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Analytical Chemistry

  • Kô Takehara, Associate Professor
The keyword of our group is to understand quantitatively the interaction between small molecules and biological systems. The current effort is focused on the physicochemical analysis of the interaction between bioactive small molecules, such as general anesthetics (GAs), and cell membrane components. For this purpose, the bioluminescence by bacterial luciferase (BL) was controlled by electrochemical method and applied it to analyze the inhibition mechanism of GAs to protein function; The binding properties of hydrophobic molecules to proteins were analyzed using spectrophotometric methods; The surface-modified electrodes were developed using self-assemble technique for the analysis of signal transfer mechanism in cell membrane.

1. Electrochemical control of the bioluminescence of BL for protein binding assay

Bioluminescence is a powerful tool for the study of biological processes because of it's high sensitivity. The bioluminescence of BL has been intensively studied and elucidated the reaction mechanism. In the present work, we developed the control method of the BL bioluminescence using the electrochemical method. The enzyme reaction of BL uses a reduced form of flavinmononucleotide (FMNH2) as a coenzyme, however, the FMNH2 is oxidized readily by the oxygen dissolved in solution. In our system, the FMNH2 is regenerated by the controlled-potential electrolysis of FMN. Figure 1 shows the schematic illustration of the flow-electrolysis bioluminescence (FEBL) system developed in our laboratory to obtain continuous BL light intensity. Figure 2 shows the time-course profile of the BL light intensity recorded with switching the electrolysis potential turned on and off. The light intensity quickly changed depending on the switch of the electrolysis potential.

With using this system, the effects of hydrophobic molecules (terminal-modified hydrocarbons, Cn-X) on the BL intensity was measured and classified the inhibition mechanism in competitive, non-competitive and mixed-competitive inhibitions, depending on the properties of Cn-X. It was clarified that the terminal charge X of the Cn-X critically affects on the inhibition mechanism, and the chain length n of the Cn-X affects on the inhibition potency. The luminescence efficiency of the FEBL system has been improved by using the polymer film to confine the BL to the electrode surface.

Figure 1
Figure 1. Schematic illustration of the FEBL system using BL. The sample solution is supplied continuously with peristaltic pump (not shown in the figure).
Figure 2
Figure 2. Time-course profile of the BL light intensity when the electrolysis potential of −0.7 V is turned on and off.

2. Binding properties of hydrophobic molecules to human serum albumin

In order to understand the action mechanism of hydrophobic agents on proteins, the binding behaviors of hydrophobic molecules to human serum albumin (HSA) has been studied with fluorescence spectroscopy.

Although GAs have been used widely for long years in clinical purpose, the action mechanism of GAs has not yet been fully clarified. For example, it remains unknown how and where GAs bind to the proteins and inhibit their enzyme function (Fig. 3). To get information on the action mechanisms of hydrophobic agents on proteins, we are using naphthalene sulfonate (NS) as the fluorescence probe in analyzing the interaction between hydrophobic agents and proteins. NS emits an intense fluorescence when bound to the hydrophobic drug-binding sites in protein. For a model protein, HSA has been used because HSA has only one tryptophan residue (Trp-214) in the domain II. Trp-214 emits the fluorescence of 340 nm maximum wavelength with the light absorption of 295 nm. On the other hand, 1,8-anilinonaphthalene sulfonate (ANS) emits the fluorescence of 470 nm maximum wavelength with the absorption of 340 nm light. If the ANS binds in the vicinity of the Trp-214, the fluorescence of Trp-214 can be reabsorbed by the ANS and then the fluorescence of ANS increases, known as the fluorescence resonance energy transfer (FRET). Figure 4 shows the effect of ANS concentration on the fluorescence spectrum of the mixture of HSA and ANS. The excitation wavelength of Fig. 4 is 295 nm, which is not absorbed by the ANS. The fluorescence at 340 nm is decreased in contrast to the increase at 470 nm with the increase of ANS concentration, indicating the FRET between Trp-214 in HAS and ANS. By analyzing the FRET signals, the information on the binding sites of ANS could be elucidated.

Figure 3
Figure 3. Binding type of enzyme inhibitor. Do the inhibitor bind to enzyme in specific or nonspecific manner?
Figure 4
Figure 4. Fluorescence spectra of HSA and ANS mixture excited at 295 nm. [HSA]: 1.0 µM, [ANS]: 0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10 µM.

3. The surface-modified electrodes for the analysis of signal transmission in cell membrane

To understand the neurotransmission mechanism in cell membrane, bilayer lipid membrane (BLM) was formed on a gold (Au) electrode surface with the combination of liposome-fusion and self-assembled monolayer (SAM) methods as illustrated in Fig. 5. Then, the electrochemical impedance spectrum (EIS) was measured to confirm the formation of the BLM on Au surface. The charge-transfer resistance increased in the order of unmodified Au (bare Au), SAM modified Au (Au/SAM) and BLM-SAM modified Au (Au/SAM-BLM) as shown in Fig. 6. This result confirms us the formation of SAM and BLM-SAM films on the Au surface. This modified electrode has been used to analyzed to the effects of GAs, such as propofol and halothane, on the cell membrane.

Figure 5
Figure 5. Schematic illustration of the preparation of BLM-SAM/Au electrode for EIS analysis.
Figure 6
Figure 6. Niquist plots of the EIS spectra of benzoquinone at bare Au, Au/SAM, and Au/SAM-BLM.