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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
FEBS Lett. Author manuscript; available in PMC Oct 29, 2009.
Published in final edited form as:
PMCID: PMC2591068
NIHMSID: NIHMS76243

Interaction of Fatty Acid with Myoglobin

Abstract

Upon titration with palmitate, the 1H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb) with fatty acid. Other detectable hyperfine shifted resonances of the heme group remain unchanged. Mb also enhances fatty acid solubility, as reflected in a more intense methylene peak of palmitate in Mb than in Tris buffer. Ligand binding analysis indicates an apparent palmitate dissociation constant (Kd) of 43 μM. These results suggest that Mb can bind fatty acid and may have a role in facilitating fatty acid transport in the cell.

Keywords: Lipid, NMR, metabolism, bioenergetics

INTRODUCTION

The physiology canon asserts that Mb serves as an O2 stores or facilitates O2 transport. Textbooks have embraced the concept; even though in vivo experiments have presented conflicting evidence [1]. Certainly, Mb can supply O2 during a transient decrease in the O2 supply, such as during a marine mammal dive or during the initiation of muscle contraction [25]. Mb concentration also appears to correlate with the aerobic capacity in different species. Yet in mammalian heart, the O2 store of Mb actually prolongs respiration in a rat heart for only a few seconds during anoxia. CO inactivation of Mb function elicits no compensating response in bioenergetics or contractile function. Moreover, Mb appears to diffuse too slowly in many physiological states to compete effectively with free O2 diffusion to transport intracellular O2 [69]. Even without any Mb, a mouse model exhibits no detectable impairment in respiration, contractile function, bioenergetics, and metabolism [10;11]. These observations have raised questions about Mb function and have spawned complex explanations about multiple compensating mechanisms as well as a controversial hypothesis about Mb scavenging of NO [12;13]. The in vivo experimental data do not definitively confirm that Mb has any overarching function as an O2 store or as O2 transporter, especially in the myocardium.

Alternatively, the experimental evidence may simply indicate that Mb has another significant function in the cell. Indeed, Mb knockout mice do exhibit a slight decrease in myocardial fatty acid metabolism relative to the wild type mouse [14]. Instead of ascribing the metabolic change to a limitation in intracellular O2 diffusion, which in vivo experimental measurements have not fully supported, we have hypothesized that the absence of Mb actually diminishes the cell’s capacity to facilitate fatty acid diffusion. That viewpoint helps clarify the emerging model of intracellular fatty acid transport, which the high affinity fatty acid binding protein (FABP) plays a prominent role [15]. To test such an idea about Mb, however, requires experimental evidence that Mb can interact with fatty acid and enhances fatty acid solubility.

Indeed, 1H NMR measurements of palmitate (PA) titration in MbCN solution reveal a selective change in the 8 heme methyl signal, consistent with a specific interaction in a localized region of Mb. Moreover, palmitate appears more soluble in Mb solution than in buffer. These observations suggest then that Mb may indeed serve as an intracellular fatty acid transporter.

MATERIALS AND METHODS

Protein Preparation

Myoglobin solution was prepared from lyophilized horse heart protein (Sigma Chemical Inc., St. Louis, MO). All the samples were prepared in 30mM Tris buffer with 1mM EDTA at pH 7.4. The pH was measured at 35°C using a calomel electrode (Orion 7110BN Micro Calomel pH, Thermo Electron Corporation). For MbCN, five times excess KCN was added to the metmyoglobin in Tris, and the pH was adjusted to 7.4

Fatty Acid-Mb Preparation

Sodium palmitate (Sigma Chemical Inc., St. Louis, MO) was dissolved in 30mM Tris buffer with 1mM EDTA at pH 8.5 at 65°C. Stock solutions of 10mM and 100mM were prepared for addition to myoglobin protein solutions and kept in a heating block (Thermolyne 17600 Dri-Bath) at 65°C. 10–100 mM palmitate (PA) was added to myoglobin to yield a final solution with different PA:Mb ratios. For the NMR experiments, two separate heating blocks maintained the PA solution at at 65°C and the Mb solution at at 35°C, respectively. Aliquots of PA in tris buffer at 65°C were added to 600ul of myoglobin solution maintained at 35°C. Immediately after the PA addition, the sample was transferred to a 5mm NMR tube. The NMR probe was also maintained at 35°C. The time between PA addition and the start of the NMR measurement was approximately 10 minutes.

