Small-footprint Absorbance System for Characterizing Heme Proteins

In this application note, we focus on the visible response of the heme group using the compact Spark spectral sensor to measure absorbance spectra for the heme proteins hemoglobin, myoglobin and cytochrome c. The heme groups found in metalloproteins generate absorbance bands that vary based on the state of the heme group. These absorbance changes make UV-Vis 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.

Experimental Setup

We used a Spark-VIS spectral sensor with direct-attach cuvette holder-white LED module to measure the visible absorbance spectra for the metalloproteins hemoglobin, myoglobin and cytochrome c diluted in solutions. Spark integration time was set at 3. 5 ms, with 50 scans to average and boxcar smoothing width at 10.  The 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.

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

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

Results

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

Conclusions

The ability to study the state of the heme group using UV-Vis absorbance spectroscopy makes it a powerful technique for education, research and even clinical diagnosis. 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.

Newsletters

Receive the latest analytical science news, personalities, education, and career development – weekly to your inbox.

I have read and understand the Privacy Notice*

Subscribe

About the Author(s)

Yvette Mattley

Yvette Mattley, Ph.D.

More Articles by Yvette Mattley