{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T12:17:16Z","timestamp":1762777036657,"version":"build-2065373602"},"reference-count":40,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T00:00:00Z","timestamp":1762732800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Department of Dermatology, Aalborg University Hospital"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>We here present a novel and sensitive methodology for determining the melting point (MP) of Bovine Serum Albumin (BSA) from micromolar to picomolar concentration levels under label-free conditions. At 1 pM we could model the melting with a sharp Gaussian. However, from the transient state observed during the melting process by using a simple exponential decay model, we determined a time constant of 67 s. We applied this methodology by studying a 3.3 pM sample of a botulinum toxin A (BoNT-A) (stabilized with 2.8 nanomolar denatured Human Serum Albumin (HSA)). We were able to determine the Tm of BoNT-A in the presence of approximately 1000-fold more concentrated HSA. This method enables the detection of protein melting transitions at picomolar concentrations without the use of a fluorescence dye. Its sensitivity and simplicity make it a valuable analytical tool for studying protein stability in diluted pharmaceutical formulations. This method is useful for correlating thermal conformational changes with catalytic function.<\/jats:p>","DOI":"10.3390\/biom15111579","type":"journal-article","created":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T11:47:27Z","timestamp":1762775247000},"page":"1579","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Observing Picomolar Protein Unfolding Using Resonance Light Scattering"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4461-6379","authenticated-orcid":false,"given":"Alain","family":"Bola\u00f1o Alvarez","sequence":"first","affiliation":[{"name":"Department of Dermatology and Venerology, Aalborg University Hospital, Hobrovej 18-20, 9000 Aalborg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4913-6756","authenticated-orcid":false,"given":"Kristian Bakke","family":"Arvesen","sequence":"additional","affiliation":[{"name":"Department of Dermatology and Venerology, Aalborg University Hospital, Hobrovej 18-20, 9000 Aalborg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6082-5096","authenticated-orcid":false,"given":"Kasper Fjellhaugen","family":"Hjuler","sequence":"additional","affiliation":[{"name":"Department of Dermatology and Venerology, Aalborg University Hospital, Hobrovej 18-20, 9000 Aalborg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3031-1390","authenticated-orcid":false,"given":"Peter","family":"Bjerring","sequence":"additional","affiliation":[{"name":"Department of Dermatology and Venerology, Aalborg University Hospital, Hobrovej 18-20, 9000 Aalborg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0739-2751","authenticated-orcid":false,"given":"Steffen B.","family":"Petersen","sequence":"additional","affiliation":[{"name":"Department of Dermatology and Venerology, Aalborg University Hospital, Hobrovej 18-20, 9000 Aalborg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1224","DOI":"10.1038\/s41467-025-56304-z","article-title":"Cryo-EM structure of the botulinum neurotoxin A\/SV2B complex and its implications for translocation","volume":"16","author":"Khanppnavar","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.ejpb.2006.09.009","article-title":"High throughput screening of protein formulation stability: Practical considerations","volume":"65","author":"Capelle","year":"2007","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3597","DOI":"10.1063\/1.1589587","article-title":"Methods of single-molecule fluorescence spectroscopy and microscopy","volume":"74","author":"Moerner","year":"2003","journal-title":"Rev. Sci. Instrum."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.bpc.2003.09.004","article-title":"Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering","volume":"107","author":"Militello","year":"2004","journal-title":"Biophys. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1002\/0471140864.ps2809s79","article-title":"Analysis of protein stability and ligand interactions by thermal shift assay","volume":"79","author":"Huynh","year":"2015","journal-title":"Curr. Protoc. Protein Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Elgert, C., R\u00fchle, A., Sandner, P., and Behrends, S. (2020). Thermal shift assay: Strengths and weaknesses of the method to investigate the ligand-induced thermostabilization of soluble guanylyl cyclase. J. Pharm. Biomed. Anal., 181.","DOI":"10.1016\/j.jpba.2019.113065"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"349","DOI":"10.2174\/1389203003381315","article-title":"The use of circular dichroism in the investigation of protein structure and function","volume":"1","author":"Kelly","year":"2000","journal-title":"Curr. Protein Pept. Sci."},{"key":"ref_9","first-page":"164","article-title":"The use of circular dichroism spectroscopy to study protein folding, form and function","volume":"3","author":"Ramos","year":"2009","journal-title":"Afr. J. Biochem. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1007\/978-3-030-28247-9_6","article-title":"Dynamic Light Scattering (DLS). Principles, Perspectives, Applications to Biological Samples","volume":"Volume 8","author":"Falke","year":"2019","journal-title":"Radiation in Bioanalysis"},{"key":"ref_11","unstructured":"Company, H. (2025). Choosing the Concentration Range for DLS Size Measurement, HORIBA."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1126\/science.7638615","article-title":"Resonance light scattering: A new technique for studying chromophore aggregation","volume":"269","author":"Pasternack","year":"1995","journal-title":"Science"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1111\/j.1423-0410.1984.tb01558.x","article-title":"Stabilization of human albumin by caprylate and acetyltryptophanate","volume":"47","author":"Yu","year":"1984","journal-title":"Vox Sang."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1093\/protein\/12.6.439","article-title":"Crystal structure of human serum albumin at 2.5 A resolution","volume":"12","author":"Sugio","year":"1999","journal-title":"Protein Eng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2450","DOI":"10.1007\/s00894-014-2450-y","article-title":"Analysis of the structure and dynamics of human serum albumin","volume":"20","author":"Guizado","year":"2014","journal-title":"J. Mol. Model."