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Photoresponse of Langmuir-Blodgett film containing bacteriorhodopsin
Photoresponse of Langmuir-Blodgett bacteriorhodopsin
film containing
J. R. Li, J. P. Wang, T. F. Chen and L. Jiang Instituie K.
of Photogruphic
S.
Institute
Chemistry,
Academiu
Sinico,
Beijing
100101 (Prople’,s
Republic
of China)
Hu, A. J. Wang and M. Q. Tan of
Biophysics,
Acudemiu
Sinica,
Be(jing
100080 (People ‘.s Rryddic
of Chino)
Abstract The photoresponse of the bacteriorhodopsin (BR) molecule in a purple membrane (PM) fragment LB film was investigated by pulsed laser excitation method. The initial results of laser flash photolysis carried out at room temperature indicated that the intermediate of the photochemical reaction is much the same as that of BR in the aqueous suspension of PM fragments. However the span of the appearance and disappearance of the intermediates for multilayers is different from that for the aqueous suspension.
1. Introduction It was reported [l] that pulsed laser excitation methods with nanosecond or picosecond time resolution can be used to detect the photoproduct of rhodopsin at physiological temperature. However the photoresponse behavior of BR, of interest in the molecular electronic research, especially in an organized assembly such as a LB film, has not been reported much. BR molecules constitute the only protein component of the PM [2] and function as light driven proton pumps [3]. BR molecules in the PM are organized into a two-dimensional hexagonal lattice of trimers which are surrounded by about 30 lipid molecules. The PM of Halobacterium halobium is a simple system for the conversion of light energy into a pH gradient across the cell membrane. On illumination, optically distinct intermediates K, L, M and 0 are known to appear [4]. In this work, PMs of H. halobium have been introduced into LB films of soybean phospholipid and the photoresponse of the PM fragment in the multilayer LB film was investigated with a camera photoflash lamp or laser flash photolysis.
2. Experimental PM fragments from N. halobium were isolated and purified as described by Oesterhet and Stoeckenius [5]. Soybean phospholipid was purified as previously described [6]. The PM fragment was directly added to hexane solution without sonication. Then the solution was spread on a subphase of distilled water (containing 0040-6090/92/$5.00
0.01 M KCl, pH 5.6). Pressure-area isotherms were measured by a Langmuir balance type HBM-ss made in Japan. Multilayer PM LB film was obtained by depositing the film from the air-subphase interface onto quartz plates or calcium fluoride plates with a hydrophilic surface at 25 mN/m. All films described here were a mixture of BR/soybean phospholipid with weight ratio of 5 to 1. The build up of films was checked by transfer ratio measurements. Deposition was carried out by vertical dipping. In most cases it was a Z-type deposition. The absorption spectra of multilayer PM LB films were measured with FTIR (Nicolet Model 20 Sx and HP 845 1A) spectrometers. Photoresponse behavior was examined by two methods. The first was to observe the decay of the intermediate with time. The other was to measure the whole spectrum of BR LB film with a Nd : YAG laser photolysis system (FWHM < 5 ns); the monitoring light source was a pulse xenon lamp (XF300) and a camera photoflash lamp with appropriate filter.
3. Results and discussion Figure for pure soybean soybean see that pholipid containing same for previous
1 shows the surface pressure-area isotherms soybean phospholipid ( 1) and PM mixed with phospholipid (2). The weight ratio of PM to phospholipid is 2: 1. From 7c- A curves we can the PM fragments mixed with soybean phoscan form a stable monolayer on a subphase 0.01 M KCl. The collapse pressure is the both films at 46 mN/m. It was reported in our work [7] that the orientation of BR in multi-
(‘ 1992
Elsevier Sequoia.
All rights
reserved
761
J. R. Li et al. / Photoresponse of LB film containing bacteriorhodopsin 0.02
i
4O
~'x
PM : Soyb~n ( weight ratio ) 0.01
"~.
......
2:1
\
2c
~o~ ( \
2b 3b 4b time ( ms )
-Y~ 4 ore(]
6
8
I0
Fig. 2. Flash-induced absorbance change of the intermediate M412. , the suspension of PM fragments; - - - , the PM fragments in LB film (100 layers).
cm'/.a.g soybeon phosphotipid I
-
Fig. 1. n A curves of pure soybean phospholipid and mixed soybean phospholipid with PM fragment.
