The original publication is available at www.springerlink.com
Gordon C. K. Roberts
Encyclopedia of Biophysics
© European Biophysical Societies' Association (EBSA) 2013
Igor V. Chizhov
Institute for Biophysical Chemistry, Hannover Medical School,
Carl-Neuberg-Straße 1, D-30625 Hannover, Germany
Pulsed-laser transient spectroscopy; Time-resolved light-induced reactions
Flash photolysis is “a technique of transient spectroscopy 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).
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 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 μs) 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 photolysis was born (Porter 1950). Further developments of the method were mainly concentrated on increasing of time resolution (down to few nanoseconds using specially 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 revolutionized the method. Nowadays, it allows transient kinetics to be recorded with a time resolution of few femtoseconds (e.g., Huber et al. 2002). The dual pulse method (now is known as pump-probe spectroscopy), initially proposed by Porter, allows this unprecedented resolution because after the splitting of a pico- or femtosecond 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 femtoseconds 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 disadvantages. Firstly, for each time point one needs the renewal of reaction system, and secondly, the temporal accuracy of kinetics is greatly determined by the stability of the pulses. An obvious benefit is the ability to record a transient spectrum at each time point. Therefore, 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 investigation of reaction kinetics in the nanosecond and slower time domain and particularly in the field of biophysics, where quite complicated multistep relaxation pathways of molecules (proteins, DNA, etc.) are often observed. The kinetic analysis of such long transients needs very high accuracy of detection. Two applications of such laser flash photolysis system are described as examples.
Flash Photolysis of Bacteriorhodopsin
Bacteriorhodopsin (BR) is a 7-alpha helical, transmembrane 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 biological 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 picosecond time domains (Dobler et al. 1988), thus giving 11 experimentally resolved transitions of the proton transportation by BR.
Fig. 1 Flash photolysis of Bacteriorhodopsin. The transient absorption changes at 650 nm were recorded from 0.5 μs 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
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 subsequent reassociation is shown. Analysis of dissociation (exponential) and association (logistic) kinetics provided important values of the apparent second-order rate constant of the actomyosin dissociation and the catalytic activity of myosin.
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 dissociation of actomyosin. After the long steady-state period of three 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
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. Dynamics 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 experiment. 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 photolysis technique with the appropriate log-time data acquisition device (digitizing PC card or digital oscilloscopes) 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 compound (NPE-ATP) under the experimental conditions. One should note however, that the flash photolysis method of caged compounds could overcome 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 (~ μs 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 μs duration and emits few tens of millijoules of energy. Such a flash lamp is adequate for feasibility studies before investing in more expensive equipment.