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An analytical technique to measure reactions in biological membranes was developed and applied to monitoring the hydrolysis reaction of phospholipids (dipalmitoylphosphatidylcholine, DPPC) by phospholipase A(2). The technique uses the time-resolved quasi-elastic laser scattering (TR-QELS) method to measure an oil/phospholipid monolayer/water membrane system by monitoring the change of interfacial tension under a noncontact condition and in real time. When the TR-QELS method is used with the newly developed oil/phospholipid monolayer/water membrane system, measurement of the hydrolysis reaction of phospholipids with long alkyl chains (C >or=16), which are the major components in biological membranes, becomes possible. The reaction progress is monitored by the increase of interfacial tension at the oil/water interface caused by the decrease of surface-active DPPC molecules due to the reaction. The characteristic phases, namely, lag, burst, and equilibrium, are observed. The relationship between the duration of the lag phase (the rate-limiting step of the reaction) and the concentration of calcium ion (an essential cofactor of the reaction) is also investigated. Increase of calcium ion concentration in the subphase is found to shorten the duration of the lag phase. In addition, the real-time measurement simplifies the estimation process for the reaction activation energy.

The correlation between the fluorescent dynamics of excited anilinonaphthalene sulfonate (ANS) and the microviscosity of solvent molecules surrounding ANS is investigated by time-resolved fluorescence spectroscopy. ANS has been widely used to probe the local hydrophobicity due to the drastic change in its intensity. It is revealed that the fluorescence lifetime from the charge transfer (CT) state of ANS sensitively reflects the microviscosity. The higher sensitivity of 2,6-ANS than of 1,8-ANS demonstrates that the spatial freedom of the rotating phenylamino group in the photoexcited ANS is an important factor that determines the sensitivity. As an application, the measurements of the microviscosity of water in biologically important systems, such as hyaluronan, gellan gum, and gelatin aqueous solutions are also presented. The present results suggest that the fluorescence lifetime of ANS enables the estimation of the solvent microviscosity and provide a useful probe molecule for fluorescence lifetime imaging microscopy.

We have developed the total internal reflection ultrafast transient lens (TIR-UTL) method to detect nonradiative chemical processes at interfaces and surfaces with subpicosecond time resolution. In the TIR-UTL measurements, the evanescent field of a pump beam irradiated under the TIR condition generates a refractive index change. The refractive index change is attributed to changes of the molecular electronic state, of density by molecular orientation/structure change, and of temperature by vibrational relaxation processes. The refractive index change is detected as a change of the power intensity of the probe beam adjusted coaxially with the pump beam. At first, we discuss a theoretical principle of a coaxial configuration in the TIR-UTL measurement. This configuration has an advantage of versatility over the established TIR configuration. Then, we evaluate time resolution of TIR-UTL and obtain a value of less than 400 fs. We measure the ultrafast molecular dynamics of the cationic chromophore Auramine O (AuO) at a silica/water interface. Two slow time constants originating from AuO adsorbed on the silica surface are detected by TIR-UTL. These are attributed to AuO, whose twisting motion is strongly hindered by adsorption on a silica surface.