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Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction.
Abstract: The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.
Publication Date: 1987-11-01 PubMed ID: 2823719DOI: 10.1016/0003-9861(87)90385-7Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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The research investigates the effects of ionic strength on the binding process of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase. This interaction was analysed using fluorescence quenching technique, and it was inferred that binding only occurs at certain ionic strengths, and is independent of the redox state of cytochrome c at specified ranges.
Background of the study
- The study focuses on the interaction between horse heart cytochrome c and yeast cytochrome c peroxidase, two proteins involved in cellular respiration and metabolism.
- The yeast porphyrin cytochrome c peroxidase is a particular type of enzyme, with its heme group replaced by protoporphyrin IX in this study.
Study Design and Methodology
- The binding of these two proteins was determined by applying a fluorescence quenching technique, which is a method used to investigate changes in molecular interactions.
- The study was carried out at a certain pH level, 6.0, using specific buffers.
- Another aspect that was varied in the experiment was the ionic strength, which was altered between the values of 3.5 mM and 1.0 M.
Main Findings
- It was noted that no binding of the proteins occurred at the ionic strength of 1.0 M, however, the decrease in fluorescence was substantial because of an effect known as the inner filter effect.
- Upon correcting for this inner filter effect, it was observed that significant fluorescence quenching took place at an ionic strength of 0.1 M and below.
- The quenching that was observed was described as a 1:1 complex formation between the two proteins.
- Additionally, the values of the equilibrium dissociation constant that were determined from this study matched those from a prior study for the same enzyme-protein complex at pH 6.0.
Additional Observations
- The binding patterns of both the ferricytochrome c and ferrocytochrome c versions to cytochrome c peroxidase were examined at pH 7.5 with varying ionic strengths.
- It was found that the binding procedure is independent of the redox state of cytochrome c at an ionic strength between 10 and 20 mM.
- Interestingly, ferricytochrome c presented a higher affinity for binding at ionic strengths of 30 mM and above.
Cite This Article
APA
Vitello LB, Erman JE.
(1987).
Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction.
Arch Biochem Biophys, 258(2), 621-629.
https://doi.org/10.1016/0003-9861(87)90385-7 Publication
Researcher Affiliations
- Department of Chemistry, Northern Illinois University, DeKalb 60115.
MeSH Terms
- Animals
- Cytochrome c Group / metabolism
- Cytochrome-c Peroxidase / metabolism
- Horses
- Hydrogen-Ion Concentration
- Kinetics
- Myocardium / metabolism
- Osmolar Concentration
- Peroxidases / metabolism
- Protein Binding
- Saccharomyces cerevisiae / enzymology
- Spectrometry, Fluorescence
Citations
This article has been cited 3 times.- Hewitt SH, Filby MH, Hayes E, Kuhn LT, Kalverda AP, Webb ME, Wilson AJ. Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins. Chembiochem 2017 Jan 17;18(2):223-231.
- Turcotte RF, Raines RT. Interaction of onconase with the human ribonuclease inhibitor protein. Biochem Biophys Res Commun 2008 Dec 12;377(2):512-514.
- Dixon DW, Hong X, Woehler SE. Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5. Biophys J 1989 Aug;56(2):339-51.
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