Photochemistry ond Phorobralogy. Vol. 29. pp. 6YY lo 702. 0 Perg,,rnun Press Lld. 1979. Printcd in Great Brilmn
ASSIGNING THE RESONANCE RAMAN SPECTRAL FEATURES OF RHODOPSIN, ISORHODOPSIN AND BATHORHODOPSIN I N BOVINE PHOTOSTATIONARY STATE SPECTRA$ MICHAIL A MAKCLS*and AARON Lkwist School of Applied and Fngineering Physics. Cornell Univcrsity. I t h x a . NY 14853 I' S A
Abstract- High resolution rcsonance Raman spectra of rhodopsin. isorhodopsin and photostationary state mixtures containing a high percentage of bathorhodopsin arc presented. New spectral features
are detected which were not obsei-ved in lower resolution studies by other workers. All of the hands in the photostationary state spcctra arc assigned based on pure rhodopsin and isorhodopsin resonance Raman results and alterations in the photostationary state mixture. The spectral features i n these spectra are invariant from 20 to 150 K indicating that retinal and protein structural alteration. consistent with a model of excitation proposed by Lewis, occurs in steady-state spectra even at 20 K. In addition, thc relative intensity of certain features i n the photostationary state spectra are altered upon D,O suspension. One explanation for these alterations is that the contributions of various intcrmediates to the photostationary state mixture are changed when mcmbranc fragments are suspended in DLO.
MATERIALS AND METHODS
INTRODUCTION
T h e a i m of this paper is t o present a consistent set of resonance R a m a n spectra a t high resolution on bovine rhodopsin. isorhodopsin and photostationary mixtures or rhodopsin, isnrhodopsin a n d bathorhodopsin. Previous investigators (Oseroff and Callender, 1974; Callender et ul., 1976; Mathies et ul., 1976) have obtained spectra of these species at relatively low resolution which varied from spectra t o spectra. However, investigations performed in o u r laboratory o n squid rhodopsin (Sulkcs et ul.. 1976, 1978) a n d bacteriorhodopsin (Marcus a n d Lewis. 1977; Marcus t't ul., 1977, 1978) havc dcrnonstrated t h a t new resolvable features occur in higher resolution spectra ( 2 cm I) which are n o t apparent in lower resolution ( - 10 c m - I ) resonance Raman spcctroscopy. As demonstrated by comparing t h e relatively high resolution spectra in this p a p e r t o previous investigations. this is also t h e case in bovine rhodopsin. On t h e basis of this data. we can now assign t o the various species present all of t h e bands in t h e low temperature photostationary state spectra. These spectra demonstrate that there a r e bathorhodopsin features previously undetected by other workers (Oseroff and Callender, 1974). Furthermore, we also demonstrate that t h e steady-state resonance R a m a n spectrum of bathorhodopsin is invariant from 20 t o 150K.
