Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin
Authors
Radomirović, Mirjana Ž.Gligorijević, Nikola
Minić, Simeon
Nikolić, Milan
Stanić-Vučinić, Dragana
Ćirković-Veličković, Tanja
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Phycobiliproteins (PBP) have been employed in numerous fluorescence-based techniques owing to highly fluorescent, covalently bound tetrapyrrole chromophores. So far, only entire PBPs have been utilized as fluorescent probes. A new method for covalent attachment of phycocyanin’s chromophore, phycocyanobilin (PCB), to potentially any protein, is proposed, relying on the ability of PCB to be attached to sulfhydryl groups of proteins. Traut’s reagent (TR, 2-iminothiolane) was exploited for introduction of sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its primary amines. Introduced sulfhydryl groups were then targeted for modification by PCB. All tested molar ratios of TR per mole of protein were successful in modification of BSA. Near-UV and far-UV circular dichroism spectroscopy revealed that a higher degree of modification by TR induces more profound alterations of BSA structure, leading at the same time to minor changes in BSA oligomerization and aggre...gation profile. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold ratio of TR. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal ratio for balancing between the effect on protein structure and the degree of labeling and thus fluorescent signal obtained. The proposed method could be used for labeling of virtually any protein, as means of either obtaining fluorescent probes for application in fluorescent techniques or functionalization of, for example, food proteins through covalent attachment of bioactive PCB.
Source:
FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts, 2021, 37-37Publisher:
- Belgrade : Faculty of Chemistry
Funding / projects:
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200168 (University of Belgrade, Faculty of Chemistry) (RS-MESTD-inst-2020-200168)
- FoodEnTwin-Twinning of research activities for the frontier research in the fields of food, nutrition and environmental omics (EU-H2020-810752)
- Serbian Academy of Sciences and Arts, grant number F-26
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Hemijski fakultet / Faculty of ChemistryTY - CONF AU - Radomirović, Mirjana Ž. AU - Gligorijević, Nikola AU - Minić, Simeon AU - Nikolić, Milan AU - Stanić-Vučinić, Dragana AU - Ćirković-Veličković, Tanja PY - 2021 UR - http://cherry.chem.bg.ac.rs/handle/123456789/6032 AB - Phycobiliproteins (PBP) have been employed in numerous fluorescence-based techniques owing to highly fluorescent, covalently bound tetrapyrrole chromophores. So far, only entire PBPs have been utilized as fluorescent probes. A new method for covalent attachment of phycocyanin’s chromophore, phycocyanobilin (PCB), to potentially any protein, is proposed, relying on the ability of PCB to be attached to sulfhydryl groups of proteins. Traut’s reagent (TR, 2-iminothiolane) was exploited for introduction of sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its primary amines. Introduced sulfhydryl groups were then targeted for modification by PCB. All tested molar ratios of TR per mole of protein were successful in modification of BSA. Near-UV and far-UV circular dichroism spectroscopy revealed that a higher degree of modification by TR induces more profound alterations of BSA structure, leading at the same time to minor changes in BSA oligomerization and aggregation profile. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold ratio of TR. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal ratio for balancing between the effect on protein structure and the degree of labeling and thus fluorescent signal obtained. The proposed method could be used for labeling of virtually any protein, as means of either obtaining fluorescent probes for application in fluorescent techniques or functionalization of, for example, food proteins through covalent attachment of bioactive PCB. PB - Belgrade : Faculty of Chemistry C3 - FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts T1 - Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin SP - 37 EP - 37 UR - https://hdl.handle.net/21.15107/rcub_cherry_6032 ER -
@conference{ author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Minić, Simeon and Nikolić, Milan and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja", year = "2021", abstract = "Phycobiliproteins (PBP) have been employed in numerous fluorescence-based techniques owing to highly fluorescent, covalently bound tetrapyrrole chromophores. So far, only entire PBPs have been utilized as fluorescent probes. A new method for covalent attachment of phycocyanin’s chromophore, phycocyanobilin (PCB), to potentially any protein, is proposed, relying on the ability of PCB to be attached to sulfhydryl groups of proteins. Traut’s reagent (TR, 2-iminothiolane) was exploited for introduction of sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its primary amines. Introduced sulfhydryl groups were then targeted for modification by PCB. All tested molar ratios of TR per mole of protein were successful in modification of BSA. Near-UV and far-UV circular dichroism spectroscopy revealed that a higher degree of modification by TR induces more profound alterations of BSA structure, leading at the same time to minor changes in BSA oligomerization and aggregation profile. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold ratio of TR. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal ratio for balancing between the effect on protein structure and the degree of labeling and thus fluorescent signal obtained. The proposed method could be used for labeling of virtually any protein, as means of either obtaining fluorescent probes for application in fluorescent techniques or functionalization of, for example, food proteins through covalent attachment of bioactive PCB.", publisher = "Belgrade : Faculty of Chemistry", journal = "FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts", title = "Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin", pages = "37-37", url = "https://hdl.handle.net/21.15107/rcub_cherry_6032" }
Radomirović, M. Ž., Gligorijević, N., Minić, S., Nikolić, M., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2021). Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin. in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts Belgrade : Faculty of Chemistry., 37-37. https://hdl.handle.net/21.15107/rcub_cherry_6032
Radomirović MŽ, Gligorijević N, Minić S, Nikolić M, Stanić-Vučinić D, Ćirković-Veličković T. Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin. in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts. 2021;:37-37. https://hdl.handle.net/21.15107/rcub_cherry_6032 .
Radomirović, Mirjana Ž., Gligorijević, Nikola, Minić, Simeon, Nikolić, Milan, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin" in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts (2021):37-37, https://hdl.handle.net/21.15107/rcub_cherry_6032 .