• Abstract
• Introduction
• Materials and Methods
• Results and Discusion
• Conclusions and Acknowledgment 
• References

The ground state absorption spectrum of Rh-123 for the UV and visible spectral region is presented in Fig.1a. The Rh-123 absorption spectrum in neutral aqueous solution consists of an intensive band having a maximum around 500 nm, a shoulder at 475 nm and less pronounced bands with maxima at 330 nm and 240 nm (Fig.1a). The fluorescence spectrum of Rh-123 in buffer solution at neutral pH has a maximum at 530 nm (Fig.1a), which is slightly shifted when pH is varied.
NADH in neutral aqueous solution is a fluorescent biomolecule with absorption maxima around 340 nm and 260 nm as well as with a broad emission band around 450 nm (Fig.1b). Only reduced NADH is excited at 340 nm, whereas both reduced NADH and oxidized NAD⁺ are excited at shorter wavelengths (excitation bands of NAD⁺: l< 260 nm). Fluorescence excited at 330-370 nm can be used to probe the excitation energy relaxation in NADH molecules.
Flavin mononucleotide (FMN) absorbs light in the UV and visible spectral region with maxima around 220, 270, 370 and 450 nm. Its emission maximum is about 513 nm (Fig.1c).
The difference absorption spectra of Rh-123, NADH and FMN in aqueous solution under the excitation by 5 ps pulses of the third harmonic (352 nm) of a Nd³⁺ glass laser are presented in Figs. 2-4. The DA spectrum of Rh-123 in aqueous solution exhibits a bleaching band with a maximum around 500 nm (Fig.2). In comparison with the absorption band of Rh-123, the bleaching band in the difference absorption spectrum is wider and slightly red-shifted. It should be concluded that the bleaching band of Rh-123 in aqueous solution reflects the depopulation of its S₀ state. This band is partially affected by the amplification of emission in the red part of the spectrum. No transient absorption (DA > 0) was detected in the visible part of the spectrum from 400 to 800 nm, probably due to very low absorption cross section of the excited state S₁ of Rh-123 in aqueous solution. The lifetime of relaxation of absorption bleaching at 500 nm in aqueous Rh-123 solution is about 3.7 ns (Fig.5) and coincides with its fluorescence lifetime.
Different DA spectra were detected in aqueous solutions of NADH and FMN. Immediately after excited state population of NADH in buffer solution at pH 7 following 352 nm excitation, the spectrum of DA (shown in Fig.3) exhibits the transient absorptionin the spectral region of 600-950 nm and the absorption bleaching at 450-600 nm. The wavelengths of absorption bleaching in the DA spectrum approximately coincide with the fluorescence band of NADH. The maximum of the bleaching band is red-shifted in comparison with the maximum of NADH fluorescence (Fig.3). The transient absorption in the spectral region of 600-950 nm does not reflect absorption from the excited state S₁ of NADH, since at the delay of 1.4 ns between pump and probe pulse the transient absorption still remains, whereas the bleaching band with the maximum of DA around 500 nm disappears (due to a transition of excited molecules to the ground state). Therefore, it appears that after excitation of NADH in aqueous solution the formation of a transient species absorbing in the spectral region of 500-900 nm takes place. This is supported by the kinetic measurements shown in Fig.6. The relaxation time of the bleaching band at 500 nm reflecting the relaxation of excited molecules to the ground state is much shorter than transient absorption at 670 nm. It seems that immediately after excitation of the S₁ state of NADH the formation of transient absorption in a wide region of the visible spectrum occurs. It is impossible to assign this transient absorption to absorption from the excited state of NADH molecules, since in this case, the relaxation time at 500 nm and 670 nm should be the same. It is also difficult to connect the transient absorption in the visible part of the spectrum with the population of a triplet state of NADH, since the transient absorption appears at the same time as the population of the S₁ state (Fig.6). Therefore, it is more likely that immediately after excitation of NADH by pulses of 5 picoseconds in aqueous solution photoproducts (ionic species, aqueous electrons, etc.) or transient states of NADH are detected. Since NADH in aqueous solution is unstable and since the formation of oxidized NAD⁺ was detected on illumination, the long-lived transient absorption might be attributed to intermediate products appearing during NADH oxidation.
Figure 4 shows the result of picosecond transient absorption measurements of FMN in aqueous solution. The DA spectra around 410-900 nm exhibit the bleaching bands around 440-480 nm (characteristic of the ground state depopulation) and around 540-650 nm (coinciding with those of the fluorescence band of FMN). The bleaching band at 540-650 nm indicates that the monitoring light pulse of the picosecond continuum is slightly amplified by induced emission from the S₁ state. On the other hand, the transient absorption at 480-540 nm and 640-900 nm (Fig.4) might be assigned to absorption by a transient species immediately formed after excitation of FMN in aqueous solution by picosecond light pulses. The decay detected at 500 nm and 750 nm was different (Fig.7). The decay of transient absorption of FMN cannot be fitted by a single-exponential function with a relaxation time of 4.7 ns (the lifetime of fluorescence decay [9]), but can be approximated by a biexponential function (the Levenberg-Marquardt iteration method) with relaxation life times t₁ around 170 ps and t₂ around 4.7 ns (l = 500 nm). The transient absorption decay at 750 nm (Fig. 7) cannot be fitted by a biexponential function. The best approximation was achieved by a single-exponential function (relaxation life time t around 170 ps) and a plateau. Obviously, absorption changes around 500 nm and around 750 nm reflect different processes occurring after excitation of FMN. The relaxation of transient absorption around 500 nm and decay of absorption bleaching around 560 nm (t₂ about 4.7) should be assigned to the singlet excited state lifetime which is close to the fluorescence lifetime reported by Ref. [9]. The remaining transient absorption plateau (Figs. 4,7) in the spectral region of 600 - 900 nm might be related to the long-lived photoproduct or transient state of FMN after excitation by picosecond light pulses.

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Fig.1 Absorption (concentration as indicated) and fluorescence spectra (measured at concentration 10^-6 M) of: (a) Rh-123, (b) NADH, (c) FMN in phosphate buffer solution, pH around 7.0, path length 10 mm.

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Fig.2. The absorption difference spectrum of Rh-123, c=10^-4 M, in PBS, path length 1 mm, excitation at 352 nm. Difference absorption spectra were measured at the time when the pump and probe pulses are overlapping (Dt=0 ps); it reflects the maximum population of the excited molecular state S₁.

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Fig.3. Absorption difference spectra of NADH, c=10^-4 M, in PBS, path length 1 mm, excitation at 352 nm. The difference absorption spectra were measured at the time when the pump and probe pulses are overlapping (Dt=0 ps) (it reflecting the maximum population of the excited molecular state S₁) and at the selected delay time Dt between pump and probe pulses.

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Fig.4. Absorption difference spectra of FMN, c=10^-4 M, in PBS, path length 1 mm, excitation at 352 nm. The difference absorption spectra were measured at the time when the pump and probe pulses are overlapping (Dt=0 ps) (it reflecting the maximum population of the excited molecular state S₁) and at the selected delay time Dt between pump and probe pulses.

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Fig.5. The absorbance kinetics of Rh-123 in PBS measured at 500 nm, excitation at 352 nm, path length 1 mm.

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Fig.6. The absorbance kinetics of NADH in PBS, excitation at 352 nm, path length 1 mm.

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Fig.7. The absorbance kinetics of FMN in PBS, excitation at 352 nm, path length 1 mm.