-
~
*Present address: Kodak Research Laboratories. Kodak Park B-81. Rochester. NY 14650, U.S.A. t To whom correspondence should be addressed. $ Contributed at a session on Visual Pigments and Response. ASP mceting. Burlington. VT. June IS. 197%
Bovine retina were purchased from George Hormel Co.. Austin. MN and stored at 77 K until ready for use. Rod outer segments (ROS) and solubilized rhodopsin (ammonyx-LO) were prepared as described previously (Applebury r f a/.. 1974). Rhodopsin was purified on a hydroxylapetite column by the procedure of Applebury et u / . (1974). Deuterated ROS samples were prepared by washing ROS pellets four times with D 2 0 pH I6.6. All operations were performed under dim red light or in the dark at 4 C . Variable temperature (2&150 K ) spectra were obtained with a Lake Shore Cryotronics Spectrim Model closed cycle helium refrigerator. The rhodopsin samples (OD 2.0 in a 1 em path length) were sealed in 1 mrn wide glass cuvettes. Laser excitation sources included Coherent Radiation Ar ion, Kr and/or rhodamine 6G dye lasers. A hackscattering geometry was used throughout this investigation. The scattered light was dispersed in a Spex 1401 double monochromator equipped with a thermoelectrically cooled RCA C31034 photomultiplier tube and photon counting electronics designed in our laboratory. Data were collected in a stepping mode (1-2 cm- ' steps) via a custom designed single channel scaler (Perreault et ( I / . - 1976). Spcctra were stored and analyzed with a Mod Comp I 1 minicomputer. Spectra of pure bovine rhodopsin were obtained by thc flow method described by Mathies c'r cri. (1976) and Ciillender et a / . (1976). A Micro-Pump model 12-41-301 wiis used in these experimcnts. Absorption measurements before and after the flow experiments vcrilied that less than 10Yc,of the sample was photoconvcrted or destroyed during the Raman measurements. In these experiments 20 mW of 590 nm dye laser light focused to 35 pm was used and thc data was collected with a I s time constant. M~iltiple runs were pooled with each run having 2 c m - ' resolution. A pump-probe dual laser beam technique first described by OserofT and Callender (1974) was used at low tcmpcratures t o vary the concentrations of rhodopsin. bathorhodopsin and isorhodopsin in the probing laser beam. With
699
MltrlAl L A M A R C Land S AARON LIWI$
700
BOVINE
x
95K
n-
CYI
"R
A) PROBE BEAM 4 8 2 5 n m
8 ) PROBE BEAM: 482 5nm
+
PUMP BEAM 580nm
I I
(cm-9
Figure 1. Resonance Raman spectra of purified bovine rhodopsin obtained at 95 K . The probe beam in each case was 3 m W of 482.5 nm excitation from a Kr ion laser. A single-beam spectrum is shown in (A) and a spectrum with the addition of an 18 m W piimp beam a t 580nm is shown in (B).Resolution is 2 em-' in each case. tration of bathorhodopsin decreases from about 60:,, in Fig. 1A to about 30% in Fig. 1B. On this basis bands at 818 c m - ' , 855 cm-',* 875 cm I , 922 cm-'. 1019cm-', 1208cm-'.* 1 3 2 4 c m - ' and 1537cm-' can he assigned to bathorhodopsin. More band assignments can be made in the photostationary state spectra (Fig. 1A and B) when the RESULTS AND DISCUSSION results on rhodopsin and isorhodopsin (Fig. 2A and B) are examined. F o r example. i t is seen in Fig. 3A Figure I A shows spectra of purified bovine rhodopand Fig. 1 that rhodopsin has a band at 84.5 cm sin at 9.5 K obtained with 3 m W of 482.5 n m exciwhich was not detected in OseroK and Callender's tation. In Fig. 1B the same excitation conditions are used but the sample is being simultaneously illu- (1974) low temperature investigation or in the bovine rhodopsin flow experiments of Callender c't ul. (1976). minated with a n 1 8 m W 5 8 0 n m p u m p beam. Thus, Mathies of ul. (1976). however. also observed this low thesc t w o spectra differ drastically in their relative frequency mode in their bovine rhodopsin flow speccontribution from