Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the pre...

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Autores principales:	Paolo Ruzza, Claudia Honisch, Rohanah Hussain, Giuliano Siligardi
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	photo-oxidation spectroscopy and biochemistry buffers protein denaturation reactive oxygen species (ROS) synchrotron radiation circular dichroism (SRCD) Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/8135630e2d3f44aebde9f298d97e787d
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spelling	oai:doaj.org-article:8135630e2d3f44aebde9f298d97e787d2021-11-11T16:48:43ZFree Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution10.3390/ijms2221113251422-00671661-6596https://doaj.org/article/8135630e2d3f44aebde9f298d97e787d2021-10-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11325https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.Paolo RuzzaClaudia HonischRohanah HussainGiuliano SiligardiMDPI AGarticlephoto-oxidationspectroscopy and biochemistry buffersprotein denaturationreactive oxygen species (ROS)synchrotron radiation circular dichroism (SRCD)Biology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11325, p 11325 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	photo-oxidation spectroscopy and biochemistry buffers protein denaturation reactive oxygen species (ROS) synchrotron radiation circular dichroism (SRCD) Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	photo-oxidation spectroscopy and biochemistry buffers protein denaturation reactive oxygen species (ROS) synchrotron radiation circular dichroism (SRCD) Biology (General) QH301-705.5 Chemistry QD1-999 Paolo Ruzza Claudia Honisch Rohanah Hussain Giuliano Siligardi Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution
description	A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.
format	article
author	Paolo Ruzza Claudia Honisch Rohanah Hussain Giuliano Siligardi
author_facet	Paolo Ruzza Claudia Honisch Rohanah Hussain Giuliano Siligardi
author_sort	Paolo Ruzza
title	Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution
title_short	Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution
title_full	Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution
title_fullStr	Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution
title_full_unstemmed	Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution
title_sort	free radical generation in far-uv synchrotron radiation circular dichroism assays—protein and buffer composition contribution
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/8135630e2d3f44aebde9f298d97e787d
work_keys_str_mv	AT paoloruzza freeradicalgenerationinfaruvsynchrotronradiationcirculardichroismassaysproteinandbuffercompositioncontribution AT claudiahonisch freeradicalgenerationinfaruvsynchrotronradiationcirculardichroismassaysproteinandbuffercompositioncontribution AT rohanahhussain freeradicalgenerationinfaruvsynchrotronradiationcirculardichroismassaysproteinandbuffercompositioncontribution AT giulianosiligardi freeradicalgenerationinfaruvsynchrotronradiationcirculardichroismassaysproteinandbuffercompositioncontribution
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Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution

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