{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T08:47:53Z","timestamp":1792399673733,"version":"4.4.0"},"reference-count":69,"publisher":"American Chemical Society (ACS)","issue":"20","license":[{"start":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T00:00:00Z","timestamp":1791331200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institutes of Health (NIH)","award":["R01HL174012"],"award-info":[{"award-number":["R01HL174012"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>This study examines bench-scale synthesis and the physicochemical and functional properties of hemoglobin (Hb) formulations, including bovine (bHb), human (hHb), and pyridoxylated (PLP, pyridoxal-5\u2032-phosphate) human Hb (PLP-hHb), along with their poly(ethylene glycol) (PEG)-conjugated counterparts. These PEGylated Hb constructs were synthesized via an inside-out strategy targeting the \u03b2-Cys93 residue of Hb using a four-arm PEG-maleimide molecule (MW = 20 kDa per arm; 80 kDa total), producing multi-arm PEG conjugated Hbs (MA-PEG-Hbs). The precursor Hbs exist as \u03b12\u03b22 tetramers (MW \u223c64\u201365 kDa; diameter \u223c5 nm), whereas MA-PEG-Hbs showed increased size (320\u2013345 kDa; \u223c14 nm), consistent with 3\u20134 Hb tetramers per PEG scaffold. This expansion reduced haptoglobin binding kinetics by \u223c6\u20139-fold, potentially prolonging circulation and reducing renal clearance. Circular dichroism confirmed preservation of \u03b1-helical structure and heme integrity, while thermal stability decreased but remained within 63\u201366 \u00b0C. Bench-scale synthesis demonstrated scalability, with MA-PEG-hHb achieving the highest yield (\u223c84 \u00b1 8%). Oxygen-binding studies showed preserved cooperativity and increased affinity (lower P50): MA-PEG-bHb (16.2 \u00b1 0.6 mm Hg), MA-PEG-hHb (6.88 \u00b1 0.10 mm Hg), and MA-PEG-PLP-hHb (8.87 \u00b1 0.27 mm Hg), with cooperativity coefficients of 2.1 \u00b1 0.1, 2.2 \u00b1 0.01, and 1.7 \u00b1 0.03. These contrast with Hemospan (P50 = 6 \u00b1 2 mm Hg; cooperativity 1.2 \u00b1 0.5). Hemospan was synthesized by surface PEGylation of thiolated hHb with 5 kDa maleimide-PEG, suggesting that high oxygen affinity HBOCs such as the MA-PEG-Hbs variants may benefit in targeting ischemic tissues with extremely low oxygen tension. Oxidative analysis showed increased auto-oxidation after PEGylation (bHb: 0.010 \u2192 0.026 h\u22121; hHb: 0.007 \u2192 0.013 h\u22121; PLP-hHb: 0.011 \u2192 0.015 h\u22121), comparable to Hemospan (0.007 \u2192 0.021 h\u22121). Overall, multi-arm PEGylation enhances the molecular size, increases oxygen affinity, and circulation potential while maintaining oxidative behavior within ranges observed for commercial HBOCs, highlighting a balance between functional performance and biochemical stability.<\/jats:p>","DOI":"10.1021\/acsabm.6c00954","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T08:01:41Z","timestamp":1791360101000},"page":"9566-9581","source":"Crossref","is-referenced-by-count":0,"title":["Comprehensive Biophysical and Biochemical Characterization of Multi-Arm Maleimide-Poly(ethylene glycol) Conjugated Bovine and Human Hemoglobin as Potential Red Blood Cell Substitutes"],"prefix":"10.1021","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4698-2332","authenticated-orcid":true,"given":"Mohd Asim","family":"Khan","sequence":"first","affiliation":[{"name":"The Ohio State University , , , ,","place":["Columbus, Ohio, United States of America, 43210"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6367-3046","authenticated-orcid":true,"given":"Griffin J.","family":"Beyer","sequence":"additional","affiliation":[{"name":"The Ohio State University , , , ,","place":["Columbus, Ohio, United States of America, 43210"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Munoz","sequence":"additional","affiliation":[{"name":"University of California San Diego , , , ,","place":["San Diego, California, United States of America, 92093"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8794-2839","authenticated-orcid":true,"given":"Pedro","family":"Cabrales","sequence":"additional","affiliation":[{"name":"University of California San Diego , , , ,","place":["San Diego, California, United States of America, 92093"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andre F.","family":"Palmer","sequence":"additional","affiliation":[{"name":"The Ohio State University , , , ,","place":["Columbus, Ohio, United States of America, 43210"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,7]]},"reference":[{"issue":"9","key":"2026101904072500100_cit1","doi-asserted-by":"publisher","first-page":"1724","DOI":"10.1093\/clinchem\/43.9.1724","article-title":"Blood substitutes: evolution and future applications","volume":"43","author":"Scott","year":"1997","journal-title":"Clin. 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