176
Biochimica et Biophysica Acta, 576 (1979) 176--191 © Elsevier/North-Holland Biomedical Press
BBA 38069
MECHANISMS OF SPECTRAL SHIFTS IN L O B S T E R CAROTENOPROTEINS THE RESONANCE R A M A N SPECTRA O F O V O V E R D I N AND THE CRUSTACYANINS
V.R. SALARES, N.M. YOUNG, H.J. BERNSTEIN and P.R. CAREY *
Divisions of Biological Sciences and Chemistry, National Research Council of Canada, Ottawa, KIA OR6 (Canada) (Received May 25th, 1978)
Key words: Carotenoprotein; Astaxanthin; Crustacyanin; Ovoverdin; (Homarus americanus)
Summary Resonance Raman data have been used to elucidate the mechanisms of the absorption spectral shifts occurring for astaxanthin u p o n binding to the carotenoproteins, ovoverdin and a-, /~- and 7-crustacyanins, from the lobster Homarus americanus. Although distinguishable on the basis of small differences in their resonance Raman spectra the binding sites of the crustacyanins, giving rise to kmax at 605 + 25 nm, are essentially the same. The large red shift in )~max for the crustacyanins compared to free astaxanthin ()~m~x 480 nm), is accounted for by a charge-polarisation mechanism in which charged groups and possibly hydrogen bonds in the binding site set up ~r electron polarisation in the ligand. Several alternate mechanisms can be eliminated. Ovoverdin is found to consist of three polypeptide chains of molecular weight 105 000, 95 000 and 78 000 which are n o t linked by disulfide bridges. The visible absorption peaks of ovoverdin at 460 and 640 nm are shown to arise from two astaxanthin molecules each b o u n d at a different site. The spectral characteristics of the 460 nm site suggest a rigid h y d r o p h o b i c environment for astaxanthin, in which no charge-ligand interactions occur. The mechanism of the spectral shift in the 640 nm site is the same as in the crustacyanins, i.e. a charge-polarisation effect. Resonance Raman spectra of ovoverdin and the crustacyanins could be obtained in situ; they were identical to the spectra of the purified proteins showing that the carotenoid sites were unperturbed by protein isolation.
* To whom
corresPondence should be addressed.
177 Introduction
While the biological role of many carotenoids remains enigmatic [1] there is considerable current interest in the mechanism of the spectral shifts observed for carotenoids in protein [2] and photosynthetic membrane [3] environments and during the visual process [4]. The carotenoid astaxanthin is responsible for the color of several carotenoproteins isolated from the lobster Homarus americanus. However depending on the protein, astaxanthin undergoes .widely different spectral shifts upon binding. The object of t h e present study was to elucidate the mechanisms for these different spectral shifts and thereby provide a basis for a more general understanding of carotenoid spectra in terms of biological sites. In the crustacyanins, a group of proteins isolated from the carapace of the lobster (H. americanus or Hormarus gammarus) astaxanthin undergoes a large red shift in its )'max from the 480 nm value for free astaxanthin to 580--640 nm in the protein-bound form. In contrast, in the 'yellow protein' isolated from the same source [5] the astaxanthin absorbance is narrowed and markedly blue shifted to 410 nm. This protein has recently been identified in the carapace in vivo [6] and it was shown that the spectral properties resulted from electronic (exciton) interactions between proximal astaxanthin molecules. A third protein, ovoverdin, which occurs in lobster eggs, has two absorption peaks, one near 480 n m essentially unshifted from free astaxanthin and the other around 640 n m resembling the absorption found in the crustacyanins. There is some disagreement as to the number of astaxanthin molecules per mol of ovoverdin, a lipoprotein of molecular weight 360 000 [7]. Stern and Salomon [8] favored one while Zagalsky [9] and Ceccaldi et al. [10] presented evidence for two astaxanthins per mol of protein. Furthermore, it was not known if the two absorption bands in ovoverdin arose from one protein-bound astaxanthin species or two and the origin of the multiple peaks in the 440--480 nm band presented a further puzzle. The new results reported herein rely heavily on the use of resonance Raman spectroscopy. This technique provides detailed vibrational spectra of chromophores in the concentration range 10-4--10 -7 M. In contrast to the other optical techniques used to study carotenoproteins, e.g. absorption, CD and ORD which are sensitive to effects in both the ground and excited electronic states, the position of features in the resonance Raman spectrum is a property solely of the ground state. This provides an opportunity to elucidate with some certainty the mechanisms of spectral shifts in carotenoproteins. A further advantage of the resonance Raman technique is that spectra can frequently be obtained from chromophores in situ, i.e. without purification. Thus the spectra of astaxanthin in the crustacyanins in the shell of the live lobster and in ovoverdin in intact eggs were compared to those of the purified proteins. Thus, if artifacts arose during the preparation of the carotenoproteins they could be readily detected. Materials and Methods