bathorhodopsin a s the concentrum, and it is interesting that this band is absent *At first glance it may not he obvious from the data in spectra of protonated SchilT bases of 1 I c i s retinal shown in Fig. I how these assignments are made. The 855 cm ~Ihand can be assiged to hathorhodopsin sincc (Mathies et ul., 1977). Thus, it is evident that bathoit is considerably weaker in Fig. IB than the isorhodopsin rhodopsin is not unique in having vibrational modes and rhodopsin vibrational modes at 956cm-' and between 800 a n d 900 c m - '. In addition examination 970cm-I. The 1208cm-l band is reduced in intensity in of the fingerprint region in Fig. 2 indicates that rhoFig. 1B to the point of being equal in intensity to the and rhodopsin I215 cm.. I band. This suggests that bathorho- dopsin has modes at 1215cm-'. 1239cm 1269 cm - I whereas isorhodopsin has hands at Jopsin has an intense hand at 1208 cm-' since the relative intensity of isorhodopsin bands in the spectrum are not 1206cm-', 1241 c m - ' , 1270cm-', 1295cm-' and reduced with the addition of a pump beam at 580nm. 1 3 2 0 c m - ' plus a doublet at 1 1 4 4 c m - ' and Thus. even though isorhodopsin does have a band in this I 1 53 cm-' (see Fig. 2B). Previous investigations have region. the reduction in the intensity of the 1 2 0 8 c m ~ ~ ' observed only a single band in the 1150cm region band with the addition of the 580 nm beam indicates that bathorhodopsin does make a strong contribution to this in isorhodopsin. This observation is significant i n peak. view of the fact that squid isorhodopsin also exhibits t Based on the isorhodopsin data obtained by previous a doublet centered around 1 1 5 0 c m - ' t . Based on workers (Oscroff and Callender. 1974: Mathies cr uI.. 1976) these results bands at 1 2 2 7 c m - ' , 1 2 7 7 c m - ' and which indicated a singlc band at I153 cm-I. Sulkes "r 01. 1 3 2 4 c m - ' which occur only in the low temperature (1976) assigned a band at I147cm-' to bathorhodopsin. However. subsequent work (Sulkes c't ui.. 19711) including photostationary statc spectra (Fig. 1) must be data presented in this paper on isorhodopsin indicates that assigned to bathorhodopsin. the 1147cm-' hand is present in isorhodopsin and was Bands in the 96@970 cm -- region havc been probably obscured in lower resolution studies (Oscroff and assigned t o C- C - H bends. Rhodopsin and isorhodopCallender. 1974; Mathies c'r u/.. 1976). Therefore. the sin exhibit intense bands in this region at - 9 7 0 c m - ' 1147 cm-' band cannot be assigned to hathorhodopsin.
482.5 nm laser excitation it was found that bathorhodopsin had a concentration of 609
ASSIGNING THE RESONANCE RAMAN SPECTRAL FEATURES OF RHODOPSIN, ISORHODOPSIN AND BATHORHODOPSIN I N BOVINE PHOTOSTATIONARY STATE SPECTRA$ MICHAIL A MAKCLS*and AARON Lkwist School of Applied and Fngineering Physics. Cornell Univcrsity. I t h x a . NY 14853 I' S A
Abstract- High resolution rcsonance Raman spectra of rhodopsin. isorhodopsin and photostationary state mixtures containing a high percentage of bathorhodopsin arc presented. New spectral features
are detected which were not obsei-ved in lower resolution studies by other workers. All of the hands in the photostationary state spcctra arc assigned based on pure rhodopsin and isorhodopsin resonance Raman results and alterations in the photostationary state mixture. The spectral features i n these spectra are invariant from 20 to 150 K indicating that retinal and protein structural alteration. consistent with a model of excitation proposed by Lewis, occurs in steady-state spectra even at 20 K. In addition, thc relative intensity of certain features i n the photostationary state spectra are altered upon D,O suspension. One explanation for these alterations is that the contributions of various intcrmediates to the photostationary state mixture are changed when mcmbranc fragments are suspended in DLO.