NMR

An Avance 500-MHz Bruker spectrometer measured the 1H signals with a 5 mm probe. The 1H 90° pulse, calibrated against the H2O signal from a 0.15 M NaCl solution or perfusate, was 9 μs. A Watergate pulse sequence was used to suppress the solvent peak. Sodium-3-(trimethylsilyl) propionate 2,2,3,3 d4 (TSP) served as the internal chemical shift and concentration reference. All samples contained 5% D2O to enable the lock during signal acquisition. All measurements were carried out at 35°C. A typical spectrum required 1024 scans and used the following signal acquisition parameters: 24kHz spectral width, 2K data points, and 107-ms recycle time. Zero-filling the free induction decay (FID) and apodizing with an exponential window function improved the spectra. A spline fit then smoothed the baseline.

Fatty Acid Binding Affinity

The curve fit used equations to express specific and non-specific binding at two sites [16]. For the first site, the equation [PA]bound=Bmax[PA]freeKd+[PA]free determined a one site specific binding of palmitate to Mb based on the signal intensity loss of the 8 heme methyl. For 2 site binding, corresponding to specific and non specific binding, the curve fit used the equation

[PA]bound=Bmax[PA]freeKd+[PA]free+Bmax[PA]freeKd+[PA]free.

Assuming Kd’ [dbl greater-than sign] [PA]

[PA]bound=Bmax[PA]freeKd+[PA]free+Ns[PA]free

Where Ns=BmaxKd. [PA] = palmitate; Kd = dissociation constant; and Bmax= maximum capacity for palmitate binding.

The analysis assumes that the specific binding of fatty acid to Mb reduces the 8 heme methyl signal and the specific and non specific binding of fatty acid to Mb also gives rise to the observed methylene peak. Subtracting the bound palmitate concentration derived from the observed methylene lipid signal from the total palmitate added to the solution yields an estimate of the free palmitate concentration.

Statistical Analysis

Statistical analysis used the Sigma Plot/Sigma Stat program (Systat Software, Inc., Point Richmond, CA) and expressed the data as mean value ± standard error (SE). Nonlinear regression analysis of the average data points determined the dissociation constant using Marquardt-Levenberg algorithm [17]. Statistical significance was determined by two-tailed student’s t-test, P<0.05.

RESULTS AND DISCUSSION

Spectral Change

Fig 1A displays the downfield region of the MbCN spectra. In the literature, many studies have used MbCN as a surrogate model for the physiological MbO2, because it shares similar structural features but exhibits observable hyperfine shifted resonances of the heme groups away from the crowded diamagnetic region [18]. Specifically the prominent peaks at 26.4, 17.9, and 13.2 ppm correspond to the 5, 1, 8 methyl groups of the heme [19]. Upon addition of PA to MbCN, only the 8 heme methyl signal intensity decreases. Fig 1B displays the MbCN spectrum with the PA: Mb ratio of 0.4: 1. The difference spectrum (Fig 1BFig 1A), reveals that palmitate induces only an 8 heme methyl signal intensity loss (fig 1C). The difference spectra with PA:Mb ratios of 0.6:1 and 0.9:1 shows a only a slight change in the 8 heme methyl signal intensity (Fig 1D–1E).

Figure 1
1H NMR spectra of 0.8mM MbCN with and without palmitate in Tris buffer at pH 7.4 35°C: A) control spectrum of 0.8mM MbCN B) spectrum of 0.8mM MbCN with 0.8 mM TSP and with PA at a PA:Mb ratio of 0.4:1 C) Difference spectrum (1A–1B) D) ...

