Photochemical reactions of bacteriorhodopsin in ...

Photochemical reactions of bacteriorhodopsin in Triton X-100 solution studied by low temperature spectrophotometry' TATSUOIWASAA N D FUMIOTOKUNAGA Department of Physics, Faculty of Science, Tohoku Utiiversity, Serzdai 980, Japan AND

TORUYOSHIZAWA? Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan Received August 16, 1984 This paper is dedicated to Professor Camille Sandorfy on the occasion of his 65th birthday

Can. J. Chem. Downloaded from www.nrcresearchpress.com by 121.12.120.246 on 08/26/15 For personal use only.

TATSUO IWASA, FUMIO TOKUNAGA, and TORUYOSHIZAWA. Can. J. Chem. 63, 189 1 (1985). The photochemical reaction of purple membrane solubilized with Triton X- 100 (T-BR) was investigated by low temperature spectrophotometry. The batho- and meta-intermediates of T-BR were observed to resemble bacteriorhodopsin in native purple membrane. Two photoproducts characteristic of the T-BR system were found, which were named the "490-nm complex" and the "380-nm complex". The 490-nm complex was in thermal equilibrium with T-BR in the dark. Cooling T-BR to low temperature favoured the 490-nm complex, which was photoinsensitive. On the other hand, the 380-nm complex was produced by warming the batho-intermediate and reverted to the original T-BR. The meta-intermediate of T-BR may possibly be in thermal equilibrium with the 380-nm complex. On the basis of the above results, the possible role of the membrane structure was discussed.

TATSUO IWASA, FUMIO TOKUNAGA et TORUYOSHIZAWA. Can. J. Chem. 63, 1891 (1985). Faisant appel a la spectrophotometrie a basse temperature, on a Ctudit la reaction photochimique de la membrane pourpre en solution dans le Triton X-100 (T-BR). Par example, on a observe les intermkdiaires batho et mCta du T-BR, comme la bactCriorhodospine dans la membrane pourpre naturelle. On a trouvC deux produits photochimiques qui sont caracteristiques du systkme T-BR; on leur a donnC les noms de "complexe 490 nm" et "complexe 380 nm". Dans I'obscuritC, le complexe 490 nm est en Cquilibre thermique avec T-BR. Le refroidissement du T-BR favorise le complexe 490 nm qui est photosensible. Par ailleurs, le complexe 380 nm est obtenu en chauffant I'intermCdiaire batho et il se retransfornle en T-BR original. I1 est possible que I'intermCdiaire mCta du T-BR soit en Cquilibre thermique avec le complexe 380 nm. A partir de ces rksultats, on discute du r6le possible de la structure de la membrane. [Traduit par le journal]

Introduction Bacteriorhodopsin (BR) is the sole protein in the purple membrane of Halobacterium halobium (1). Like animal rhodopsin, BR has a retinal as its chromophore which binds to the €-amino group of lysine residue of the protein moiety through a Schiff base linkage (2, 3). The retinylidene chromophore of light-adapted BR (BRL) is in an all-trans form. On absorption of light, BRL (trans-bR) converts to several intermediates such as batho- (K), lumi- (L), and meta-trans-BR (M) and eventually reverts to the original pigment (4, 5). This photochemical cycle forms an electrochemical gradient of protons across the membrane (1). According to an electron diffraction analysis, BR molecules form a trirner structure alignec in a hexagonal lattice with the lattice distance of about 62 A (6). The retinylidene chromophores of BR in the trimer exhibit a typical circular dichroic (cd) band owing to an exciton coupling. The purple membrane solubilized with Triton X-100 (T-BR), however, displays no exciton cd band because of dissociation of the trimer structure, though it has a small positive cd band due to an intrinsic one (7, 8). This indicates that T-BR should be in monomeric form (9). Now the question is whether or not such a lattice structure has any physiological meaning. To answer this question, we have tried to investigate the photoreaction of T-BR. Irradiation ' A part of this work was presented at the U.S.-Japan Cooperative Seminar on "The purple membrane of Halobacteria" held in May 1980 in Japan. ' ~ u t h o rto whom all correspondence should be addressed.

of T-BR produced photointermediates, similar to the bathoand meta-intermediates in purple membrane, but the metaintermediate of T-BR was more stable than that in purple membrane. We also found two new molecular species named the "490-nm complex" and the "380-nm complex", which are characteristic of the T-BR system.