layer LB film o f 60 layers m e a s u r e d by linear d i c h r o i s m has a preferential o r i e n t a t i o n with an angle 0 close to the value in native P M , a n d the p e a k shape a n d position (2 = 570 nm) o f P M LB film in visible a b s o r p t i o n spectra were the same as in native P M suspension. T a b l e 1 shows the frequency o f the p e a k s in the I R a b s o r p t i o n s p e c t r u m o f P M LB film (32 layers) a n d P M suspension. The results indicate t h a t the B R molecules did not tend to be d e n a t u r e d in o u r m u l t i l a y e r LB films. F i g u r e 2 shows the flash-induced a b s o r b a n c e c h a n g e o f the i n t e r m e d i a t e Mal 2. P h o t o l y s i s o f the P M fragm e n t in 100 layer LB film by the m o n i t o r i n g light was m i n i m i s e d by using short (10 3 s) exposures ( o b t a i n e d with a s y n c h r o n i s e d c a m e r a shutter) a n d c o m b i n a t i o n o f 2 = 568 n m glass filter. It has been f o u n d that the decay rate o f the M412 o f the P M f r a g m e n t in 100 layer LB film is slower t h a n that in a q u e o u s suspensions o f PM. F i g u r e 3 shows the transient a b s o r b a n c e s p e c t r u m after pulsed N d : Y A G laser excitation ()~ = 532 nm, 170 m J / c m 2) o f 100 layer P M LB film at r o o m temperature. D a t a were r e c o r d e d at 500 ns ( ), 9 5 0 n s ( .... ) a n d 5.4 ps ( . . . . . . . ); the angle ~ between the film p l a n e a n d the i l l u m i n a n t direction was 30 ° . T h r e e
500 ns 40£ ~'E._ 300
;~.
.....
950 ns
.......
5.4 ,~s
.~ 2O0 \ ' ~ .
~
IO0 450
5 0
5 0
600
650
X, Into )
Fig. 3. Transient absorbance spectrum after pulsed Nd:YAG laser excitation (532 nm, 170 mJ/cm 2) of PMLB film (100 layers) at room temperature. Delay time 500 ns ( ), 950 ns ( . . . . ), 5.4 ~ts ( . . . . . ).
transient a b s o r b a n c e p e a k s at 620, 550 a n d 420 n m were observed. It is similar to the characteristic s p e c t r u m o f p h o t o m e d i a t e r K625, L550 a n d M412 in the p h o t o reaction cycle in an a q u e o u s suspension o f P M at 77 K [8, 9]. The i n t e r m e d i a t e K decreases with increasing delay time whereas the i n t e r m e d i a t e M increases. The study showed that n o t only at low t e m p e r a t u r e , but also at r o o m t e m p e r a t u r e c o u l d p h o t o i n t e r m e d i a t e s be detected. But the times o f a p p e a r a n c e a n d d i s a p p e a r a n c e
TABLE 1. Frequency of peaks in IR absorption spectrum of purple membrane (in cm t)~ Amide I
Amide II
CH stretching vibration
N H stretching vibration
PMLB film
1659
1547
2952 (va(CH3)) 2921 (va(CH2)) 2869 (vs(CH3)) 2852 (vs(CH2))
3302
PM suspension
1658
1545
2957 (v"(CH 3)) 2926 (v"(CH2)) 2870 (vs(CH3))
3298
aThe substrates for all PM samples were CaF 2 plates. The transmission infrared ratio spectra (sample/blank) were recorded on a Nicolet Model 20 Sx Fourier transform spectrophotometer equipped with a TGS detector; 100 interferograms for PM suspension samples and 500 interferograms for PMLB film samples were collected at a resolution of 4 cm-L
762
J. R. Li et al. / Photore~ponse o f LB film containing bacteriorhodopsin
for intermediates K, L and M are different from those in the aqueous suspension. There appears to be a relatively longer lifetime for intermediates in a multilayer PM LB film. The results provide a new approach to monitor the photoresponse time of PM materials.
Acknowledgment We thank the National Natural Science Foundation of China (NSFC) for financial support.
4. Conclusions
References
The photoresponse of BR of PM has been detected at room temperature by a pulsed laser excitation method. The intermediates K, L and M can be observed in the PM LB film at room temperature. The lifetime of the photocycle intermediate is longer in a multilayer PM LB film than in an aqueous suspension of PM, providing a means to control the lifetime of the intermediate species and investigate the primary process in visual excitation.
1 2 3 4 5 6 7 8 9
Ch. R. Goldschmidt et al., Nature, 263 (1976) 169. J. Bridgen and I. D. Walker, Biochemistry, 15(1976) 792. E. Racker and W. Stoeckenius, J. Biol. Chem., 249 (1974) 662. W. Stoeckenius et al., Biochim. Biophys. Acta, 505 (1979) 2. D. Oesterhelt and W. Stoeckenius, Methods Enzymol., 31 (1974) 667. K. S. Hu et al., Acta Biophys. Sin., 5(1989) 241. A. J. Wang et al., Aeta Biophys. Sin., in press. M. Braiman and R. Mathies, Biochemistry, 19 (1980) 5421. S. O. Smith et al., J. Membr. Biol., 85 (1985) 95.
film containing
J. R. Li, J. P. Wang, T. F. Chen and L. Jiang Instituie K.
of Photogruphic
S.
