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In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart.

Brazhe NA, Treiman M, Faricelli B, Vestergaard JH, Sosnovtseva O - PLoS ONE (2013)

Bottom Line: Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions.Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb).The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Department, Biological faculty, Moscow State University, Moscow, Russia.

ABSTRACT
We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50[Formula: see text] under hypoxia conditions.

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FCCP application.Raman spectra of perfused heart in control and under the application of protonophore FCCP (10 μM).
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pone-0070488-g003: FCCP application.Raman spectra of perfused heart in control and under the application of protonophore FCCP (10 μM).

Mentions: If the protonmotive force is dissipated by an increased proton re-entry to the mitochondrial matrix (such as in ADP-stimulated respiration or uncoupling), the electron flow along the ETC increases with a concomitant decrease of the reduction of electron carriers including cytochromes [28], [29]. Accordingly, we expected an addition of FCCP to the perfusion solution to cause a decrease in the intensity of cytochromal peaks. Fig. 3 shows that peaks at 750, 1127, 1310 and 1337 cm−1 all diminished in amplitude following FCCP application. In contrast, there were no changes in the intensities of peaks at 1377, 1587 and 1640 cm−1 because of constant amount of oMb. Peak heights at 750 and 1127 cm−1, expressed relative to the sum of all intensities within the spectra (Table 2) were significantly decreased at 5 and 10 min of FCCP treatment. We conclude that the behavior of the designated mitochondrial cytochromal Raman peaks following FCCP treatment was in agreement with the expected dependence of the reduction state of c-and b-type cytochromes on the protonmotive force.


In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart.

Brazhe NA, Treiman M, Faricelli B, Vestergaard JH, Sosnovtseva O - PLoS ONE (2013)

FCCP application.Raman spectra of perfused heart in control and under the application of protonophore FCCP (10 μM).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757006&req=5

pone-0070488-g003: FCCP application.Raman spectra of perfused heart in control and under the application of protonophore FCCP (10 μM).
Mentions: If the protonmotive force is dissipated by an increased proton re-entry to the mitochondrial matrix (such as in ADP-stimulated respiration or uncoupling), the electron flow along the ETC increases with a concomitant decrease of the reduction of electron carriers including cytochromes [28], [29]. Accordingly, we expected an addition of FCCP to the perfusion solution to cause a decrease in the intensity of cytochromal peaks. Fig. 3 shows that peaks at 750, 1127, 1310 and 1337 cm−1 all diminished in amplitude following FCCP application. In contrast, there were no changes in the intensities of peaks at 1377, 1587 and 1640 cm−1 because of constant amount of oMb. Peak heights at 750 and 1127 cm−1, expressed relative to the sum of all intensities within the spectra (Table 2) were significantly decreased at 5 and 10 min of FCCP treatment. We conclude that the behavior of the designated mitochondrial cytochromal Raman peaks following FCCP treatment was in agreement with the expected dependence of the reduction state of c-and b-type cytochromes on the protonmotive force.

Bottom Line: Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions.Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb).The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Department, Biological faculty, Moscow State University, Moscow, Russia.

ABSTRACT
We developed a Raman spectroscopy-based approach for simultaneous study of redox changes in c-and b-type cytochromes and for a semiquantitative estimation of the amount of oxygenated myoglobin in a perfused rat heart. Excitation at 532 nm was used to obtain Raman scattering of the myocardial surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling mitochondrial respiration. Conversely, transient hypoxia causes the reversible increase in the intensity of peaks assigned to cytochromes c and c1, reflecting electron stacking proximal to cytochrome oxidase due to the lack of terminal electron acceptor O2. Intensities of peaks assigned to oxy- and deoxyhemoglobin were used for the semiquantitative estimation of oMb deoxygenation that was found to be of approximately 50[Formula: see text] under hypoxia conditions.

Show MeSH
Related in: MedlinePlus