Materials and methods The purple membrane was prepared from Halobacteriunz halobium R I by a slight modification (5) of the method described by Oesterhelt and Stoeckenius ( l o ) , and suspended in 10 mM phosphate buffer (pH 6.8). T o solubilize the membrane, a Triton X-100 solution (10% v/v) was mixed with the membrane suspension to give a final concentration of 1% (v/v) (about lo3 moles/mole BR). The mixture (16 p M BR) was used for spectroscopic and cd measurements at room temperature. The cd spectra were measured with a recording spectropolarimeter (Jasco 5-20), An optical cell containing the sample (10-mm light path; I OD) was placed close to the window of the detector to reduce the scattering of the measuring light. For low temperature spectrophotometry, glycerol in a final concentration of 75% (v/v) was added to the mixture (160 p M BR, 50- 100 moles of Trixon X-100/mole BR) and a recording spectrophotometer (Hitachi 323, 320 or Shimadzu MPS 5000) was equipped with a cryostat described by Yoshizawa ( I I). The sample was placed in an optical cell composed of two quartz plates and a rubber ring (1or 2-mm light path; 1 OD) under dim red light and incubated in the dark at least for 8 h to be fully dark-adapted. The temperature of the sample was monitored continuously with a copper vs. constantan thermocouple attached to the sample holder made of copper. A 500-W xenon lamp (Ushio) was used as a light source for irradiation of the sample. The wavelength of the actinic light was selected by a combination of interference and (or) cut-off filters (Toshiba).


CAN. J . CHEM. VOL. 63, 1985

Wavelength (nm) FIG. 1. (a) Photoreaction of T - B R ~at 20°C. Curve I: T - B R ~(--lo3 moles of Triton X-100/mole BR). Curves 2- 10: products of the successive irradiation of T - B R ~at 500 nm for 10, 10, 20, 40, 80, 160, 320, 640, and 1280 s, respectively. Curve 10 is a mixture of T-BRL and the 380-nm complex. (b) Dark-reversion at 20°C. Curve 1: the mixture redrawn from curve 10 in (a). Curves 2-7: products of the successive dark incubation of the mixture. The spectra were measured at 5 , 15, 40, 90, 585, and 955 rnin from the beginning of the dark incubation, respectively. Curve 8: a product of the dark incubation for more than 30 h. The extraction of the chromophoric retlnal from BR or T-BR was performed according to Tsuda et al., (12). The sample (nearly 1 OD) was mixed with an equivolume of CHrClz and the mixture was sonicated for a total of 5 min in an ice bath. After addition of 3 volumes of petroleum ether, 10-20 pL of the upper phase was injected to the hplc system (Jasco Tri-Rotar 11 with porous silica gel SS-05 column). All procedures were carried out at O°C under dim red light and repeated 3 times.

Results Photoreaction of dark-adapted T-BR and its thermal reversion; appearance of the "380-nm complex" Upon addition of Triton X-100 to dark-adapted BR (BRD, A,,: 560 nm) the scattering of the measuring light decreased within 1 min, and the A,, shifted to shorter wavelength (about 10 nm) within 7 min (data not shown), probably owing to some destruction of the membrane structure. The time course of these changes depends on the molecular ratio of Triton X-100 to BR molecules (13). The dark-adapted T-BR ( T - B R ~ thus ) obtained is shown in Fig. l a as curve 1. An irradiation of T-BRDwith 500-nm light for 40 s at 20°C caused a bathochromic shift of the A,, from 550 nm to 560 nm with a small decrease of absorbance at the A,, (curves 2-4 in Fig. la). Since this spectral change can be

