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Biochemistry 1987, 26, 1422-1427
Grellmann, K. H., Schmitt, U., & Weller, H. (1982) Chem. Phys. Lett. 88, 40-45. Heihoff, K., Braslavsky, S. E., & Schaffner, K. (1987) Biochemistry (third paper of three in this issue). Holzwarth, A. R., Wendler, J., Ruzsicska, B. P., Braslavsky, S . E., & Schaffner, K. (1984) Biochim. Biophys. Acta 791, 265-27 3. Inoue, Y., & Furuya, M. (1985) Plant Cell Physiol. 26, 8 13-8 19. Inoue, Y., Konomi, K., & Furuya, M. (1982) Plant Cell Physiol. 23, 731-736. Jabben, M., Heihoff, K., Braslavsky, S. E., & Schaffner, K. (1984) Photochem. Photobiol. 40, 361-367. Kelly, J. M., & Lagarias, J. C. (1985) Biochemistry 24, 6003-6010. Kendrick, R. E., & de Kok, J. (1983) Sci. Prog. (Oxford) 68, 475-486. Kwart, H. (1982) Acc. Chem. Res. 15, 401-408. Lachish, U., Shafferman, A,, & Stein, G. (1976) J . Chem. Phys. 64, 4205-421 1. Land, E. J. (1979) Photochem. Photobiol. 29, 483-487. Linschitz, H., & Kasche, V. (1967) Proc. Natl. Acad. Sci. U.S.A.58, 1059-1064.
Linschitz, H., Kasche, V., Butler, W. L., & Siegelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403. Matta, M. S., & Andracki, M. E. (1985) J. Am. Chem. SOC. 107, 6036-6039. Moon, D. K., Jeen, G. S., & Song, P. S. (1985) Photochem. Photobiol. 42, 633-641. Neumann, M. G., Matthews, J. I., & Braslavsky, S. E. (1984) Photochem. Photobiol. 39, 3 1-36. Pratt, L. H., & Butler, W. L. (1970) Photochem. Photobiol. 11, 361-369. Pratt, L. H., Shimazaki, Y., Inoue, Y . , & Furuya, M. (1982) Photochem. Photobiol. 36, 471-477. Pratt, L. H., Inoue, Y., & Furuya, M. (1984) Photochem. Photobiol. 39, 241-246. Ruzsicska, B. P., Braslavsky, S. E., & Schaffner, K. (1985) Photochem. Photobiol. 41, 681-688. Shimazaki, Y., Inoue, Y., Yamamoto, K. T., & Furuya, M. (1980) Plant Cell Physiol. 21, 1619-1625. Swain, C. G., & Bader, R. F. W. (1960) Tetrahedron 10, 182-199. Thiimmler, F., & Riidiger, W. (1983) Tetrahedron 39, 1943-195 1.
Study of 124-Kilodalton Oat Phytochrome Photoconversions in Vitro with Laser-Induced Optoacoustic Spectroscopyt Klaus Heihoff, Silvia E. BraSlavsky,* and Kurt Schaffner Max-Planck-Institut fur Strahlenchemie, 0-4330 Mulheim an der Ruhr, West Germany Received June 19, 1986; Revised Manuscript Received November 5, 1986
(P,) of 124-kDa oat phytochrome were studied by laser-induced optoacoustic spectroscopy. The use of poly(viny1idene fluoride) foil as a broad-frequency-band piezoelectric detector permitted the time-resolved measurement of the pressure wave produced after the absorption of the 580-, 660-, and 695-nm laser flashes. The prompt heat dissipation is the same for 124- and 60-kDa P, when determined for the same transit time of the acoustic wave across the laser beam radius. This supports the concept that the primary photochemical process is confined to the tetrapyrrole chromophore. A t shortest possible observation time (which minimizes transient contributions) 150% of the absorbed light energy is released as heat, and the quantum yield of the phototransformation of P, to the first set of intermediates (I$9o)is 250%. These data correct earlier values obtained from the heat dissipation a t longer observation times. ABSTRACT: The radiationless deactivation processes undergone by the excited red-absorbing form
previous studies of the fluorescence lifetimes of the P,‘ form of phytochrome (Holzwarth et al., 1984; Wendler et al., 1984) and the kinetic behavior of the two primary photoproducts (the I$oo’s),which arise in the transformationpf PI into the P, form [see the preceding paper by Aramendia et al. (1987) and references cited therein] ,2 have been carried out with oat phytochrome preparations of various molecular sizes (“small” = 60 kDa, “large” = 1141118 kDa, and “native” = 124 kDa). The results indicated that the transformations upon irradiation of PI, affording the 1300’sand the next set of intermediates (I:,) (Kendrick & de Kok, 1983), are mainly restricted to the bilitriene chromophore and are not affected by the molecular This work was supported by a fellowship award to K.H. from the Alfried Krupp von Bohlen und Halbach Stiftung. * Correspondence should be addressed to this author.
