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Flash Photolysis

Chapter · January 2013

DOI: 10.1007/978-3-642-16712-6_63

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Flash Photolysis
Sowa Y, Berry RM. Bacterial flagellar motor. Q Rev Biophys.
2008;41(2):103–32.
Thomas DR, Francis NR, et al. The three-dimensional structure
of the flagellar rotor from a clockwise-locked mutant of
Salmonella enterica serovar Typhimurium. J Bacteriol.
2006;188(20):7039–48.
Flagellum
▶ Bacterial Flagellar Motor: Overview
Flash Photolysis
Igor V. Chizhov
Institute for Biophysical Chemistry, Hannover
Medical School, Hannover, Germany
Synonyms
Pulsed-laser transient spectroscopy; Time-resolved
light-induced reactions
Definition
Flash photolysis is “a technique of transient spectros-
copy and transient kinetic studies in which a light pulse
is used to produce transient species. Commonly, an
intense pulse of short duration is used to produce
a sufficient concentration of a transient species suitable
for spectroscopic observation” (IUPAC 1997).
Introduction
In 1967 Ronald Norrish, George Porter, and Manfred
Eigen were awarded the Nobel Prize for studies of
extremely fast chemical reaction, effected by
disturbing the equilibrium by means of very short
impulses of energy. The flash photolysis method is
one those famous relaxation techniques which were
developed by laureates and many other scientists in
the last half of twentieth century. As it happens quite
often in history of science, a technique developed in
wartime has been applied in unexpected way. In 1948,
George Porter realized that high intensity flash tubes
765 F
designed for nighttime aerial photography during World
War II can generate a concentration of chemical species
(free radicals, transient triplet states, etc.) large enough
to be observed spectroscopically. Moreover, he decided
to use two flash tubes: one powerful 2 ms flash for
excitation of reaction and a second weaker but shorter
(ca 50 ms) flash of light which passed the reaction
volume after particular delay time. This light then hits
detector (a spectrograph with photographic plate) and
allowed the determination of the transient concentration
of reaction species. Thus, the technique of flash photol-
ysis was born (Porter 1950). Further developments of
F
the method were mainly concentrated on increasing of
time resolution (down to few nanoseconds using spe-
cially designed coaxial flash lamps), improvement of
detection techniques, and an extension of application to
reactions in liquid and solid phases.
The advent of the laser in early 1960s revolution-
ized the method. Nowadays, it allows transient kinetics
to be recorded with a time resolution of few femtosec-
onds (e.g., Huber et al. 2002). The dual pulse method
(now is known as pump-probe spectroscopy), initially
proposed by Porter, allows this unprecedented resolu-
tion because after the splitting of a pico- or femtosec-
ond laser pulse to give the strong (pumping) and weak
(probing) beams, the latter can arrive to the sample
after precisely defined delay time from few femtosec-
onds to few nanoseconds. One should bear in mind that
the light propagates about 1 mm in air within 3 ps.
The optical delay unit provides this variable time.
Today, this method of ultrafast flash photolysis
spectroscopy is one of the most active experimental
approaches in physics, chemistry, and biology.
In 1999, Ahmed H. Zewail received the Nobel Prize
in Chemistry for his pioneering work in this field.
However, this double pulse method has some dis-
advantages. Firstly, for each time point one needs the
renewal of reaction system, and secondly, the temporal
accuracy of kinetics is greatly determined by the sta-
bility of the pulses. An obvious benefit is the ability to
record a transient spectrum at each time point. There-
fore, in parallel with Porter’s first flash photolysis
system, a setup with a single flash tube was developed
in his lab. Here the probe light was provided by
a continuous xenon arc or tungsten lamp, which was
passed through a monochromator, and detected with
a photomultiplier tube connected to oscilloscope. This
configuration is most popular nowadays for investiga-
tion of reaction kinetics in the nanosecond and slower
F 766
Flash Photolysis,
Fig. 1 Flash photolysis of
Bacteriorhodopsin. The
transient absorption changes at
650 nm were recorded from
ΔmOD at 650 nm
0.5 ms to 2 s after the laser
pulses at six different
temperatures (0–50 C, step 10).
Experimental data (circles) and
eight exponential fits (lines) are
plotted
time domain and particularly in the field of biophysics,
where quite complicated multistep relaxation path-
ways of molecules (proteins, DNA, etc.) are often
observed. The kinetic analysis of such long transients
needs very high accuracy of detection. Two applica-
tions of such laser flash photolysis system are
described as examples (▶ Protein Dynamics: Time-
Resolved Spectroscopic Studies).