regarded as that corresponding to the light-adaptation of the native BR, the product was designated as T-BRL. Further irradiation with the same light caused a decrease of the absorbance around the A,, and an increase in the wavelength region shorter than 440 nm, resulting in formation of a photoproduct having A,, at 380 nm. Since the A,, is located at a wavelength about 10 nm longer than that of free all-trans retinal, the chromophore seems to attach to the protein moiety. It is plausible that the chromophore may be located in the binding site, because the subsequent dark incubation slowly recovered the absorbance at 570 nm, with disappearance of the peak at 380 nm (Fig. 1b). Therefore, we designated the product as the "380-nm complex". This slow "dark-reversion" is composed of two sequential reaction processes; one is the reversion from the 380-nm complex to T - B R ~and the other is the dark-adaptation of T-BRL to T-BRD.The reason is that the spectrum of the early stage in the spectral change (curve 3 - curve 1 in Fig. lb) was similar in shape to that between the 380-nm complex and T-BRL (curve 10 - curve 7 in Fig. la), indicating that the 380-nm complex reverted to T-BR1-. Also, the difference spectrum of the later stage (curve 7 - curve 4 in Fig. lb) displayed a different shape from that of the early stage; the former showed the maximum in the shorter wavelength region and a smaller ratio of absorb-


lWASA ET AL.

Wavelength (nm) FIG. 2. Cooling effect on spectra of T-BR" and T-BRL.(a) T-BR' (- lo3 moles of Triton X- 100/mole BR). Curve 1 : 0°C. Curve 2: - 190°C. (b) T-BRL(- 10' moles of Triton X-100/mole BR). Curve 1: 0°C. Curve 2: - 190°C. (c) Spectral change of T-BRLon warming from - 190°C

to 0°C. Curves 1-7 were measured at -30, -25, -20,

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15, - 10, -5, and O0C, respectively.

ance between the maximum and minimum than the latter. In addition, the semi-log plot of the absorbance change at 550 nm against the incubation time did not display a single exponential curve but, rather, the kinetics composed of two sequential thermal reactions, i.e. dark-reversion of the 380-nm complex to T - B R ~and dark-adaptation of T-BRL to T-BRD. The absorption spectrum of the final product of the dark-reversion (curve 8 in Fig. lb) was smaller in absorbance than that of the initial one (curve 1 in Fig. la), presumably due to the destruction of the pigment during the slow dark-reversion.

Cooling effrcts on spectra of T-BRDand T-BRL;appearance of the "490-nm complex" On cooling T-BRD to - 190°C, a shoulder around 490 nm appeared in the absorption spectrum (Fig. 2a). Upon warming to 0°C in the dark, the spectrum recovered its original shape. This spectral change was perfectly reversible. A similar experiment was performed on T-BRLwhich had been prepared by irradiating T-BRDwith 500-nm light for 5 min at 0°C (Fig. 2b). It should be noted that the shoulder of T - B R ~at - 190°C was more prominent than that of T - B R ~(Figs. 2a and b). On

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FIG. 3. Absorption spectra of mixtures composed of T-BRL, 380-nm complex, and 490-nm complex. (a) Formation of the 380-nm complex by irradiation of T-BR~(-10' moles of Triton X-100/mole BR) (curve 1) with 500-nm light for 17, 40, and 70 min (curves 2-4), respectively. (b) Low temperature spectra of the products. Each product was cooled to -190°C and then spectra were measured. Curves 2' and 4' show low temperature spectra of the products designated as curves 2 and 4 in (a), respectively. warming T-BRL above -30"C, the absorbance at the shoulder decreased, with formation of an isosbestic point at 505 nm (Fig. 2c). This result indicates the presence of a thermal equilibrium between T-BRL and a molecular species named the "490-nm complex"; low temperatures favoured the 490-nm complex. A similar spectral change was observed in warming T-BRD from - 190°C. The amount of 490-nm complex in the light-adapted sample was more than in dark-adapted one. The amount of 490-nm complex on cooling depends on the molar ratio of Triton X-100 to BR; the larger the ratio, the more 490-nm complex is formed. A possibility that the 380-nm complex might convert to the 490-nm complex on cooling was excluded by the following experiment: T-BRL was irradiated at 0°C for given periods in order to prepare the sample containing different amounts of 380-nm complex (Fig. 3a), and then their spectra were measured at - 190°C. As shown in Fig. 3b, the absorption band characteristic of the 380-nm complex shifted to around 400 nm at - 190°C. Thus it is clear that the 380-nm complex can not convert to the 490-nm complex on cooling. Photoreactions a t - 190°C; formation of batho-intermediates The photoreaction of T-BR was first examined by use of the sample shown in Figs. 2 and 3. The spectral changes of T-BRL in the sample at - 190°C were very small because of the large amount of 490-nm complex, which was photo-insensitive or less photosensitive (see below), present in the sample. In order to reduce the amount of 490-nm complex in the sample, a lesser amount of Triton X-100 was added to the purple membrane

