Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited

Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the...

Descripción completa

Detalles Bibliográficos
Autores principales: Schleeger, Michael, Wagner, Christoph, Vellekoop, Michiel J., Lendl, Bernhard, Heberle, Joachim
Formato: Texto
Lenguaje:English
Publicado: Springer-Verlag 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709881/
https://www.ncbi.nlm.nih.gov/pubmed/19521691
http://dx.doi.org/10.1007/s00216-009-2871-0
_version_ 1782169329239851008
author Schleeger, Michael
Wagner, Christoph
Vellekoop, Michiel J.
Lendl, Bernhard
Heberle, Joachim
author_facet Schleeger, Michael
Wagner, Christoph
Vellekoop, Michiel J.
Lendl, Bernhard
Heberle, Joachim
author_sort Schleeger, Michael
collection PubMed
description Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the elucidation of the functional mechanisms of proteins, this technique is currently limited to repetitive systems undergoing a rapid photocycle. To overcome this obstacle, we developed a flow-flash experiment in a miniaturised flow channel which was integrated into a step-scan FT-IR spectroscopic setup. As a proof of principle, we studied the rebinding reaction of CO to myoglobin after photodissociation. The use of microfluidics reduced the sample consumption drastically such that a typical step-scan experiment takes only a few 10 ml of a millimolar sample solution, making this method particularly interesting for the investigation of biological samples that are only available in small quantities. Moreover, the flow cell provides the unique opportunity to assess the reaction mechanism of proteins that cycle slowly or react irreversibly. We infer that this novel approach will help in the elucidation of molecular reactions as complex as those of vectorial ion transfer in membrane proteins. The potential application to the oxygen splitting reaction of cytochrome c oxidase is discussed.
format Text
id pubmed-2709881
institution National Center for Biotechnology Information
language English
publishDate 2009
publisher Springer-Verlag
record_format MEDLINE/PubMed
spelling pubmed-27098812009-07-15 Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited Schleeger, Michael Wagner, Christoph Vellekoop, Michiel J. Lendl, Bernhard Heberle, Joachim Anal Bioanal Chem Original Paper Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the elucidation of the functional mechanisms of proteins, this technique is currently limited to repetitive systems undergoing a rapid photocycle. To overcome this obstacle, we developed a flow-flash experiment in a miniaturised flow channel which was integrated into a step-scan FT-IR spectroscopic setup. As a proof of principle, we studied the rebinding reaction of CO to myoglobin after photodissociation. The use of microfluidics reduced the sample consumption drastically such that a typical step-scan experiment takes only a few 10 ml of a millimolar sample solution, making this method particularly interesting for the investigation of biological samples that are only available in small quantities. Moreover, the flow cell provides the unique opportunity to assess the reaction mechanism of proteins that cycle slowly or react irreversibly. We infer that this novel approach will help in the elucidation of molecular reactions as complex as those of vectorial ion transfer in membrane proteins. The potential application to the oxygen splitting reaction of cytochrome c oxidase is discussed. Springer-Verlag 2009-06-12 2009-08 /pmc/articles/PMC2709881/ /pubmed/19521691 http://dx.doi.org/10.1007/s00216-009-2871-0 Text en © The Author(s) 2009
spellingShingle Original Paper
Schleeger, Michael
Wagner, Christoph
Vellekoop, Michiel J.
Lendl, Bernhard
Heberle, Joachim
Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
title Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
title_full Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
title_fullStr Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
title_full_unstemmed Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
title_short Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited
title_sort time-resolved flow-flash ft-ir difference spectroscopy: the kinetics of co photodissociation from myoglobin revisited
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709881/
https://www.ncbi.nlm.nih.gov/pubmed/19521691
http://dx.doi.org/10.1007/s00216-009-2871-0
work_keys_str_mv AT schleegermichael timeresolvedflowflashftirdifferencespectroscopythekineticsofcophotodissociationfrommyoglobinrevisited
AT wagnerchristoph timeresolvedflowflashftirdifferencespectroscopythekineticsofcophotodissociationfrommyoglobinrevisited
AT vellekoopmichielj timeresolvedflowflashftirdifferencespectroscopythekineticsofcophotodissociationfrommyoglobinrevisited
AT lendlbernhard timeresolvedflowflashftirdifferencespectroscopythekineticsofcophotodissociationfrommyoglobinrevisited
AT heberlejoachim timeresolvedflowflashftirdifferencespectroscopythekineticsofcophotodissociationfrommyoglobinrevisited