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image medical diagnostics 4 624x434

Small-footprint Absorbance System for Characterizing Heme Proteins

Heme proteins play many vital roles in mammal physiology, including the storage and transport of oxygen and the transport of electrons in the mitochondria, where the high-energy molecule ATP (adenosine trisphosphate) is synthesized. The presence of the heme prosthetic group in these proteins gives rise to UV-Vis absorbance spectra that vary with the state of the heme group. In this application note, we focus on the visible response of the heme group using the compact Ocean OpticsSpark spectral sensor to measure absorbance spectra for the heme proteins hemoglobin, myoglobin and cytochrome c.

Background

Heme proteins are metalloproteins with an iron atom found at the center of a porphyrin prosthetic group. The heme group is bound to the protein and is absolutely required for the protein to carry out its critical biological function. The metalloproteins hemoglobin and myoglobin are very similar proteins that function to store and transport oxygen in mammals. The metalloprotein cytochrome c plays a critical role as part of the electron transport chain in mitochondria where the ATP that powers life is synthesized.

The heme groups found in metalloproteins generate absorbance bands that vary based on the state of the heme group. These spectral changes make UV-Vis absorbance spectroscopy a very powerful tool for studying heme proteins. Absorbance measurements can be used to study changes to critical protein parameters like protein conformation and to provide information on the binding and oxidation state of the heme prosthetic group.

Spark VIS image 960x833

To demonstrate this, we used the Spark-VIS spectral sensor with direct-attach sampling accessories to measure the visible absorbance spectra for the metalloproteins hemoglobin, myoglobin and cytochrome c. The Spark-VIS is the first Ocean Optics spectral sensor to use a solid state optical component in place of a traditional diffraction grating. Spark fills the price-performance niche between diode filter-based devices and spectrometers, adding a new dimension both in size – which is about the same as a small microcontroller — and in cost reduction, but with the ability to perform full spectral analysis from 380-700 nm. Its detection limits and response make Spark useful for absorbance, fluorescence and color measurements in educational, lab and industrial settings.

Experimental Setup

Hemoglobin (Sigma H-7379), myoglobin (Sigma M-0630) and cytochrome c (Sigma C-3131) solutions were prepared in water at a concentration of approximately 1 mg/mL. The samples were diluted as necessary to provide spectral data below 1 AU and pipetted into disposable cuvettes (CVD-VIS1M). We made absorbance measurements using the Spark-VIS and direct-attach cuvette holder with white LED module. Integration time was set at 3. 5 ms, with 50 scans to average and boxcar smoothing width at 10.

Results

The absorbance spectra measured for the metalloproteins are shown in Figure 1.

Figure 1 absorbance of heme proteins 480x358

Figure 1: The Spark-VIS spectral sensor and its direct-attach cuvette holder with white LED module measured absorbance spectra of heme proteins.

The similarities observed in the spectral features for each protein result from the presence of the heme group in these proteins. Based on the shape of these spectra — which change with the state of the heme group — the iron atom in the heme groups of these proteins appears to have been oxidized and resulted in spectra consistent with methemoglobin and metmyoglobin (Figure 2), and oxidized cytochrome c (Figure 3). The spectra measured for these proteins would be very different if the iron atom was in a different oxidation state or had oxygen or some other gas bound to it. The ability to study the state of the heme group using UV-Vis absorbance spectroscopy makes absorbance a powerful technique for education, research and even clinical diagnosis. 

 Figure 2 at left Methemoglobin spectrum 480x261

 Figure 2 at right Metmyoglobin spectrum 480x261

Figure 2: Oxidation of the iron atom in the heme groups of metalloproteins resulted in spectra consistent with methemoglobin (above) and metmyoglobin (below). 

Figure 3 Oxidzed Cytochrome c spectrum 480x261

 Figure 3: The absorbance spectrum of cytochrome c after oxidation was measured with the Spark spectral sensor.

Conclusions

 Absorbance spectroscopy is a powerful tool for characterizing proteins and other life science samples. In the case of metalloproteins, the UV-Vis absorbance spectra provide detailed information about the heme prosthetic group required for the proteins to carry out their critical biological functions. The compact size and low cost of the Spark spectral sensor make life science measurements more accessible and enable simpler use in education, research and clinical laboratory settings. 

 

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