suspension (about 50 moles of Triton X-100/mole BR). As shown in Figs. 4 and 5, the amount of 490-nm complex in the sample was about 114 of that in Figs. 2 and 3. The shoulder around 520 nm seen in Figs. 4 and 5 was due to non-flatness of the baseline. It was confirmed that the shape of spectral change of photoreactions of T-BR at - 190°C was independent of the molar ratio of Triton X-100 to BR, except for the amount of the spectral change. Now the sample containing T-BRDwas irradiated at - 190°C with light at 500 nm (curve 1 in Fig. 4a). The spectrum shifted to longer wavelengths. The absorbances in the range between 410 nm and 563 nm decreased and simultaneously those at longer wavelengths than 563 nm increased (curves 2-8 in Fig. 4a), indicating formation of a bathochromic intermediate (T-batho-BRD). A prolonged irradiation yielded a photosteadystate mixture (curves 7 and 8 in Fig. 4a) designated as T-stateD. Then, another irradiation of T-stateD with red light at 718 nm, which was absorbed by mainly T-batho-BRD,caused it to revert to T-BRD (Fig. 4b). During the irradiation, the curveintersection point of the spectra shifted from 585 nm to 550 nm (curves 1 - 10). On further irradiation with light of wavelengths longer than 640 nm (>640 nm), all the T-batho-BRD reverted to the original T-BRD (curve I I). In the case of irradiation of BRD in purple membrane at -190°C, two kinds of batho-intermediates, namely bathotrans-BR and batho-13-cis-BR, were formed (14), because BRD in purple membrane is an equimolar mixture of trans-BR and 13-cis-BR (15, 16). The same was true in the T-BRD system, because the hplc analysis of retinal isomers extracted from T-BRDrevealed that T-BRDwas a mixture of T-BR' (5 1.3 ? 3%) and T-BRI3 (48.7 ? 3%), though the spectral change did not display a clear distinction between the photoreaction due to trans-pigment (T-BR1) and that due to 13-cis-pigment (T-BR"). ' In order to prepare T-BRL, the T-BRD(curve 11 in Fig. 4b) was warmed to 0°C and then irradiated with 500-nm light for 5 min. After cooling it to - 190°C, the absorption spectrum was measured, which displayed ,A at 568 nm and a shoulder due to the 490-nm complex (curve 1 in Fig. 5a). Irradiation of the preparation with 500-nm light caused a bathochromic shift with an isosbestic point at 584 nm (curves 2-7 in Fig. 5a), and finally reached a photosteady state, designated as T-stateL (curves 6 and 7 in Fig. 5a). Irradiation of T-stateL (curve 1 in Fig. 5b) with light at 7 18 nm caused a photoreversion to T - B R ~ (curves 2-7 in Fig. 5b). For the complete reversion of the batho products, another irradiation with light of shorter wavelengths (>640 nm) was required. The spectrum of the final photoproduct of the irradiation (curve 8 in Fig. 5b) was in good agreement with the original T-BRL (curve 1 in Fig. 5a). Thus, T-BRL showed a photoreversible change at - 190°C, like BRL in purple membrane. The curve-intersection points among a series of spectra, however, shifted from 590 nm to 570 nm (Fig. 5b), suggesting that T-BRL would be a mixture of T-BR' and T-BRI3. In fact, the hplc analysis of retinal isomers extracted from T-BRL demonstrated that the amount of 13-cis retinal (13.0 ? 2% of total amount) in T-BRLwas pretty large in comparison with that in BRL (8.9 ? 2% of the total amount). A question whether or not the 490-nm complex might be photosensitive was examined. The preparations containing the 490-nm complex (-lo3 or -50 moles of Triton X-loo/ mole BR) were irradiated at - 190°C with various lights of wavelengths shorter than 500 nm. Their spectral changes due to the photoreactions were calculated, and were similar to