In the upfield region, PA titration also produces distinct spectral changes. Fig. 2 shows the MbCN spectra with different ratios of PA: Mb ratio. The control spectrum of MbCN appears in fig 2A. Fig 2B shows 0.8mM MbCN with 0.8 mM PA and 0.8 mM sodium-3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP). TSP serves as the chemical shift (0 ppm) and concentration reference. The difference spectra (Fig 2B–2A) shows the palmitate signal with the prominent methylene (-CH2-)12 peak at 1.17 ppm (Fig 3C). Even though above a PA: Mb ratio of 0.6:1, the 8 heme methyl peak decreases insignificantly with additional PA, the palmitate methylene peak, in contrast, continues to grow. Between PA:Mb ratios of 2:1 and 4:1, the palmitate signal increases noticeably (Fig 2D and 2E).

Figure 2
1H NMR spectra of the upfield region of 0.8mM MbCN with and without PA in Tris buffer at pH 7.4 and at 35°C: A) Control spectrum of 0.8mM without PA or TSP B) 0.8mM MbCN with 0.8mM TSP and with PA at PA:Mb of 1:1 C) Difference spectrum (2A–2B) ...
Figure 3
1H NMR spectra of PA in Tris, MbCN, and lysozyme at pH 7.4 and at 35°C: A) Spectrum of 0.4mM PA in Tris buffer B) Difference spectrum of MbCN with and without 0.4 mM PA at PA:Mb ratio of 2:1 C) Difference spectrum of lysozyme with and without ...

Fig. 3 shows the difference spectra of buffer, MbCN, and lysozyme with and without 0.4 mM palmitate in 30 mM Tris and 1 mM EDTA buffer at pH 7.4 at 35°C. In the spectrum of 0.4 mM PA in Tris buffer, the PA methylene peak resonates at 1.28 ppm._ Fig 3B shows the difference spectrum of 0.2mM of MbCN with and without 0.4mM PA. A more intense palmitate CH2 signal appears at 1.17 ppm, 0.1 ppm upfield of the corresponding position in buffer. Fig 3C shows the difference spectrum of 0.2mM lysozyme with and without 0.4 mM palmitate. No detectable signal of palmitate appears. Calibrated against the TSP signal, the spectra indicate that NMR detects only 0.8% of the added PA in Tris buffer, 31% in MbCN, and 0% in lysozyme. In MbCN, the detected PA signal indicates that palmitate has about 40 times greater solubility in Mb than in Tris buffer. Table 1 compares the palmitate solubility in different solutions.

Table 1
Solubility of Palmitate

Specific vs. Non-Specific Interaction

Because the chemical shifts of the PA methylene signal in buffer and Mb differ by 0.1 ppm, the analysis has assumed the observed PA signal arises from a fatty acid interaction with Mb. Such assumption agrees with literature report that fatty acid bound to the 15 kD FABP exhibits a well resolved peak, distinct from the corresponding 13C signal in buffer [20].

A plot of the free PA concentration vs. the bound PA in Mb solution shows a non-linear relationship, with a faster rising component at low PA concentration. Analyzing the curve with a specific and a non-specific site yields an apparent Kd of 0.035 ±0.008 mM and the coefficient, Ns=0.33.

Fig. 4 plots the free and bound PA based on the analysis of the 8 heme methyl Mb signal intensity in three independent sets of experiments (n=3). The analysis envisions that the 8 heme methyl signal intensity loss arises from a local PA binding to Mb. With palmitate: Mb ratio above 2:1, the signal intensity loss plateaus around 50%. A curve fit to an equation for one specific binding site yields an apparent dissociation constant (Kd) of 0.043 ±0.013 mM.

Figure 4Figure 4
Graph of bound and observed PA vs. actual PA based on the 8 heme methyl and the palmitate methylene signals based on 3 independent sets of experiments (n=3): A) The bound PA concentration derives the signal of the 8 heme methyl during PA titration and ...