MATERIALS AND METHODS
INTRODUCTION
T h e a i m of this paper is t o present a consistent set of resonance R a m a n spectra a t high resolution on bovine rhodopsin. isorhodopsin and photostationary mixtures or rhodopsin, isnrhodopsin a n d bathorhodopsin. Previous investigators (Oseroff and Callender, 1974; Callender et ul., 1976; Mathies et ul., 1976) have obtained spectra of these species at relatively low resolution which varied from spectra t o spectra. However, investigations performed in o u r laboratory o n squid rhodopsin (Sulkcs et ul.. 1976, 1978) a n d bacteriorhodopsin (Marcus a n d Lewis. 1977; Marcus t't ul., 1977, 1978) havc dcrnonstrated t h a t new resolvable features occur in higher resolution spectra ( 2 cm I) which are n o t apparent in lower resolution ( - 10 c m - I ) resonance Raman spcctroscopy. As demonstrated by comparing t h e relatively high resolution spectra in this p a p e r t o previous investigations. this is also t h e case in bovine rhodopsin. On t h e basis of this data. we can now assign t o the various species present all of t h e bands in t h e low temperature photostationary state spectra. These spectra demonstrate that there a r e bathorhodopsin features previously undetected by other workers (Oseroff and Callender, 1974). Furthermore, we also demonstrate that t h e steady-state resonance R a m a n spectrum of bathorhodopsin is invariant from 20 t o 150K.
-
~
*Present address: Kodak Research Laboratories. Kodak Park B-81. Rochester. NY 14650, U.S.A. t To whom correspondence should be addressed. $ Contributed at a session on Visual Pigments and Response. ASP mceting. Burlington. VT. June IS. 197%
Bovine retina were purchased from George Hormel Co.. Austin. MN and stored at 77 K until ready for use. Rod outer segments (ROS) and solubilized rhodopsin (ammonyx-LO) were prepared as described previously (Applebury r f a/.. 1974). Rhodopsin was purified on a hydroxylapetite column by the procedure of Applebury et u / . (1974). Deuterated ROS samples were prepared by washing ROS pellets four times with D 2 0 pH I6.6. All operations were performed under dim red light or in the dark at 4 C . Variable temperature (2&150 K ) spectra were obtained with a Lake Shore Cryotronics Spectrim Model closed cycle helium refrigerator. The rhodopsin samples (OD 2.0 in a 1 em path length) were sealed in 1 mrn wide glass cuvettes. Laser excitation sources included Coherent Radiation Ar ion, Kr and/or rhodamine 6G dye lasers. A hackscattering geometry was used throughout this investigation. The scattered light was dispersed in a Spex 1401 double monochromator equipped with a thermoelectrically cooled RCA C31034 photomultiplier tube and photon counting electronics designed in our laboratory. Data were collected in a stepping mode (1-2 cm- ' steps) via a custom designed single channel scaler (Perreault et ( I / . - 1976). Spcctra were stored and analyzed with a Mod Comp I 1 minicomputer. Spectra of pure bovine rhodopsin were obtained by thc flow method described by Mathies c'r cri. (1976) and Ciillender et a / . (1976). A Micro-Pump model 12-41-301 wiis used in these experimcnts. Absorption measurements before and after the flow experiments vcrilied that less than 10Yc,of the sample was photoconvcrted or destroyed during the Raman measurements. In these experiments 20 mW of 590 nm dye laser light focused to 35 pm was used and thc data was collected with a I s time constant. M~iltiple runs were pooled with each run having 2 c m - ' resolution. A pump-probe dual laser beam technique first described by OserofT and Callender (1974) was used at low tcmpcratures t o vary the concentrations of rhodopsin. bathorhodopsin and isorhodopsin in the probing laser beam. With
699
MltrlAl L A M A R C Land S AARON LIWI$
700
BOVINE
x
95K
n-
CYI
"R
A) PROBE BEAM 4 8 2 5 n m
8 ) PROBE BEAM: 482 5nm
+
PUMP BEAM 580nm
I I
(cm-9
