柳澤 幸子

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.