Ovoverdin was extracted from undeveloped eggs of the lohster, H. arnericanus. Female mature lobsters were caught from Eastern Canadian Waters
178 in July, 1976, June 1977 and December 1977. The ovaries were used immediately or covered with 0.5 M NaC1/5 mM EDTA (pH 5) buffer and deep frozen. The extraction of crude ovoverdin was based on the procedure of Stern and Salomon [8]. The ovaries were ground with sand, extracted with cold 0.5 M NaC1/5 mM EDTA (pH 5) and precipitated with (NH4)2SO4. Purification was performed by dialysis against 45% {NH4}2SO4 solution followed by gel filtration [7] on an 8% agarose column (Bio-l~ad Laboratories Inc., l~ichmohd, Calif.), 2.5 × 90 cm, run in the above buffer. The determination of astaxanthin and protein content was carried out on samples purified by agarose gel filtration, and dialyzed against distilled water. For astaxanthin measurement, I ml of the solution was treated with an equal volume of acetone. The astaxanthin was extracted and dissolved in pyridine. The a m o u n t of astaxanthin was calculated using a molar extinction coefficient of 1.15 • l 0 s [11]. The protein content was determined b y s p e c t r o p h o t o m e t r y using the extinction coefficient A280nm 1~ = 0.85 [7]. The ~-, ~- and ~/-crustacyanins were purified from lobster shells as previously described [6]. Absorption spectra were measured in a Cary 14 spectrophotometer. Spectra of solutions above room temperature were taken with the aid of a circulating bath. The procedure for measuring Raman spectra has been described previously [12,13]. Excitation lines were provided b y the emission lines of a carbostyril 165 dye laser (430.0--460.0 nm), a Spectra Physics model 185 He-Cd laser (441.6 nm), Spectra-Physics 164 or Coherent Radiation CR-12 argon ion lasers {454.5, 457.9, 465.9, 472.6, 476.5, 488.0, 496.5, 501.7, 514.5 nm), Rhodamine 6G dye laser {590.0--640.0 nm), l~hodamine B dye laser ( 6 3 6 . 0 - ~ 7 8 . 0 nm) and a Spectra-Physics 164 Kr ÷ laser (647.1 nm). Resonance Raman excitation profiles were measured from solutions of ovoverdin in 0.05 M phosphate buffer (pH 7), 2 M sodium formate. The intensities of the Raman bands in the 1500 and 1158 cm -1 regions were measured relative to the formate band at 1354 cm -1. The spectra were taken using static cells. There was no evidence for variation of the Raman spectra with length of irradiation time, or changes in absorbance using 5--50 mW laser powers. Electrophoresis in the presence of sodium dodecyl sulfate was carried out by the m e t h o d of Weber and Osborn [14] using gels prepared from a stock acrylamide solution containing 16.7 g acrylamide and 0.225 g methylene bisacrylamide per 100 ml {i.e. a 7.52% gel with 'half cross-linker'). The gels were stained with Coomassie Brilliant Blue, and after removal of excess stain, the bands were quantitated b y photometric scanning on a Gilford 240 system. The proteins employed as standards were ~-galactosidase, phosphorylase a, bovine serum albumin, and a commercial cross-linked protein mixture containing species of molecular weights 53~000, 106 000 and 159 000 (B.D.H. Chemicals Ltd.). The reaction b e t w e e n astaxanthin and a strong base was studied using a 1 cm diameter glass tube provided with a side arm which permitted de-aeration of the reactants. Solutions of astaxanthin in tert-butanol and either tert-butanol saturated with KOH or 1"--3% potassium butoxide (prepared by reacting potassium metal with tert.butanol) were placed in separate compartments of the tube, thoroughly evacuated, sealed and mixed. Absorption measurements
179 on the sealed tube were made a b o u t half an h o u r or longer after mixing. Resonance Raman spectra were obtained b y backscattering. Results
Characterisation of ovoverdin Purification of ovoverdin by gel filtration on 8% agarose gels resulted in a single slightly asymmetric peak of protein in which the astaxanthin absorbance was skewed to the high molecular weight side of the peak. The ratio A640nm/ A46onm was constant across t h e peak, b u t the A 2 8 o n m / A 6 4 o n m increased. Fractions with A28onm/A48onm between 5.2 and 6.5 were combined for further characterisation. Rechromatography of this material gave a single peak with little change in the A28onm/A46onm ratio, On disc gel electrophoresis the green colour of the protein changed to orange and one protein band was observed (Fig. 1) which migrated with the orange colour. The ~ability of the blue (640 nm) chromophore to revert to the orange appearance of native astaxanthin without dissociation from the. protein is in accord with other denaturation experiments (see below). Gel electrophoresis in the presence of sodium dodecyl sulfate showed the protein contained t h r e e major polypeptide chains all of considerable size, as well as a minor c o m p o n e n t close to the third band. Comparison with the migration of standard proteins yielded molecular weights of 105 000, 95 000 and 78 000 for the major bands, and 83 000 for the minor band. There were no
Fig. 1. P o l y a c r y l a m i d e gel electrophoresis of o v o v e r d i n . I n s e t (a), disc gel electrophoresis of the purified p r o t e i n , 5% gel, p H 8.9; (b) e l e c t r o p h o r e s i s in 0.1% s o d i u m d o d e c y l sulfate; a P h o t o m e t r i c s c a n of t h i s gel is also s h o w n .