Flash Photolysis of Bacteriorhodopsin
Bacteriorhodopsin (BR) is a 7-alpha helical, trans-
membrane protein that acts as a light-driven proton
pump. The protein absorbs the visible light (max
570 nm) and transfers the hydrogen ions through a bio-
logical membrane. This transport occurs in a multistep
manner and is accompanied by changes of absorption
spectrum (so-called photocycle of BR). Therefore,
time-resolved absorption spectroscopy of BR excited
by laser pulse is one of the most common experimental
methods of investigation of this protein. In Fig. 1, an
example of such traces is depicted in which a 5 ns,
5 mJ, 532 nm laser pulse from the Nd-YAG laser was
used and the UV-Vis transient traces measured. Global
kinetic analysis of these traces together with other
wavelengths in the UV-Vis part of spectrum revealed
the eight rate constants, their temperature dependence,
and five distinct spectral states of the BR photocycle
(Chizhov et al. 1996).
Note that the ultrafast pump-probe experiments
detect another three rate constants in the femto- and
Flash Photolysis
60
40
20
10–6 10–5 10–4 10–3 10–2 10–1 100
time, s
picosecond time domains (Dobler et al. 1988), thus
giving 11 experimentally resolved transitions of the
proton transportation by BR.
Flash Photolysis of Caged ATP and
Actomyosin Kinetics
Two major motor proteins, actin and myosin
are responsible for the muscle contraction. Myosin
hydrolyses ATP molecule and converts the energy
of hydrolysis to the mechanical force imposed on the
actin filaments. The minimal reaction mechanism of
the actomyosin reaction contains 15 states and 44 rate
constants between them (Geeves et al. 1984). Most
of the kinetic investigations of this reaction were
performed using rapid mixing technique (▶ stopped-
flow, ▶ quenched-flow, and ▶ relaxation methods).
Flash photolysis method can substitute some of these
measurements with better economy of sample (Weiss
et al. 2000). The 5 ns, up to 20 mJ, 355 nm laser pulses
have been used from essentially the same Nd-YAG
laser as for BR flash photolysis studies. These pulses
photolyzed the inactive precursor of ATP molecule
(caged ATP) thus initiating the reaction of free ATP
with actomyosin. In Fig. 2, transient light scattering
traces indicate actomyosin dissociation, and its subse-
quent reassociation is shown. Analysis of dissociation
(exponential) and association (logistic) kinetics pro-
vided important values of the apparent second-order
rate constant of the actomyosin dissociation and the
catalytic activity of myosin.
Flash Photolysis
1.0
0.8
LS
0.6
0.4
10–2 10–1 100
Flash Photolysis, Fig. 2 Flash photolysis experiments of
dissociation and association kinetics of actomyosin. The light
scattering signal decreased after the laser-induced release of free
ATP molecules from a caged compound, indicating the dissoci-
ation of actomyosin. After the long steady-state period of three
Concluding Remarks
Only two examples from numerous flash photolysis
experiments that can be found in the literature are
described here. They underlined some basic features
of the method related to biophysical research. Dynam-
ics of active biological molecules is characterized by
very wide spectrum of relaxation times spanning the
range from nanoseconds to seconds and slower. Time
constants shorter than nanoseconds are usually
assigned to the electron dynamics of the photo-excited
chromophores and studied by pump-probe transient
spectroscopy. These nine orders of magnitude of time
are equivalent to the time span of 1 s to 1 Gs (i.e.,

32 years). Therefore, the long life of the biological
molecule can be recorded in the flash photolysis exper-
iment. In the first example, the transient kinetics were
recorded over the seven time decades (Fig. 1), and in
the second one over the five (Fig. 2). The flash photol-
ysis technique with the appropriate log-time data
acquisition device (digitizing PC card or digital oscil-
loscopes) allows recording of such transients.
The actomyosin kinetics (Fig. 2) is shown starting
from 10 ms. This is the time constant of free ATP
release defined by photochemistry of used caged com-
pound (NPE-ATP) under the experimental conditions.
One should note however, that the flash photolysis
method of caged compounds could overcome
767 F
F
101 102 103
time, s
time decades, the reformation of actomyosin complex and
corresponding restoring of light scattering signal was observed.
Experimental data (circles) are plotted together with theoretical
fit (lines). Five consequent laser shots of different energy
(2–5 mJ) have been applied to the single sample
potentially the time resolution of the stopped-flow
apparatus (
1 ms) and allow the observation of the
kinetics of bimolecular reactions limited only by the
time of diffusion (
ms or shorter).
One of the most expensive parts of the modern flash
photolysis device is the laser. The low budget digital
photo camera is equipped nowadays with a flash lamp
that produces a light pulse of 10–30 ms duration and
emits few tens of millijoules of energy. Such a flash
lamp is adequate for feasibility studies before investing
in more expensive equipment.