0006-2960/87/0426-1422$01.50/0
weight of the protein (in the range 1 6 0 kDa). The dissipation of the excitation energy is mainly governed by radiationless processes, since the fluorescence quantum yield of P, is very low (for the 124-kDa form: q5f = 2.9 X Holzwarth et al., 1984) and the quantum yield for the Pr PfI transformation has a maximum value of 0.15 [Kelly & Lagarias, 1985; see also Holzwarth et al. (1984)l. These
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I Abbreviations: I&, (set of) first intermediates formed from photoexcited P,; Iil, (set of) intermediates into which the I;w’s are transformed in a ground-state reaction; LIOAS, laser-induced optoacoustic spectroscopy; P, and PI,, red and far-red light absorbing forms of phytochrome, respectively; PVF2, poly(viny1idene fluoride) (foil detector); PZT, Pb-Zr-Ti (ceramic detector); T ~ acoustic , transit time; /,, observed acoustic transit time. The set of first intermediates can be composed of more than two 1:”s; e.g., Furuya (1983) has found that three such photoproducts are formed from pea P,.
0 1987 American Chemical Society
VOL. 26, NO. 5, 1987
OPTOACOUSTIC S T U D Y OF PHYTOCHROME PHOTOCONVERSIONS
radiationless processes have been studied for 60-kDa phytochrome by LIOAS in order to understand the first light-driven steps in the photoconversion P, PfI (Jabben et al., 1984). The transducer then used to detect the acoustic wave (generated by the heat evolved in the sample) was a PZT resonant piezoelectric ceramics. Although this detector did not follow the course of the pressure signal in real time, the action spectrum of the heat emitted by the phytochrome solutions sufficed for Jabben et al. (1984) to demonstrate that a photoequilibrium between P, and the I$,,,’S [for its first observation on a slower time scale, see Pratt et al. (198411 is established within the duration of the 15-11s laser flash [for in vivo systems, see Inoue and Furuya (1985) and Scheuerlein and Braslavsky (1985)l. From the same spectrum, an internal energy content of 140 kJ-mol-’ was calculated for the I$,, intermediates (see below for a revision). We describe now a study of the radiationless processes of 124-kDa PI by LIOAS using a broad-band PVF, detection system, which permits a time-resolved measurement of the heat dissipation on a real time scale. The results are compared with those previously obtained with 60-kDa P,. The new method of time-resolved LIOAS is applied here to a biological system of considerable molecular complexity. Its reliability in the determination of transit lifetimes has been satisfactorily established in a study of the known properties of a more simple molecule (Heihoff, 1986; Heihoff & Braslavsky, 1986).
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MATERIALS AND METHODS For the isolation and characterization of 124-kDa P,, see Brock et al. (1987). The temperature of the sample solutions was kept constant at 296 f 1 K for all measurements. The photoreversibility and molecular weight of phytochrome remained unchanged throughout the experiments, as ascertained before and after each measurement by back-irradiation (Pf, PI:XexC1 700, 3-mm RG-9 filter from Schott. P, Pf,: Xexc = 660 nm, interference filter from Schott) and absorption spectroscopy and by discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis [cf. Laemmli (1970)]. For the LIOAS measurements, CuC12.2H20 (Merck z.A.) was used as a reference (Braslavsky et al., 1983), dissolved either in 25% ethylene glycol, 0.1 M tris(hydroxymethy1)aminomethane hydrochloride buffer, and 5 mM sodium ethylenediaminetetraacetate (pH 7.8) or only in a 25:75 (v/v) ethylene glycol/water mixture. The LIOAS signals for CuC1, were the same in the presence and absence of the buffer. The concentrations of P, (< 10 pM)and CuCl, (< 10 mM) were small enough not to influence the thermoelastic properties of the solvents. The second harmonic of a Nd-YAG laser was used to pump a dye laser (JK Lasers, System 2000). The 15-11s laser flash was directed through a round diaphragm and focused (see below for the size of the focal point) into a 1-cm standard quartz cuvette. The flash energy was adjusted by neutral density filters (Schott) in the range from 20 to 200 pJ and measured by a pyroelectric energy meter (Rj-7100 and RjP735, Laser Precision Corp.). The dyes were Rhodamin B for 580 nm, DCM for 660 nm, and Pyridin 1 for 695 nm (Lambda Physik, Radiant Dyes Chemie). Continuous fiber optic background light (Oriel 77501 illumination system, with a 5-mm RG-9 filter of 715 nm) from the rear of the cuvette and a flash repetition rate of only 1/3 H z guaranteed that only P, was present when the laser flash was fired. The pressure pulse, created by the heat evolved after laser excitation, was detected by a piezoelectric PVF, foil (Kureha, Japan) directly attached to the external bottom side of the
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-
1423
oluminiu foil
stainless steel plexig lass aluminium teflon steel springs
ca. l c m
FIGURE 1: Essential parts of the PVF, detector, preamplifier, and attachment to the sample cuvette (A = first step of impedance converter). Note that the inner Plexiglass part with aluminum head is spring-loaded.
