Human Plasma and Recombinant Hemopexins: Heme Binding Revisited
Abstract
:1. Introduction
2. Results and Discussion
2.1. Characterization of rhHPX
2.2. UV/Vis Titration
2.3. Heme Transfer from Methemalbumin to HPX
2.4. CD Titration
2.5. Heme Accessibility to Hydrogen Peroxide
2.6. Temperature-Induced CD Assessment
2.7. SEC-HPLC Assessment
3. Material and Methods
3.1. Materials
3.2. Generation of rhHPX Expression Plasmid and Transient Transfections
3.3. Purification of rhHPX
3.4. PEG-HPX Preparation
3.5. Stock Solutions of Heme, rhHPX, and Plasma HPX
3.6. Titration of Plasma HPX and rhHPX with Heme
3.7. UV/Vis Measurements
3.8. CD Measurements
3.9. Stability to Hydrogen Peroxide
3.10. SEC-HPLC
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
CD | Circular dichroism |
CE | Cotton effect |
DMSO | Dimethylsulfoxide |
EC | Exciton coupling |
HA | Human albumin |
HA/Heme | Methemalbumin, HA/Heme complex |
Heme | Iron-protoporphyrin IX |
HPX | Hemopexin |
HPX/Heme | Hemopexin/Heme complex |
L/P | Ligand-to-protein (molar) ratio |
λmax | Wavelength of absorption maximum |
PBS | Phosphate buffer saline |
SEC-HPLC | Size-exclusion high-performance liquid chromatography |
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Karnaukhova, E.; Owczarek, C.; Schmidt, P.; Schaer, D.J.; Buehler, P.W. Human Plasma and Recombinant Hemopexins: Heme Binding Revisited. Int. J. Mol. Sci. 2021, 22, 1199. https://doi.org/10.3390/ijms22031199
Karnaukhova E, Owczarek C, Schmidt P, Schaer DJ, Buehler PW. Human Plasma and Recombinant Hemopexins: Heme Binding Revisited. International Journal of Molecular Sciences. 2021; 22(3):1199. https://doi.org/10.3390/ijms22031199
Chicago/Turabian StyleKarnaukhova, Elena, Catherine Owczarek, Peter Schmidt, Dominik J. Schaer, and Paul W. Buehler. 2021. "Human Plasma and Recombinant Hemopexins: Heme Binding Revisited" International Journal of Molecular Sciences 22, no. 3: 1199. https://doi.org/10.3390/ijms22031199