Cross-References
▶ Protein Dynamics: Time-Resolved Spectroscopic
Studies
▶ Quenched-Flow Methods
▶ Relaxation Dispersion
▶ Stopped-Flow Techniques
▶ Transient-State Kinetic Methods
References
Chizhov I, Chernavskii DS, Engelhard M, M€ uller KH, Zubov
BV, Hess B. Spectrally silent transitions in the bacteriorho-
dopsin photocycle. Biophys J. 1996;71:2329–45.
Dobler J, Zinth W, Kaiser W, Oesterhelt D. Excited-state reac-
tion dynamics of bacteriorhodopsin studied by femtosecond
spectroscopy. Chem Phys Lett. 1988;144:215–20.
 F 768
Geeves MA, Goody RS, Gutfreund H. Kinetics of acto-S1 inter-
action as a guide to a model for the crossbridge cycle.
J Muscle Res Cell Motil. 1984;5(4):351–61.
Huber R, Moser J-E, Graetzel M, Wachtveitl J. Real-time obser-
vation of photoinduced adiabatic electron transfer in strongly
coupled dye/semiconductor colloidal systems with a 6 fs time
constant. J Phys Chem B. 2002;106:6494–9.
IUPAC. IUPAC compendium of chemical terminology, 2nd ed.
Oxford: Blackwell Science; 1997.
Porter G. Flash photolysis and spectroscopy – A new method for
the study of free radical reactions. Proc R Soc Lond Ser A.
1950;200:284–300.
Weiss S, Chizhov I, Geeves MA. A flash photolysis fluorescence/
light scattering apparatus for use with sub microgram quanti-
ties of muscle proteins. J Muscle Res Cell Motil. 2000;21:
423–32.
Flavin Mononucleotide
▶ Flavin Mononucleotide-Binding Fluorescent Pro-
teins
▶ Flavins
Flavin Mononucleotide-Binding
Fluorescent Proteins
Aba Losi and Cristiano Viappiani
Department of Physics, University of Parma,
Parma, Italy
Synonyms
Flavin mononucleotide; Fluorescent protein (FP);
FMN-binding fluorescent proteins (FbFPs); Green
fluorescent protein (GFP); Light, oxygen, or voltage
domains (LOV); Riboflavin 50 -Phosphate (FMN)
Definition
Flavin mononucleotide (FMN)-binding fluorescent
proteins (FbFPs) are novel reporters for cell micros-
copy, outperforming green fluorescent proteins (GFP)
and derivatives in anaerobic or microaerobic environ-
ments (Fig. 1). Furthermore, they are better suited
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Flavin Mononucleotide
for incorporation into small-sized genomes and for
following viral infections, protein expression, and
protein maturation in real time (Figs. 2, 3). FbFPs are
derived from photoreceptors of the LOV (light,
oxygen, or voltage) superfamily and exhibit fluores-
cence in the green region of the spectrum with
quantum yield of ca. 0.2–0.3. FbFPs have been
introduced as fluorescent reporters in 2007 and have
been ever since exploited for a number of selected
applications, including real-time biosensing of
changing oxygen level within living cells.
Basic Characteristics
Development of FbFPs for Fluorescence Imaging
in Anaerobic/Microaerobic Systems
Flavin mononucleotide (FMN)-binding fluorescent
proteins (FbFPs) are fluorescent proteins (FP)
engineered from LOV (light, oxygen, or voltage)
domains, photoresponsive protein units of ca. 110
amino acids in length (▶ LOV proteins:
photobiophysics; Losi and G€artner 2012). In LOV
domains the FMN chromophore (▶ Flavins) is
noncovalently bound in the dark adapted state and
brightly fluorescent with quantum yield FF ca. 0.2–0.3
(▶ Fluorescence: General Aspects; ▶ Fluorescence and
FRET in Membranes). Blue-light photoexcitation leads
to the reversible formation of a FMN-cysteine covalent
adduct, with complete loss of fluorescence. FbFPs have
been originally designed such that the reactive cysteine
is mutated into alanine and the system retains perma-
nent fluorescence (Drepper et al. 2007). This mutation
can also increase FF up to 0.39. Different from green
fluorescent protein (GFP), the FMN fluorophore
is formed also in anaerobic and microaerobic environ-
ments (Vizcaino-Caston et al. 2012). FbFPs are
thus ideally suited as fluorescent reporters (▶ Optical
Fluorescence Microscopy) of bacterial location or gene
expression in obligate anaerobes, for example the
opportunistic pathogens Porphyromonas gingivalis
(Choi et al. 2011) (Fig. 1) and Bacteroides fragilis
(Lobo et al. 2011), and the facultative anaerobe
Rhodobacter capsulatus (Drepper et al. 2007). FbFPs
have also been proposed as fluorescent tools to
study fungal colonization of hypoxic niches during
infections, based on model studies on gene expression
in Candida albicans and Saccharomyces cerevisiae
(Tielker et al. 2009).