cuvette as shown in Figure 1. In contrast to the design used with the PZT transducers, the PVF, foil was not held inside a stainless steel housing in order to improve the sensitivity. The voltage signal was treated by a homemade impedance converter (50 Hz to 35 MHz) and fed into a preamplifier (2 Comlinear CLC-103, 20 or 40 dB, DC to 100 MHz), connected to a Biomation 8100 transient recorder (10-11s sample period). Signal averaging of 10-50 traces for each measurement and data evaluation were performed with a PDP 11/04-VAX 11/780 computer system. The 7, values (see Results and Discussion) were varied in the submicrosecond range by the use of laser beam diameters in the cuvette ranging from 60 to 1100 pm. The variation was achieved with different diameters of the pinholes and focal lengths of the lenses. By varying the distance of the laser beam to the detector (Patel & Tam, 1981), the sound velocity u, = 1465 ms-’ (f5%) was determined in the water/ethylene glycol (75:25) mixture (using CuC1, as an absorber). The integrated absorbance over the 15-ns laser flash was determined for Xexc = 660 and 695 nm as follows. The transmitted flash energies with buffer only and with a phytochrome solution were measured under the LIOAS geometrical conditions, at several different beam diameters, laser energies, and absolute absorbance values. The transmission (or absorbance) values were then compared with the absorbance measured in the spectrophotometer (Perkin-Elmer 356) before and after each experiment. RESULTSAND DISCUSSION LIOAS Signal Analysis. The voltage amplitude of the first signal deflection H was used as a measure of the pressure pulse created in the solution (Braslavsky et al., 1983; Jabben et al., 1984; Patel & Tam, 1981). Figure 2 shows the signal trace obtained for three different beam diameters (signal widths as indicated in the caption). For a dilute solution the amplitude H can be described by eq 1, where E, is the incident laser energy, A the absorbance H = KaEO(1 -
(1)
1424 B I O C H E M I S T R Y
HEIHOFF ET A L .
I
Benzophenone + KI
500
r rI
f -
300
2
.P cn 100
100
300
Beam radius R, p m
2
3
4
5
6
Time, ps 2: Voltage-time profiles obtained for a reference system (benzophenone + 5 3 mM KI in acetonitrile; Heihoff, 1986; Heihoff & Braslavsky, 1986) with the PVFz detector at laser beam diameters affording full widths at l / e of signal maximum of (A) =2.2 f i s , (B) 720 ns, and (C) 120 ns (individual amplitudes are arbitrary). XeXC = 355 nm.
FIGURE 3: Full width at l / e of the maximum signal (PVF2detector) for an ethanol solution of CuC12vs. beam radius (W) (broken line = W/u,, see text); Xcxc = 660 nm; A660= 0.14.
FIGURE
of the sample, and K a proportionality constant, including the thermoelastic properties of the solvent and the geometrical parameters (Bonch-Bruevich et al., 1977; Braslavsky et al., 1983; Jabben et al., 1984). Factor a is the fraction of the absorbed laser energy dissipated as prompt heat. The time scale of the prompt heat emission is governed by the transit time 7, (=R/v,, Le., the time that the sound pulse requires to traverse the laser beam radius; R = Gaussian laser beam radius in the sample, Le., l / e of signal maximum). For a short laser flash duration T~ ( T < ~ 7,;Lai & Young, 1982), all radiationless processes faster than 7, contribute to the prompt heat and are detected in the first signal amplitude H . When the broad-band PVF2 foil is used instead of the previous ceramic detector, the signal form is no longer governed by ringing and it reflects the real time course of the pressure wave (Tam & Coufal, 1983; Tam & Leung, 1984; Kuo & Patel, 1984; Kuo et al., 1984). The sensitivity of the time resolution can be appreciated from Figure 2, where three signals with different widths at l / e of signal maximum of a reference system are depicted. These signal differences were generated by using different laser beam diameters. The smaller signal appearing after the main signal C results from multiple reflections of the acoustic wave within the bottom wall of the cuvette (Heihoff, 1986). Since the evaluation of the lifetime of the heat-storing species is based on a logarithmic plot of the heat stored (Le., 1 - a) vs. transit time (Figure 8), the latter values now have to be more precise than in the previous study by Jabben et al. (1 984). For the reference CuCI2,the full width at 1/e of the signal maximum correlated with W as shown in Figure 3. Since this width i= 1.47W/v, ( W = 1/e2 of signal maximum), the lifetime of the species contributing to the prompt heat dissipation should, in our case, be
