pathways to generate ATP. Whether or not metabolites from these pathways impact Mb-O2 associations remains to be established. In the present study, we employed isothermal titration calorimetry, O2 kinetic studies, and UV-Vis spectroscopy to evaluate the LAC affinity toward Mb (oxy- and deoxy-Mb) and the effect of LAC on O2 release from oxy-Mb in varying pH conditions (pH 6.0-7.0). Our results show that LAC avidly binds to both oxy- and deoxy-Mb (only at acidic pH for the latter). Similarly, in the presence of LAC, increased release of O2 from oxy-Mb was detected. This suggests that with LAC binding to Mb, the structural conformation of the protein (near the heme center) might be altered, which concomitantly triggers the release of O2. Taken together, these novel findings support a mechanism where LAC acts as a regulator of O2 management in Mb-rich tissues and/or influences the putative signaling roles for oxy- and deoxy-Mb, especially under conditions of LAC accumulation and lactic acidosis.

Keywords: binding; lactate; lactic acidosis; myoglobin; oxygen release.

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Conflict of interest statement

The author(s) declare no potential conflict of interest with respect to the research, authorship, and/or publication of this article. S.H.A. is the founder and principal of XenoMed, LLC, which is focused on research and discovery that has no connection to the current project. XenoMed had no part in the research design, funding, results, or writing of the manuscript.

Figures

Figure 1
Figure 1
Representative ITC plots of binding of LAC with (a) oxy-Mb at pH 7.0, (b) deoxy-Mb at pH 7.0, (c) oxy-Mb at pH 6.4, (d) deoxy-Mb at pH 6.4, (e) oxy-Mb at pH 6.0, and (f) deoxy-Mb at pH 6.0. Raw data (upper panels) and integrated data (lower panels) represent titration of reactants with time (min) or molar ratios on the x-axis and the energy released or absorbed per injection on the y-axis. The solid lines in the bottom panels represent the best fit of experimental data using a one set of binding sites model provided by the manufacturer’s software (Microcal PEAQ-ITC software). The lower graphs clearly differentiate that at pH 7.0, Mb-LAC binding was predominantly exothermic (downward slope) driven by hydrophilic interactions with large negative enthalpic (∆H) values (details are given in Results and Discussion). In contrast, at acidic pH (pH 6.4 and pH 6.0), the Mb-LAC binding was endothermic (upward slopes) and is mostly favored by hydrophobic interactions and positive ∆H values. All the ITC experiments were repeated 5 times (n = 5) to obtain the thermodynamic properties. We have only shown one representative dataset from a single experiment per condition. Statistical analysis was performed with one-tail and two-tail t-test paired two sample for means. All statistical differences in the test data are displayed in superscripts.
Figure 1
Figure 1
Representative ITC plots of binding of LAC with (a) oxy-Mb at pH 7.0, (b) deoxy-Mb at pH 7.0, (c) oxy-Mb at pH 6.4, (d) deoxy-Mb at pH 6.4, (e) oxy-Mb at pH 6.0, and (f) deoxy-Mb at pH 6.0. Raw data (upper panels) and integrated data (lower panels) represent titration of reactants with time (min) or molar ratios on the x-axis and the energy released or absorbed per injection on the y-axis. The solid lines in the bottom panels represent the best fit of experimental data using a one set of binding sites model provided by the manufacturer’s software (Microcal PEAQ-ITC software). The lower graphs clearly differentiate that at pH 7.0, Mb-LAC binding was predominantly exothermic (downward slope) driven by hydrophilic interactions with large negative enthalpic (∆H) values (details are given in Results and Discussion). In contrast, at acidic pH (pH 6.4 and pH 6.0), the Mb-LAC binding was endothermic (upward slopes) and is mostly favored by hydrophobic interactions and positive ∆H values. All the ITC experiments were repeated 5 times (n = 5) to obtain the thermodynamic properties. We have only shown one representative dataset from a single experiment per condition. Statistical analysis was performed with one-tail and two-tail t-test paired two sample for means. All statistical differences in the test data are displayed in superscripts.
Figure 2
Figure 2
Autodock results displaying LAC interaction with oxy-Mb residues at (a) pH 7.0, (b) pH 6.4, and (c) pH 6.0. At pH 7.0, LAC shows interaction with residues K45, K63, and D60, while at pH 6.4, LAC shows interaction with residues K41, H96, and K97, and at pH 6.0, LAC shows interaction with residues K56 and E59. LAC (brown), heme center (pink), and the residues (green) interacting with LAC are displayed as sticks. Mb protein (cyan) is displayed as ribbon structure an oxygen (red) in spheres. Possible hydrogen bond interactions between side chains of residues and LAC are displayed as dashed yellow lines with bond length.
Figure 3
Figure 3
Autodock results displaying LAC interaction with deoxy-Mb residues. Irrespective of the change in acidic pH (pH 6.4 and pH 6.0), LAC is docked near to proximal His side of heme center of deoxy-Mb, interacting with the residues H96 and S92, except at pH 7.0, where no LAC binding was observed. LAC (brown), heme center (pink), and the residues (green) interacting with LAC are displayed as sticks. Mb protein (cyan) is displayed as ribbon structure. Possible hydrogen bond interactions between side chains of residues and LAC are displayed as dashed yellow lines with bond length.
Figure 4
Figure 4
Effect of lactate binding to oxy-Mb and O2 release kinetics. Oxygen kinetics is studied using Oxytherm+ respirometer. All experiments were performed with 50 mM sodium phosphate buffer (pH 7.0, pH 6.4, and pH 6.0) containing 150 µM equine oxy-Mb and varying concentrations of lactate (0.625 mM to 2.5 mM) and oxy-Mb alone as a control. (a) A representative graph showing addition of lactate eliciting a rapid release of O2 from oxy-Mb at pH 6.4. Arrows in the figure indicate the point of addition of oxy-Mb and LAC into buffer during the run. (b) Rate of release of O2 from Mb against lactate concentrations with buffers (pH 7.0, pH 6.4, and pH 6.0). Inlet chart displays rate of release of O2 from deoxy-Mb alone (pre-LAC) conditions. All O2 kinetic experiments were repeated 3 times (n = 3). Statistical analysis was performed using one-way ANOVA and the Tukey–Kramer post hoc test. Statistical analysis revealed no significant difference in O2 release from oxy-Mb alone (without LAC) at pH 7.0 and pH 6.0. Additionally, no significant difference in O2 release was observed at pH 7.0 and pH 6.0 with 1.25 mM LAC condition. Moreover, with the addition of 0.625 mM LAC to oxy-Mb at pH 6.0, no significance difference in O2 release was observed when compared to oxy-Mb alone at pH 6.0. Among all the test groups, only statistically not significant groups are shown with horizontal lines and two asterisks.
Figure 5
Figure 5
The O2 release at varying concentrations (0.0 mM, 0.625 mM, 1.25 mM, and 2.5 mM) of LAC at (a) pH 7.0, (b) pH 6.4, and (c) pH 6.0. The rate of O2 release was calculated from the linear portion of the graphs immediately after addition of LAC to oxy-Mb. Representative data from a single experiment per condition are shown here. All O2 kinetic experiments were repeated 3 times (n = 3).
Figure 6
Figure 6
A representative UV-Vis spectra of freshly prepared (a) oxy-Mb and (b) deoxy-Mb samples each at pH 7.0, pH 6.4, and pH 6.0. Preparation of protein samples is detailed in the Materials and Methods section. All the spectra were repeated 3 times (n = 3).
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