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FIG.4. Photoreaction of T-BRDat - 190°C. (a) Curve 1: T-BRD(-50 moles of Triton X-100/mole BR) at - 190°C. Curves 2-8: products of the successive irradiations of T - B R ~with 500-nm light for 5, 5, 10, 20, 40, 80, and 160 s, respectively. Curves 7 and 8 correspond to photosteady-state mixture designated as state^. (b) Curve I : p state^ redrawn from curve 8 in (a). Curves 2- 10: products of the successive irradiations of T-stateDwith light at 71 8 nm for 10, 10, 20, 40, 80, 160, 320, 640, and 1280 s, respectively. Curve 1 1 was a product of further irradiation with red light (>640 nm) for 5 min, and in good agreement with curve I in (a). those shown in Fig. 5. This fact indicates that the 490-nm complex at -190°C is photo-insensitive or has very low photosensitivity. Photoreaction at -50°C; formation of the meta-intermediate Irradiation of T-BRLat -50°C with orange light (>580 nm) yielded a meta-like intermediate designated as T-meta-BR', which was stable at -50°C in the dark; a prolonged irradiation converted all the T-BRL into T-meta-BRL(curve 1 in Fig. 6a). The formation of T-meta-BRL is composed of fast and slow phases. These results indicate that at -50°C the 490-nm complex slowly converted to T-BRL in the dark. It should be noted that meta-BR' (A,,,: 418 nm) in the purple membrane is not stable at -50°C under the same conditions (10 mM phosphate buffer, pH 6.8) (5). The thermal conversion of T-meta-BR' consisted of three elementary steps (Fig. 6). Warming from -50°C to -45°C

decreased the absorbances around 410 nm and simultaneously increased those around 565 nm, forming an isosbestic point at 458 nm (curves 1-3 in Fig. 6a and circles in Fig. 6b). The difference spectrum between curves 3 and 1 is similar to that between meta-BR' and BRL (5) except for the fact that the wavelengths of the maximum and minimum in the difference spectrum and the isosbestic point are shorter by about 10 nrn than those in the BR' system. This shift is comparable to the shift of A,, on addition of Triton X-100. Therefore, this spectral change should be regarded as a decay of T-meta-BR' to T-BRL. On warming from -45°C to -35°C the absorbance around 380 nm decreased and that around 565 nm increased (curves 3-5 in Fig. 6a and triangles in Fig. 6b). The difference spectrum of this spectral change was similar in shape to that between the 380-nm complex and T-BRLshown in Fig. lb. In both spectra the isosbestic points were located at about 445 nm,


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Wavelength (nm) FIG. 5. The photoreaction of T-BRLat - 190°C. (a) Curve 1: T-BRL (-50 moles of Triton X-100/mole BR) at - 190°C. Curves 2-7: products of the successive irradiations of T-BRL with 500-nm light for 10, 10, 15, 20, 40, and 80 s, respectively. Curves 6 and 7 represent a photosteady-state mixture designated as ph state^. (b) Curve 1: state^ redrawn from curve 7 in (a). Curves 2-7: products of the successive irradiations of T-stateL (curve 1) with 718-nm light for 10, 10, 20, 40, 80, and 160 s, respectively. Curve 8: a product of the further irradiation with red light (>640 nm) for 5 min. Curve 8 is in good agreement with curve 1 in (a).

and the ratios in absorbance between the difference maximum and minimum were 0.37. These facts indicate that the spectral change may correspond to a conversion of the 380-nm complex to T-BRL. It is not clear yet whether the 380-nm complex may be formed from T-meta-BR1or from other earlier intermediates. Above -25"C, the absorbance around 460 nm decreased and that around 565 nm increased (curves 7- 12 in Fig. 6a and squares in Fig. 6b). This spectral change should be due to the decay of the 490-nm complex, because both the shape of the spectral change and the temperature range in which it was observed were in good agreement with those of the change of 490-nm complx to T-BRL (Fig. 2c).

