Materials
and Methods
Rh-123, NADH and FMN were obtained from
Fluka (Neu-Ulm, Germany) and were used without further purification. The
crude of the chemicals were dissolved in aqueous solution or phosphate
buffer solution (PBS) at pH 7.0. There was no differences whether phosphate
buffer or aqueous solution were used in our experiments.
Picosecond time resolved absorption spectrophotometer
is based on the Nd3+ glass laser as a driving generator. A single
pulse selected from a pulse train is amplified, then two beams are formed.
The first beam, after its frequency has been triplicated, is used for sample
excitation. A picosecond continuum source of D2O pumped by the
second beam of the main irradiation was used for sample probing. As a result,
a sensitivity of >10-3 optical density units can be obtained,
the sample absorption being ~ 1 unit of optical density units. The time
resolution of the spectrophotometer is up to 3 ps. The measurements were
made in a 1 mm cuvette. The absorption of the sample at the excitation
wavelength (third harmonic of neodymium glass laser ( l=
352 nm)) varied from 0.1 to 0.8 in optical density. The duration of pump
and probe pulses was about 5 ps.
The measurements of the difference absorption
spectra DA
are based on the detection of absorption spectra of buffer solutions at
different delay times between pump and probe pulses:
D A(l
,t)=A(l
,0) - A(l
,t),
where A(l
,0) is the absorption spectrum
of the sample before excitation and A(l
,t) is the absorption spectrum
at the selected delay time t between pump and probe pulses. Generally,
the spectrum DA(l
,t) includes the effects of absorption
bleaching due to a decrease of the ground state population, induced absorption
from the excited state of biomolecules, as well as amplification of theprobing
pulse by stimulated emission. |