Local perturbation

The selective intensity loss of the 8 heme methyl signal supports the hypothesis that PA interacts in a local structural region of Mb. That interaction presumably reduces the heme methyl mobility, which alters the relaxation rate and broadens the peak beyond solution state NMR detection. Such a molecular picture agrees with Mb solution and crystallographic studies, which depict the heme 7 propionate adjacent to the 8 methyl as a flexible group with the carboxyl group exposed to the solvent. The adjacent 6 propionate next to the 5 heme methyl also faces the solvent. However, the hydrogen bonding to amino acid residue, such as the FG3 histidine, reduces the accessibility to the 5 heme methyl region and introduces a molecular gate in a proposed O2 pathway to the heme [21]. Indeed, the 5 heme methyl signal shows no signal intensity change upon the addition of PA. The 1 methyl group residing inside the hydrophobic heme pocket has no exposure to solvent. It and other observable hyperfine peaks show no PA induced change.

Unfortunately, the 1D NMR analysis cannot distinguish a partial loss of signal intensity of the 1 proton heme propionate signals in MbCN spectra, which resonate in the overcrowded spectral region from −0.07 to 1.56 ppm at 35° C [22]. Additional NMR experiments must map in detail the specific Mb structural interaction with fatty acid interaction.

Palmitate Binding to Mb

The experimental data suggest that Mb can bind to PA and broaden the monolithic view that only fatty acid binding protein (FABP) traffics fatty acid in the cell. Rat heart has about 50 μM of FABP with a Kd of 14 nM but has 260 μM of Mb [2325]. The present study shows an apparent Kd of 35–43 μM. The apparent Mb Kd, however, derives from the assumption that all PA in solution can interact with Mb. Clearly with 0.8 mM MbCN with a PA:Mb ratio of 1:1, the detection of only 31% of the total signal and no detection of any signal with a chemical shift observed in buffer, indicates only a fractional amount PA may actually participate in binding to Mb, and the different PA states, involving most likely, the solution NMR undetectable lamellar state, probably exist in dynamic equilibrium.

Moreover, the characteristic upfield shifted palmitate signal in Mb suggests a non-specific interaction that exceeds the stoichiometric amount required to saturate the Mb binding site. Despite the lower binding affinity, the higher concentration of Mb and the enhanced fatty acid solubility may still allow Mb to compete effectively with FABP as a carrier of intracellular fatty acid.

Mb and Fatty Acid Transport

Imputing a fatty acid transport role for Mb agrees with previous literature studies and casts a different perspective on the biochemical mechanism maintaining metabolic homeostasis in the cell. Early experiments found 14C labeled oleic acid binding to a rat heart cytosolic fraction of 16 kD and implicated a Mb role in fatty acid binding [26]. However, the study did not distinguish the potential contribution from the 15 kD FABP. Subsequent experiments, however, showed Mb has approximately 40 times lower binding capacity than albumin on a per mole basis, which based on the presented evidence would extrapolate to a Mb Kd of 12.2 to 48 μM, in agreement with NMR determined apparent Kd value [27].

It might appear that low binding affinity of Mb for fatty would militate against any role in transporting fatty acid, currently ascribed almost exclusively to FABP with its high PA binding affinity. Dissociating the fatty acid from FABP, however, usually invokes a complex release mechanism [28]. With Mb, the lower affinity, higher protein concentration, and increased fatty acid solubility, may well skirt the need for such complex mechanism. Moreover, FABP mobility in the cell remains uncertain, whereas Mb diffusion in the cell can compete with free O2 diffusion under certain physiological conditions [8;9].

Conclusion

The selective change in the 1H NMR signal of the 8 heme methyl of MbCN during palmitate titration and the increased palmitate solubility in the presence of Mb points to a role for Mb in transporting fatty acid and sets the basis for future experiments, which must confirm its role in regulating fatty acid metabolism. Such a role would broaden the cell’s ability to meet a range of metabolic demands and suggests an alternative explanation of how exercise, which can increase Mb concentration in skeletal muscle, might regulate the system glucose and lipid metabolism [29;30]. The present study then has established a basis to explore the potential role of Mb in fatty acid binding and transport.

Acknowledgments

We gratefully acknowledge funding support from NIH GM 58688 and Philip Morris 005510.

Footnotes

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