Gas-Binding Studies of Class 1 Sugar Beet Phytoglobin and C86A Mutant Using Isothermal Spectral Shifts in High-Precision Microliter Assay
(2025) In International Journal of Molecular Sciences 26(17).- Abstract
Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. Standard assays often require large sample volumes and lack sensitivity for high-affinity, low-abundance proteins like hexacoordinated Pgbs. Here, we present a microscale capillary-based fluorescence assay for the high-precision measurement of protein–gas binding. Fluorophore-labeled proteins are loaded into gas-saturated capillaries and analyzed via dual-wavelength fluorescence to monitor isothermal spectral shifts... (More)
Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. Standard assays often require large sample volumes and lack sensitivity for high-affinity, low-abundance proteins like hexacoordinated Pgbs. Here, we present a microscale capillary-based fluorescence assay for the high-precision measurement of protein–gas binding. Fluorophore-labeled proteins are loaded into gas-saturated capillaries and analyzed via dual-wavelength fluorescence to monitor isothermal spectral shifts upon ligand binding. Phosphate-buffered saline with Tween20 (PBS-T20) ensures gas stability and minimizes nonspecific adsorption. Using this approach, we characterized CO and NO binding to the recombinant wildtype (rWT) of Beta vulgaris Pgb 1.2 (BvPgb 1.2) and its C86A mutant. CO titrations revealed biphasic binding, with EC50 ~400 nM and ~700 μM (rWT) and ~500 nM and ~400 μM (C86A). NO binding showed KD values of ~1600 nM (rWT) and ~400 nM (C86A), implicating Cys86 in ligand affinity. This assay provides a robust, low-volume method for high-affinity protein–gas studies and shows biphasic dynamics in BvPgbs.
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- author
- Groth, Leonard LU and Bülow, Leif LU
- organization
- publishing date
- 2025-09
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- carbon monoxide, gas-binding assay, hexacoordinated heme, ligand binding, microscale fluorescence, nitric oxide, phytoglobin, protein-ligand interactions, spectral shift assay, Tween20
- in
- International Journal of Molecular Sciences
- volume
- 26
- issue
- 17
- article number
- 8240
- publisher
- MDPI AG
- external identifiers
-
- pmid:40943164
- scopus:105015618562
- ISSN
- 1661-6596
- DOI
- 10.3390/ijms26178240
- language
- English
- LU publication?
- yes
- id
- 749f47d9-3f1d-4eb0-8373-8010b46d7d3e
- date added to LUP
- 2025-10-15 15:35:31
- date last changed
- 2026-01-08 12:56:26
@article{749f47d9-3f1d-4eb0-8373-8010b46d7d3e,
abstract = {{<p>Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. Standard assays often require large sample volumes and lack sensitivity for high-affinity, low-abundance proteins like hexacoordinated Pgbs. Here, we present a microscale capillary-based fluorescence assay for the high-precision measurement of protein–gas binding. Fluorophore-labeled proteins are loaded into gas-saturated capillaries and analyzed via dual-wavelength fluorescence to monitor isothermal spectral shifts upon ligand binding. Phosphate-buffered saline with Tween20 (PBS-T20) ensures gas stability and minimizes nonspecific adsorption. Using this approach, we characterized CO and NO binding to the recombinant wildtype (rWT) of Beta vulgaris Pgb 1.2 (BvPgb 1.2) and its C86A mutant. CO titrations revealed biphasic binding, with EC<sub>50</sub> ~400 nM and ~700 μM (rWT) and ~500 nM and ~400 μM (C86A). NO binding showed K<sub>D</sub> values of ~1600 nM (rWT) and ~400 nM (C86A), implicating Cys86 in ligand affinity. This assay provides a robust, low-volume method for high-affinity protein–gas studies and shows biphasic dynamics in BvPgbs.</p>}},
author = {{Groth, Leonard and Bülow, Leif}},
issn = {{1661-6596}},
keywords = {{carbon monoxide; gas-binding assay; hexacoordinated heme; ligand binding; microscale fluorescence; nitric oxide; phytoglobin; protein-ligand interactions; spectral shift assay; Tween20}},
language = {{eng}},
number = {{17}},
publisher = {{MDPI AG}},
series = {{International Journal of Molecular Sciences}},
title = {{Gas-Binding Studies of Class 1 Sugar Beet Phytoglobin and C86A Mutant Using Isothermal Spectral Shifts in High-Precision Microliter Assay}},
url = {{http://dx.doi.org/10.3390/ijms26178240}},
doi = {{10.3390/ijms26178240}},
volume = {{26}},
year = {{2025}},
}