Discussion We have described several properties of the T-BR system different from the BR system: the formation of the 490-nm and 380-nm complexes, and the stabilization of meta-intermediates. Needless to say, these differences can be attributed to the difference in the environment around the BR molecule. BR molecules in the purple membrane are in the lattice structure and form a "trimer" structure. The molecules in the trimer interact with each other as demonstrated in the energy transfer (17), the exciton cd band (7, 8), and the rate of dark-adaptation (18). According to the cd measurements, the solubilization of purple membrane with a detergent causes the lattice structure to dissociate, resulting in loss of the interaction among the mole-

IWASA ET AL.

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FIG. 7. A reaction cycle of T-BR. Trans-BR and 13-cis-BR in Triton X-100 solution, designated as T-BR' and T-BR", respectively, formed batho-intermediates,i.e., T-batho-BR'and T-batho-BR". The meta-intermediate was formed only from T-BR'. Whether or not a lumi-like intermediate was formed has not yet been clarified, because the spectral complexity due to formation of the 490-nm complex. The 490-nrn and 380-nm complexes are both characteristic of the T-BR photocycle.

by destruction of the Schiff base linkage, but by deprotonation of the Schiff base by some change of the microenvironment around the chromophore.

Wavelength (nm) FIG.6. (a) The thermal decay of T-meta-BR' obtained by irradiation of T-BRLat -50°C for 70 rnin wlth orange light (>580 nrn). T-meta-BR' was gradually warmed in the dark. The spectra were measured at -50, -48, -45°C (curves 1 , 2, and 3) and then at intervals of 5°C (curves 4- 12). (b) Circles (0-0): the difference spectrum between the spectra measured at -45 and -50°C (3 - 1). Triangles (A-A): the difference spectrum between -35 and -45°C (5 - 3). Squares (m-m): the difference spectrum between 0 and -25°C (12 - 7).

cules. An ultra-centrifugation experiment (9) showed that BR molecules in 2% Triton X-100 solution, probably in a micelle composed of Triton X- 100 and several lipids, were in a monomeric form. Thus, the properties of T-BR different from the BR system mentioned above (see Fig. 7) should be attributed to some differences in lipid-protein, protein-protein, and detergent-protein interactions. The 380-nm complex The irradiation of T-BR brought an increase of absorbance at 380 nm (refs. 18, 19, and the present work). If the formation of the 380-nm complex was an indication of destruction of the Schiff base linkage between retinal and the €-amino group of the lysine residue, the dark recovery would be a regeneration of T-BR from all-trans retinal and bacterioopsin. In order to test this possibility, all-trans retinal was mixed with bacterioopsin in 1% Triton X-100 solution. No pigment formation was observed. Thus, the 380-nm complex is a product formed not

The 490-nm conzplex The 490-nrn complex was formed only by cooling to a temperature lower than 0°C. Its amount increased with decrease of temperature and reached a steady level at -40°C. The spectral change on cooling may possibly be due to change of pK value of a certain charged group or the phase transition of the glycerol-Triton-lipid rnicelle containing BR molecules. We did not observe a molecular species similar to the 490-nm complex at 25OC in the pH range between 2.96 and 10.65. Below pH 4 a decrease of absorbance at A,,, an increase at longer wavelengths (-650 nrn), and an increase at shorter wavelengths (-360 nm) were observed. At pH higher than 9, a decrease of the absorbance at X,,,, and an increase of that around 365 nm were observed. In the case of BR in purple membrane, an increase of absorbance around 470 nm can be observed in the pH region higher than 1 1, due to deprotonation of the Schiff base (20). However, such a molecular species having A,,,, around 470 nm has never been observed in pH titration experiments of Triton-treated BR. According to Lozier et al. (21), BR in a phospholipid vesicle at pH 8.6 displays the A,, at 480 nrn. Such a blue shift of the A,, , has never been observed in anv cell envelope vesicles. BR molecules in a phospholipid vesicle do not exhibit the exciton cd band, because BR molecules become monomeric in a phospholipid vesicle under these conditions. Because of similarities between the 480-nm absorbing species and the 490-nm complex, in conditions for their formation and their absorption maxima, it is indicated that both pigments may have a retinylidene chromophore in a similar microenvironment. A few years ago, we reported the formation of a molecular species absorbing around 500 nm which was produced from acid-induced species with orange light irradiation (22). The isomeric form of the retinal extracted from this product was 9-cis retinal. When 9-cis retinal was mixed with bacterioopsin under neutral pH conditions, no formation of the pigment was observed, as reported by Oesterhelt and Schuhmann (23). It is not plausible that the 490-nm complex has 9-cis retinal as its chromophore. Further information about the structure of these molecular species would give some clue to explaining the coloration of BR.


