Superparamagnetic iron oxide--myoglobin as potential nanoparticle: iron oxide--myoglobin binding properties and magnetic resonance imaging marker in mouse imaging

Figures & data

Figure 1. Low-frequency resonance Raman spectra of sol--gel encapsulated deoxyMb. The top spectrum (a) is at pH 7.0. After the jump of pH from 7.0 to 2.6, spectra were taken at (b) 5 min, (c) 15 min, and (d) 100 min. Each spectrum shows the data collected at 30 sec time intervals for spectra (a), (b), and (c) and 15 min for spectrum (d). For better clarity, the intensity of spectrum at Raman shifts in the range of wave numbers 200–1700 cm⁻¹ for spectra (a--d) is vertically displaced (on left) and (a--b) (on right). The intensity of spectrum (d) is normalized to the same collection time (30 sec). Modified from the Reference with permission 7.

Figure 2. A detailed edge view geometry of the FeO₂ bond in the myoglobin molecule in the top row. The bound dioxygen is bent, held end-on and forms a hydrogen bond to the distal (E7) histidine at position 64, with the angles of ⁺Fe–O–O = 115° and angle between O–O–H = 96°. A simplified molecular orbital (MO) representation of the FeO₂ molecule is shown at the bottom. Modified from reference with permission 7.

Figure 3. Schematic representation of the basal autoxidation process in water with the rate constant kH₂O and the protoncatalyzed process with the rate constant kH H₂O in neutral pH range. For sperm whale MbO₂, k_H2O = 0:18 × 10⁻⁴ h⁻¹M⁻¹ and  = 0:20 × 10⁴ h⁻¹M⁻² in 0.1 M buffer at 25°C. The aqua-met Mb formed by each process can be isomerized into the other. Modified from reference with permission 8.

Table 1. Low-frequency resonance Raman spectra intensity (in arbitrary units or a.u.) of sol--gel encapsulated deoxyMb represented as base value (100) and reduced intensity value (in%) from base 100 a.u. at different pHs (column 1) and duration (column 2). The Raman shifts show more sensitivity at excitation frequency λ_ex = 441 nm* (shown here) than λ_ex = 413 nm (not shown). The reduced intensities of spectral peaks at low pH after jump and duration of time are notable due to Fe--His F8 linkage disruption and conformational fluctuations resulting in heme pocket unfolding or opening and more protonated His pocket in myoglobin. The formation and existence of intermediates are shown as Raman shifts or wave number (cm⁻¹) in columns 3–7 represented as spectral peaks in figure 1.

		Raman shift* (cm^−1)
pH (1)	Time (min) (2)	(220 cm^−1) (3)	(370 cm^−1) (4)	(672 cm^−1) (5)	(787 cm^−1) (6)	(1355 cm^−1) (7)
7.0	0	100	100	100	100	100
2.6	0	75	60	80	90	80
2.6	5	70	40	80	80	--
2.6	15	40	30	60	60	--
2.6	100	20	30	50	--	70

Figure 4. Role of the globin moiety in stabilizing the FeO₂ bond in myoglobin. Notice the globin chain folding in relation to time. Redrawn from the reference with permission 8.

Figure 5. (On leftmost): A electron microscopic TEM image of Fe₂O₃; (on second left) SEM of albumin microspheres; (on third panel) TEM image of polyethylene coated microspheres; (on rightmost panel): A SEM image of polystyrene coated microsphere (shown with arrow) attached to red blood cell. Redrawn from references 1, 13.

Figure 6. The figure represents the scheme of two-site immunoassay to develop magnetic immunoassay using anti-myoglobin biotinylated antibodies and tagged biomarker to measure myoglobin. Redrawn from reference 15.

Figure 7. (On left panel) The figure represents MRI images of a capillary filled with iron oxide--myoglobin at pH 7.4 in presence of phosphate buffered saline pH7.4. In the background, mice body cross-sectional single slice image is shown with heart image (at the botton on right) without contrast agent. (On right panel) Same capillary with myoglobin at pH 4.0 in presence of acetate buffer pH 4.0 shows loss of signal of iron--oxide--myoglobin shown with asterisk. The images were generated using multislice multiecho spin echo proton density weighted technique at scan parameters TE = 15 ms, TR = 1500 ms, matrix 256 × 256, NEX = 2, FOV = 2.5 cm at 11.7 T or 500 MHz MRI imager. In the background, a same cross-sectional slice image of mice image after anti-myoglobin-iron oxide superparamagnetic contrast injection with cardiac territories are shown by arrows and details of different heart chambers (marked as RA, LA, LV, RV) can be seen in insert. Notice the enhancement of vascular wall regions rich with anti-myoglobin bound superparamagnetic particles after injection at pH 7.4 while the same imaging contrast agent in the capillary turned darker at pH 4.0.

Table 2. The relationship of Raman spectra peaks and MRI signal intensity of phantom is represented to show reduced intensities by both Raman spectrum and MRI imaging. The reason for the reduced Raman spectra peaks of iron oxide--myoglobin in phantom was the low. pH while the reduced MRI signal was due to both low pH and dephasing with phase shifting characteristics of superparamagnetic iron oxide molecule. Notice the increased MRI signal of mouse body* (marked A) after injection of iron-oxide myoglobin contrast agent due to relative loss of signal at solid tissue mass while vascular or fluid mass (marked B) appeared brighter as shown in this table and figure 4 by arrows.

			MRI signal intensity (a.u.)
				Body*
pH	% Reduced Raman 672 cm^−1	Spectra peaks 1355 cm^−1	Phantom	A	B
7.4			100	100	100
7.4*				255 (2.5 fold)	60 (0.6 fold)
7.0	100	100
4.0			40
2.7	50	80

Tang , Q , Kalsbeck , WA , Olson , JS and Bocian , DF . 1998 . Disruption of the heme iron-proximal histidine bond requires unfolding of deoxymyoglobin . Biochemistry , 37 : 7047

 Google Scholar

Shikama , K . 1988 . Stability properties of dioxygen--iron(II) porphyrins: an overview from simple complexes to myoglobin . Coordinate Chem. Rev. , 83 : 73

 Google Scholar

Chatterjee , J , Haik , Y and Chen , CJ . 2001 . Synthesis and characterization of heat-stabilized albumin magnetic microspheres . Colloid Polym. Sci. , 279 ( 11 ) : 1073

 Google Scholar

Chatterjee , J , haik , Y and Chen , CJ . 2001 . Modification and characterization of polystyrene based magnetic micrpspheres and its composition with albumin based magnetic microspheres . J. Magn. Mag. Mat. , 225 : 21

 Web of Science ®Google Scholar

Cardovaz , M . 2001 . Point of care diagnosis of acute myocardial infarction using magnetic immunoassays , Master's thesis, Florida State University .

 Google Scholar