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1006\/biol.1998.0163","article-title":"Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents","volume":"26","author":"Tarelli","year":"1998","journal-title":"Biologicals"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1661","DOI":"10.1001\/archopht.121.11.1661-a","article-title":"Botulinum toxin and human serum albumin","volume":"121","author":"Malhotra","year":"2003","journal-title":"Arch. Ophthalmol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3426","DOI":"10.1128\/aem.58.10.3426-3428.1992","article-title":"Stabilization of botulinum toxin type A during lyophilization","volume":"58","author":"Goodnough","year":"1992","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.toxicon.2019.07.005","article-title":"The role of human serum albumin and neurotoxin associated proteins in the formulation of BoNT\/A products","volume":"168","author":"Kutschenko","year":"2019","journal-title":"Toxicon"},{"key":"ref_20","unstructured":"Johnson, M. (1994). Recovery of Type A Botulinal Toxin Following Lyophilization. ACS Symposium Series, ACS."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1023\/A:1026386710428","article-title":"Endoproteinase activity of type A botulinum neurotoxin: Substrate requirements and activation by serum albumin","volume":"16","author":"Schmidt","year":"1997","journal-title":"J. Protein Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"67","DOI":"10.2165\/11584780-000000000-00000","article-title":"Content of botulinum neurotoxin in Botox\u00ae\/Vistabel\u00ae, Dysport\u00ae\/Azzalure\u00ae, and Xeomin\u00ae\/Bocouture\u00ae","volume":"10","author":"Frevert","year":"2010","journal-title":"Drugs R D"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"277","DOI":"10.5826\/dpc.0804a05","article-title":"New trends in botulinum toxin use in dermatology","volume":"8","author":"Guida","year":"2018","journal-title":"Dermatol. Pract. Concept."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1319","DOI":"10.2147\/CCID.S332247","article-title":"Utilities of Botulinum Toxins in Dermatology and Cosmetology","volume":"14","author":"Naik","year":"2021","journal-title":"Clin. Cosmet. Investig. Dermatol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1021\/cr60091a003","article-title":"The Solubility of Gases in Liquids","volume":"28","author":"Markham","year":"1941","journal-title":"Chem. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"17882","DOI":"10.1021\/jacs.7b08570","article-title":"Excited-State Aromatic Interactions in the Aggregation-Induced Emission of Molecular Rotors","volume":"139","author":"Sturala","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Borzova, V.A., Markossian, K.A., Chebotareva, N.A., Kleymenov, S.Y., Poliansky, N.B., Muranov, K.O., Stein-Margolina, V.A., Shubin, V.V., Markov, D.I., and Kurganov, B.I. (2016). Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0153495"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1038\/nbt.1641","article-title":"Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations","volume":"28","author":"Rissin","year":"2010","journal-title":"Nat. Biotechnol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1016\/S0009-2614(02)02046-8","article-title":"Understanding the variable fluorescence quantum yield of tryptophan in proteins using QM-MM simulations. Quenching by charge transfer to the peptide backbone","volume":"369","author":"Callis","year":"2003","journal-title":"Chem. Phys. Lett."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1016\/0006-3002(62)90869-7","article-title":"The effect of temperature on the fluorescence of some aromatic amino acids and proteins","volume":"60","author":"Gally","year":"1962","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Lakowicz, J.R. (2006). Principles of Fluorescence Spectroscopy, Springer. [3rd ed.].","DOI":"10.1007\/978-0-387-46312-4"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1111\/j.1470-8744.1995.tb00346.x","article-title":"Enzyme thermostabilization by bovine serum albumin and other proteins: Evidence for hydrophobic interactions","volume":"22","author":"Chang","year":"1995","journal-title":"Biotechnol. Appl. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1208\/s12249-014-0076-0","article-title":"The role of caprylate ligand ion on the stabilization of human serum albumin","volume":"15","author":"Faroongsarng","year":"2014","journal-title":"AAPs PharmSciTech"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"S65","DOI":"10.1016\/j.ajic.2007.10.007","article-title":"Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Health Care Settings","volume":"35","author":"Siegel","year":"2007","journal-title":"Am. J. Infect. Control"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/j.jaad.2011.10.008","article-title":"Recommendations and current practices for the reconstitution and storage of botulinum toxin type A","volume":"67","author":"Liu","year":"2012","journal-title":"J. Am. Acad. Dermatol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"157","DOI":"10.3988\/jcn.2013.9.3.157","article-title":"Effect of the refrigerator storage time on the potency of botox for human extensor digitorum brevis muscle paralysis","volume":"9","author":"Park","year":"2013","journal-title":"J. Clin. Neurol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"eads7379","DOI":"10.1126\/sciadv.ads7379","article-title":"Protein misfolding involving entanglements providesa structural explanation for the origin of stretched-exponential refolding kinetics","volume":"11","author":"Jiang","year":"2025","journal-title":"Sci. Adv."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"12046","DOI":"10.1038\/ncomms12046","article-title":"Photon-efficient imaging with a single-photon camera","volume":"7","author":"Shin","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_39","unstructured":"Hamamatsu Photonics, K.K. (2007). Photomultiplier Tubes\u2014Basics and Applications, Hamamatsu Photonics K.K."},{"key":"ref_40","first-page":"843","article-title":"Resonance light scattering spectroscopy can directly characterize protein solubility","volume":"40","author":"Chen","year":"2020","journal-title":"Nan Fang Yi Ke Da Xue Xue Bao"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/11\/1579\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T12:11:43Z","timestamp":1762776703000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/11\/1579"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,10]]},"references-count":40,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["biom15111579"],"URL":"https:\/\/doi.org\/10.3390\/biom15111579","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,10]]}}}