{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:37:54Z","timestamp":1760060274024,"version":"build-2065373602"},"reference-count":43,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,8,25]],"date-time":"2025-08-25T00:00:00Z","timestamp":1756080000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Formas","award":["2019-02496"],"award-info":[{"award-number":["2019-02496"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats: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\u2013gas 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 \u03bcM (rWT) and ~500 nM and ~400 \u03bcM (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\u2013gas studies and shows biphasic dynamics in BvPgbs.<\/jats:p>","DOI":"10.3390\/ijms26178240","type":"journal-article","created":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T06:26:31Z","timestamp":1756189591000},"page":"8240","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Gas-Binding Studies of Class 1 Sugar Beet Phytoglobin and C86A Mutant Using Isothermal Spectral Shifts in High-Precision Microliter Assay"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7041-4178","authenticated-orcid":false,"given":"Leonard","family":"Groth","sequence":"first","affiliation":[{"name":"Division of Pure and Applied Biochemistry, Department of Chemistry, Lund University, 221 00 Lund, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leif","family":"B\u00fclow","sequence":"additional","affiliation":[{"name":"Division of Pure and Applied Biochemistry, Department of Chemistry, Lund University, 221 00 Lund, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1093\/pcp\/pcu027","article-title":"Differential Expression Patterns of Non-Symbiotic Hemoglobins in Sugar Beet (Beta vulgaris ssp. vulgaris)","volume":"55","author":"Pin","year":"2014","journal-title":"Plant Cell Physiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"112393","DOI":"10.1016\/j.plantsci.2025.112393","article-title":"Differential Regulation of the \u201cPhytoglobin-Nitric Oxide Respiration\u201d in Medicago Truncatula Roots and Nodules Submitted to Flooding","volume":"352","author":"Chammakhi","year":"2025","journal-title":"Plant Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"111984","DOI":"10.1016\/j.plantsci.2024.111984","article-title":"Regulation of Nitric Oxide by Phytoglobins in Lotus japonicus Is Involved in Mycorrhizal Symbiosis with Rhizophagus irregularis","volume":"340","author":"Fukudome","year":"2024","journal-title":"Plant Sci."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Igamberdiev, A.U., and Bykova, N.V. 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