Institute
Chemistry,
Academiu
Sinico,
Beijing
100101 (Prople’,s
Republic
of China)
Hu, A. J. Wang and M. Q. Tan of
Biophysics,
Acudemiu
Sinica,
Be(jing
100080 (People ‘.s Rryddic
of Chino)
Abstract The photoresponse of the bacteriorhodopsin (BR) molecule in a purple membrane (PM) fragment LB film was investigated by pulsed laser excitation method. The initial results of laser flash photolysis carried out at room temperature indicated that the intermediate of the photochemical reaction is much the same as that of BR in the aqueous suspension of PM fragments. However the span of the appearance and disappearance of the intermediates for multilayers is different from that for the aqueous suspension.
1. Introduction It was reported [l] that pulsed laser excitation methods with nanosecond or picosecond time resolution can be used to detect the photoproduct of rhodopsin at physiological temperature. However the photoresponse behavior of BR, of interest in the molecular electronic research, especially in an organized assembly such as a LB film, has not been reported much. BR molecules constitute the only protein component of the PM [2] and function as light driven proton pumps [3]. BR molecules in the PM are organized into a two-dimensional hexagonal lattice of trimers which are surrounded by about 30 lipid molecules. The PM of Halobacterium halobium is a simple system for the conversion of light energy into a pH gradient across the cell membrane. On illumination, optically distinct intermediates K, L, M and 0 are known to appear [4]. In this work, PMs of H. halobium have been introduced into LB films of soybean phospholipid and the photoresponse of the PM fragment in the multilayer LB film was investigated with a camera photoflash lamp or laser flash photolysis.
2. Experimental PM fragments from N. halobium were isolated and purified as described by Oesterhet and Stoeckenius [5]. Soybean phospholipid was purified as previously described [6]. The PM fragment was directly added to hexane solution without sonication. Then the solution was spread on a subphase of distilled water (containing 0040-6090/92/$5.00
0.01 M KCl, pH 5.6). Pressure-area isotherms were measured by a Langmuir balance type HBM-ss made in Japan. Multilayer PM LB film was obtained by depositing the film from the air-subphase interface onto quartz plates or calcium fluoride plates with a hydrophilic surface at 25 mN/m. All films described here were a mixture of BR/soybean phospholipid with weight ratio of 5 to 1. The build up of films was checked by transfer ratio measurements. Deposition was carried out by vertical dipping. In most cases it was a Z-type deposition. The absorption spectra of multilayer PM LB films were measured with FTIR (Nicolet Model 20 Sx and HP 845 1A) spectrometers. Photoresponse behavior was examined by two methods. The first was to observe the decay of the intermediate with time. The other was to measure the whole spectrum of BR LB film with a Nd : YAG laser photolysis system (FWHM < 5 ns); the monitoring light source was a pulse xenon lamp (XF300) and a camera photoflash lamp with appropriate filter.
3. Results and discussion Figure for pure soybean soybean see that pholipid containing same for previous
1 shows the surface pressure-area isotherms soybean phospholipid ( 1) and PM mixed with phospholipid (2). The weight ratio of PM to phospholipid is 2: 1. From 7c- A curves we can the PM fragments mixed with soybean phoscan form a stable monolayer on a subphase 0.01 M KCl. The collapse pressure is the both films at 46 mN/m. It was reported in our work [7] that the orientation of BR in multi-
(‘ 1992
Elsevier Sequoia.
All rights
reserved
761
J. R. Li et al. / Photoresponse of LB film containing bacteriorhodopsin 0.02
i
4O
~'x
PM : Soyb~n ( weight ratio ) 0.01
"~.
......
2:1
\
2c
~o~ ( \
2b 3b 4b time ( ms )
-Y~ 4 ore(]
6
8
I0
Fig. 2. Flash-induced absorbance change of the intermediate M412. , the suspension of PM fragments; - - - , the PM fragments in LB film (100 layers).
cm'/.a.g soybeon phosphotipid I
-
Fig. 1. n A curves of pure soybean phospholipid and mixed soybean phospholipid with PM fragment.