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Another noteworthy point is that the amount of the 490-nm complex formed from T-BRLwas larger than that from T-BRD (Fig. 2). In the case of BR in the purple membrane, BRL has only all-trans retinal as its chromophore but BRD has both all-trans and 13-cis retinals in equimolar amount (15, 16). The present experiment showed that the ratios of 13-cis to all-trans retinals in T-BRLand T-BRDwere 13.0 2 2187.0 2 2 and 51.3 t 3148.7 -C 3, respectively. It should be noted that a part of T-BRL has 13-cis retinal as its chromophore. The existence of 13-cis retinal in T-BRL has already been reported by Casadio and Stoeckenius (18), but their result (38 ? 2% of the total amount) is much larger than ours. It is not yet clear why they are so different from each other. In any case, the species having all-trans retinal (T-BR') is more abundant in T - B R ~than in T-BRD. Thus it may be safe to say that the 490-nm complex comes from T-BR1 rather than T-BR". Stabilization of the meta-intermediate The meta-intermediate was stabilized under several conditions: Oesterhelt and Hess (24) found that the 4 12-nm complex was easily formed by irradiating BR suspended in about 3.6 M NaCl solution saturated with ether, and mentioned that the ether could stabilize the 412-nm complex. Becher and Ebrey (25) reported that the high salt and high pH conditions (1 M NaCl, pH 10) could stabilize the meta-intermediate. Yoshida et al. (26) showed that guanidine hydrochloride prolonged the decay time of the meta-intermediate, and this effect depended on the concentration of guanidine hydrochloride. Even 8 M solution did not saturate this effect. Recently we investigated the photoreaction of tyrosine-iodinated BR at low temperature, and found meta-intermediates of tyrosine-iodinated BR stable at -65°C (27). As shown in the present paper and by Lam and Packer (28), the meta-intermediate also is more stable in Triton X-100 solution than under native conditions. The stabilization effects of guanidine hydrochloride, high salt - high pH conditions, and iodination of tyrosine on the meta-intermediate were probably due to change in the interaction between the retinylidene chromophore and charges on the bacterioopsin. This idea may be supported by the fact that the stabilization effect was also observed in replacement of guanidine hydrochloride by arginine hydrochloride. The effect resulting from ether or Triton X-100, however, was not explained clearly, because structural information about BR in both solutions is limited. The tertiary structure of the protein was changed with Triton X-100, as shown in the cd spectrum, and probably the secondary structure would be changed. These structural changes would result in charge replacement which might stabilize the meta-intermediate. Whether or not such an explanation can be applied to the effect needs further investigation, in order to obtain detailed information on the structure of the metaintermediate and the protein moiety in such a micelle structure or organic solvent. The investigation of solubilized BR revealed that the destruction of the lattice structure resulted in formation of byproducts of the photoreaction cycle of BR, such as the 490-nm and 380-nm complexes; the former is probably formed from T-BR', and the latter slowly reverts to the original pigment. It seems probable that the lattice structure can stabilize the BRLby preventing it from formation of by-products and can play a role in rapid rotation of the photoreaction cycle. Of course the possibility that the results mentioned above may be due to direct interaction between Triton X-100 molecule and BR rather than destruction of the lattice structure can not be ex-

cluded. Further investigation of the structure of BR in the micelle must be made. Such investigation may provide important information regarding structure and function of membrane proteins extracted with the aid of detergents.

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