Spectroscopic Measurement of the Redox Potential of Cytochrome c for the Undergraduate Biochemistry Laboratory

Craig, Douglas B.; Nichols, Ellert R.

doi:10.1021/ed083p1325

Cited by 12 publications

(14 citation statements)

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“…4 and Additional file 3: Figure S3). The redox potential measurements for human and horse cyt c with 263.43 and 268.64 mV, respectively, were in close agreement with earlier reported values validating our technique [18, 19]. The redox potential measurements of native and recombinant R. arrhizus cyt c were 266.90 and 270.04 mV and statistically the same.…”

Section: Resultssupporting

confidence: 90%

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Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus

et al. 2015

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BackgroundMembers of Rhizopus species are the most common cause of mucormycosis, a rare but often fatal fungal infection. Host induced pathogen apoptosis and pathogen induced host cell apoptosis are often involved in fungal infections. In many organisms, the release of mitochondrial cytochrome c can trigger apoptosis by activating caspase proteases, but the role of fungal cytochrome c in apoptosis remains unknown.ResultsDNA sequence encoding Rhizopus arrhizus cytochrome c was cloned and expressed in E. coli. Both native and recombinant cytochrome c were purified using ion exchange followed by gel filtration chromatography. The identities of purified proteins were confirmed by MALDI-MS and UV-Visible spectroscopy. For the first time, we demonstrated that Rhizopus arrhizus cytochrome c could activate human capspase-3 in HeLa cell extracts. We also found that Rhizopus arrhizus cytochrome c has redox potential, peroxidase activity, and spectral properties similar to human and horse cytochrome c proteins.ConclusionsRhizopus arrhizus cytochrome c can activate human caspase-3 in HeLa cell extracts and it possesses similar physical and spectral properties as human and horse cytochrome c. This protein was found to have a previously unknown potential to activate human caspase-3, an important step in the apoptosis cascade.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0050-9) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 90%

“…The redox potential of R. arrhizus cyt c was determined as described [18] with some minor modifications. Briefly, 100 mM stock solutions of potassium ferrocyanide (ferroCN), potassium ferricyanide (ferriCN), and (+)-sodium L-ascorbate were prepared in 100 mM potassium phosphate buffer at pH 7.0.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus

et al. 2015

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“…These values are near that of HRC (262 ± 2 mV) 52 and are comparable to other cyt c proteins. 69–71 These findings are, once again, support His/Met ligation to the heme iron.…”

Section: Resultssupporting

confidence: 81%

“…52 Slopes of the linear fit of these dependencies (Figure S2 in the Supporting Information ) were used to calculate the cyt c reduction potentials.…”

Section: Methodsmentioning

confidence: 99%

Recombinant Expression, Biophysical Characterization, and Cardiolipin-Induced Changes of TwoCaenorhabditis elegansCytochromecProteins

et al. 2013

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Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of C. elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein’s denatured state. Both C. elegans proteins bind to CL-containing liposomes and these interactions promote the proteins’ peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved FRET in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms.

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“…Cytochrome c is a well‐characterized protein which has become a standard used in teaching undergraduate biochemistry students the protein purification procedures and biological assays to fully characterize the protein . A variety of biological assays have been conducted in conjunction with the standard set of cytochrome c protein purification laboratories.…”

Section: Introductionmentioning

confidence: 99%

Circular dichroism spectroscopy: Enhancing a traditional undergraduate biochemistry laboratory experience

Lewis

Seal

Lorts

et al. 2017

Biochem Molecular Bio Educ

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The undergraduate biochemistry laboratory curriculum is designed to provide students with experience in protein isolation and purification protocols as well as various data analysis techniques, which enhance the biochemistry lecture course and give students a broad range of tools upon which to build in graduate level laboratories or once they begin their careers. One of the most common biochemistry protein purification experiments is the isolation and characterization of cytochrome c. Students across the country purify cytochrome c, lysozyme, or some other well-known protein to learn these common purification techniques. What this series of experiments lacks is the use of sophisticated instrumentation that is rarely available to undergraduate students. To give students a broader background in biochemical spectroscopy techniques, a new circular dichroism (CD) laboratory experiment was introduced into the biochemistry laboratory curriculum. This CD experiment provides students with a means of conceptualizing the secondary structure of their purified protein, and assessments indicate that students' understanding of the technique increased significantly. Students conducted this experiment with ease and in a short time frame, so this laboratory is conducive to merging with other data analysis techniques within a single laboratory period. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):515-520, 2017.

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Spectroscopic Measurement of the Redox Potential of Cytochrome c for the Undergraduate Biochemistry Laboratory

Cited by 12 publications

References 1 publication

Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus

Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus

Recombinant Expression, Biophysical Characterization, and Cardiolipin-Induced Changes of TwoCaenorhabditis elegansCytochromecProteins

Circular dichroism spectroscopy: Enhancing a traditional undergraduate biochemistry laboratory experience

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