Figure 1. Resonance Raman spectra of purified bovine rhodopsin obtained at 95 K . The probe beam in each case was 3 m W of 482.5 nm excitation from a Kr ion laser. A single-beam spectrum is shown in (A) and a spectrum with the addition of an 18 m W piimp beam a t 580nm is shown in (B).Resolution is 2 em-' in each case. tration of bathorhodopsin decreases from about 60:,, in Fig. 1A to about 30% in Fig. 1B. On this basis bands at 818 c m - ' , 855 cm-',* 875 cm I , 922 cm-'. 1019cm-', 1208cm-'.* 1 3 2 4 c m - ' and 1537cm-' can he assigned to bathorhodopsin. More band assignments can be made in the photostationary state spectra (Fig. 1A and B) when the RESULTS AND DISCUSSION results on rhodopsin and isorhodopsin (Fig. 2A and B) are examined. F o r example. i t is seen in Fig. 3A Figure I A shows spectra of purified bovine rhodopand Fig. 1 that rhodopsin has a band at 84.5 cm sin at 9.5 K obtained with 3 m W of 482.5 n m exciwhich was not detected in OseroK and Callender's tation. In Fig. 1B the same excitation conditions are used but the sample is being simultaneously illu- (1974) low temperature investigation or in the bovine rhodopsin flow experiments of Callender c't ul. (1976). minated with a n 1 8 m W 5 8 0 n m p u m p beam. Thus, Mathies of ul. (1976). however. also observed this low thesc t w o spectra differ drastically in their relative frequency mode in their bovine rhodopsin flow speccontribution from bathorhodopsin a s the concentrum, and it is interesting that this band is absent *At first glance it may not he obvious from the data in spectra of protonated SchilT bases of 1 I c i s retinal shown in Fig. I how these assignments are made. The 855 cm ~Ihand can be assiged to hathorhodopsin sincc (Mathies et ul., 1977). Thus, it is evident that bathoit is considerably weaker in Fig. IB than the isorhodopsin rhodopsin is not unique in having vibrational modes and rhodopsin vibrational modes at 956cm-' and between 800 a n d 900 c m - '. In addition examination 970cm-I. The 1208cm-l band is reduced in intensity in of the fingerprint region in Fig. 2 indicates that rhoFig. 1B to the point of being equal in intensity to the and rhodopsin I215 cm.. I band. This suggests that bathorho- dopsin has modes at 1215cm-'. 1239cm 1269 cm - I whereas isorhodopsin has hands at Jopsin has an intense hand at 1208 cm-' since the relative intensity of isorhodopsin bands in the spectrum are not 1206cm-', 1241 c m - ' , 1270cm-', 1295cm-' and reduced with the addition of a pump beam at 580nm. 1 3 2 0 c m - ' plus a doublet at 1 1 4 4 c m - ' and Thus. even though isorhodopsin does have a band in this I 1 53 cm-' (see Fig. 2B). Previous investigations have region. the reduction in the intensity of the 1 2 0 8 c m ~ ~ ' observed only a single band in the 1150cm region band with the addition of the 580 nm beam indicates that bathorhodopsin does make a strong contribution to this in isorhodopsin. This observation is significant i n peak. view of the fact that squid isorhodopsin also exhibits t Based on the isorhodopsin data obtained by previous a doublet centered around 1 1 5 0 c m - ' t . Based on workers (Oscroff and Callender. 1974: Mathies cr uI.. 1976) these results bands at 1 2 2 7 c m - ' , 1 2 7 7 c m - ' and which indicated a singlc band at I153 cm-I. Sulkes "r 01. 1 3 2 4 c m - ' which occur only in the low temperature (1976) assigned a band at I147cm-' to bathorhodopsin. However. subsequent work (Sulkes c't ui.. 19711) including photostationary statc spectra (Fig. 1) must be data presented in this paper on isorhodopsin indicates that assigned to bathorhodopsin. the 1147cm-' hand is present in isorhodopsin and was Bands in the 96@970 cm -- region havc been probably obscured in lower resolution studies (Oscroff and assigned t o C- C - H bends. Rhodopsin and isorhodopCallender. 1974; Mathies c'r u/.. 1976). Therefore. the sin exhibit intense bands in this region at - 9 7 0 c m - ' 1147 cm-' band cannot be assigned to hathorhodopsin.
482.5 nm laser excitation it was found that bathorhodopsin had a concentration of 609
