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Improved stopped‐flow time‐resolved resonance Raman spectroscopy device for studying enzymatic reactions
Author(s) -
Yanagisawa Sachiko,
Deshpande Megha Subhash,
Hirota Shun,
Nakagawa Tatsuo,
Ogura Takashi
Publication year - 2017
Publication title -
journal of raman spectroscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.748
H-Index - 110
eISSN - 1097-4555
pISSN - 0377-0486
DOI - 10.1002/jrs.5100
Subject(s) - resonance raman spectroscopy , myoglobin , chemistry , raman spectroscopy , spectroscopy , heme , resonance (particle physics) , analytical chemistry (journal) , ferric , substrate (aquarium) , hydrogen peroxide , reaction rate constant , photochemistry , nuclear magnetic resonance , kinetics , inorganic chemistry , enzyme , chromatography , organic chemistry , optics , atomic physics , physics , oceanography , quantum mechanics , geology
An improved stopped‐flow resonance Raman spectroscopy device was constructed using a stopped‐flow mixer with a dead time of 3 ms and a mixing volume of 0.1 mL. The device was tested using myoglobin, where the formation reaction of a high‐valent heme species, ferryl‐oxo heme, was monitored by time‐resolved resonance Raman spectroscopy after mixing a ferric myoglobin solution with a hydrogen peroxide solution. The ferryl‐oxo heme formation rate constant obtained by Raman spectroscopy is in good agreement with the rate constant obtained by conventional stopped‐flow absorption spectroscopy for the same reaction under the same conditions. It is proved by these results that the present device is generally applicable to enzyme–substrate reactions with a significantly higher time resolution than previously reported. Copyright © 2017 John Wiley & Sons, Ltd.