layer LB film o f 60 layers m e a s u r e d by linear d i c h r o i s m has a preferential o r i e n t a t i o n with an angle 0 close to the value in native P M , a n d the p e a k shape a n d position (2 = 570 nm) o f P M LB film in visible a b s o r p t i o n spectra were the same as in native P M suspension. T a b l e 1 shows the frequency o f the p e a k s in the I R a b s o r p t i o n s p e c t r u m o f P M LB film (32 layers) a n d P M suspension. The results indicate t h a t the B R molecules did not tend to be d e n a t u r e d in o u r m u l t i l a y e r LB films. F i g u r e 2 shows the flash-induced a b s o r b a n c e c h a n g e o f the i n t e r m e d i a t e Mal 2. P h o t o l y s i s o f the P M fragm e n t in 100 layer LB film by the m o n i t o r i n g light was m i n i m i s e d by using short (10 3 s) exposures ( o b t a i n e d with a s y n c h r o n i s e d c a m e r a shutter) a n d c o m b i n a t i o n o f 2 = 568 n m glass filter. It has been f o u n d that the decay rate o f the M412 o f the P M f r a g m e n t in 100 layer LB film is slower t h a n that in a q u e o u s suspensions o f PM. F i g u r e 3 shows the transient a b s o r b a n c e s p e c t r u m after pulsed N d : Y A G laser excitation ()~ = 532 nm, 170 m J / c m 2) o f 100 layer P M LB film at r o o m temperature. D a t a were r e c o r d e d at 500 ns ( ), 9 5 0 n s ( .... ) a n d 5.4 ps ( . . . . . . . ); the angle ~ between the film p l a n e a n d the i l l u m i n a n t direction was 30 ° . T h r e e
500 ns 40£ ~'E._ 300
;~.
.....
950 ns
.......
5.4 ,~s
.~ 2O0 \ ' ~ .
~
IO0 450
5 0
5 0
600
650
X, Into )
Fig. 3. Transient absorbance spectrum after pulsed Nd:YAG laser excitation (532 nm, 170 mJ/cm 2) of PMLB film (100 layers) at room temperature. Delay time 500 ns ( ), 950 ns ( . . . . ), 5.4 ~ts ( . . . . . ).
transient a b s o r b a n c e p e a k s at 620, 550 a n d 420 n m were observed. It is similar to the characteristic s p e c t r u m o f p h o t o m e d i a t e r K625, L550 a n d M412 in the p h o t o reaction cycle in an a q u e o u s suspension o f P M at 77 K [8, 9]. The i n t e r m e d i a t e K decreases with increasing delay time whereas the i n t e r m e d i a t e M increases. The study showed that n o t only at low t e m p e r a t u r e , but also at r o o m t e m p e r a t u r e c o u l d p h o t o i n t e r m e d i a t e s be detected. But the times o f a p p e a r a n c e a n d d i s a p p e a r a n c e
TABLE 1. Frequency of peaks in IR absorption spectrum of purple membrane (in cm t)~ Amide I
Amide II
CH stretching vibration
N H stretching vibration
PMLB film
1659
1547
2952 (va(CH3)) 2921 (va(CH2)) 2869 (vs(CH3)) 2852 (vs(CH2))
3302
PM suspension
1658
1545
2957 (v"(CH 3)) 2926 (v"(CH2)) 2870 (vs(CH3))
3298
aThe substrates for all PM samples were CaF 2 plates. The transmission infrared ratio spectra (sample/blank) were recorded on a Nicolet Model 20 Sx Fourier transform spectrophotometer equipped with a TGS detector; 100 interferograms for PM suspension samples and 500 interferograms for PMLB film samples were collected at a resolution of 4 cm-L
762
J. R. Li et al. / Photore~ponse o f LB film containing bacteriorhodopsin
for intermediates K, L and M are different from those in the aqueous suspension. There appears to be a relatively longer lifetime for intermediates in a multilayer PM LB film. The results provide a new approach to monitor the photoresponse time of PM materials.
Acknowledgment We thank the National Natural Science Foundation of China (NSFC) for financial support.
4. Conclusions
References
The photoresponse of BR of PM has been detected at room temperature by a pulsed laser excitation method. The intermediates K, L and M can be observed in the PM LB film at room temperature. The lifetime of the photocycle intermediate is longer in a multilayer PM LB film than in an aqueous suspension of PM, providing a means to control the lifetime of the intermediate species and investigate the primary process in visual excitation.
1 2 3 4 5 6 7 8 9
Ch. R. Goldschmidt et al., Nature, 263 (1976) 169. J. Bridgen and I. D. Walker, Biochemistry, 15(1976) 792. E. Racker and W. Stoeckenius, J. Biol. Chem., 249 (1974) 662. W. Stoeckenius et al., Biochim. Biophys. Acta, 505 (1979) 2. D. Oesterhelt and W. Stoeckenius, Methods Enzymol., 31 (1974) 667. K. S. Hu et al., Acta Biophys. Sin., 5(1989) 241. A. J. Wang et al., Aeta Biophys. Sin., in press. M. Braiman and R. Mathies, Biochemistry, 19 (1980) 5421. S. O. Smith et al., J. Membr. Biol., 85 (1985) 95.