180 major differences between mercaptoethanol-reduced and non-reduced samples, hence the chains are not linked by disulfide bridges. Quantitation of the relative amounts of the chains by p h o t o m e t r y of the gels (Fig. 1) gave ratios of 1 : 1.3 : 1, the minor band being included with the third component. Gels with higher load and longer running times gave ratios of 1 : 1.2 : 1.16. These data suggest the chains are present in equimolar amounts, which is borne o u t by their combined weight, 278 000 dalton, being close to the weight of polypeptide in the lipoprotein, calculated from the data of Wallace et al. [7] to be 260 000 dalton. Quantitative analyses for the astaxanthin content of ovoverdin consistently gave values over 1 mol of astaxanthin per mol of protein, the range being 1.05-1.27 for duplicate determinations on t w o samples. The content was determined b y denaturing the protein and extracting the chromophore with organic solvent, the protein concentration in the sample being obtained spectrophotometrically. Some of the variations between the absorbance ratios obtained here and those of other workers (Table I) may arise from species differences. The absorption spectrum of the purified ovoverdin is shown in Fig. 2. In the visible region, ~ m a x is at 460 nm, with additional maxima at 433 and 483 nm, and a weaker, broad absorption centred approximately at 637 nm. In the ultraviolet region there is an inflexion at 320 nm and a maximum at 278 nm. The protein seems to have a low t r y p t o p h a n content from the appearance of the ultraviolet region, which would be consistent with its low ultraviolet extinction coefficient (A280nm 1~ = 0.85 [7]}. The absorption spectrum was unchanged in the pH range 4.0--9.0. As a test for free binding sites on ovoverdin, 0.1 ml 2 - 1 0 - 4 M astaxanthin in acetone was added to 4 ml ovoverdin At60,m = 0.76 in 0.5 M NaC1/5 mM EDTA buffer, pH 5.0. No evidence for additional binding was obtained. Resonance Raman spectra Thin-layer chromatography of the chromophore extracted from ovoverdin showed that it consisted entirely of astaxanthin. However, two distinct resonance Raman spectra can be obtained by irradiating ovoverdin with a blue or red laser line. The spectrum obtained with blue excitation at 488.0 nm is shown in Fig. 3a.
TABLE I A B S O R B A N C E R A T I O S O F O V O V E R D I N B A N D S IN D I F F E R E N T P R E P A R A T I O N S A278nm/A 460nm
A 460nm/A640nm
Species
Reference
2.00
2.95 2.76 2.21
H. a m e r i c a n u s H. gamrnarus H. g a m m a r u s
8 21 9 a n d 24
1.54--2.28 5.15 4.59 2.46
1.48--2.81 2.95 2.45 2.55
H. H. H. H.
10 7 This w o r k * This w o r k * *
gammarus arnericanus arnericanus arnericanus
* P~epared b y repeated precipitation w i t h a m m o n i u m sulfate, t h e n dialysis [ 8 ] . * * Pu_~ified b y gel filtration o n 8% aga~ose.
181
8.0
0.8
'E
0.6
o
ua (_) ~
~ ~~ ~
~
6 0 _z z ,,~
~0
~ ~
~ • • ~ ~
~ ~0~
Biochimica et Biophysica Acta, 576 (1979) 176--191 © Elsevier/North-Holland Biomedical Press
BBA 38069
MECHANISMS OF SPECTRAL SHIFTS IN L O B S T E R CAROTENOPROTEINS THE RESONANCE R A M A N SPECTRA O F O V O V E R D I N AND THE CRUSTACYANINS
V.R. SALARES, N.M. YOUNG, H.J. BERNSTEIN and P.R. CAREY *
Divisions of Biological Sciences and Chemistry, National Research Council of Canada, Ottawa, KIA OR6 (Canada) (Received May 25th, 1978)
Key words: Carotenoprotein; Astaxanthin; Crustacyanin; Ovoverdin; (Homarus americanus)
Summary Resonance Raman data have been used to elucidate the mechanisms of the absorption spectral shifts occurring for astaxanthin u p o n binding to the carotenoproteins, ovoverdin and a-, /~- and 7-crustacyanins, from the lobster Homarus americanus. Although distinguishable on the basis of small differences in their resonance Raman spectra the binding sites of the crustacyanins, giving rise to kmax at 605 + 25 nm, are essentially the same. The large red shift in )~max for the crustacyanins compared to free astaxanthin ()~m~x 480 nm), is accounted for by a charge-polarisation mechanism in which charged groups and possibly hydrogen bonds in the binding site set up ~r electron polarisation in the ligand. Several alternate mechanisms can be eliminated. Ovoverdin is found to consist of three polypeptide chains of molecular weight 105 000, 95 000 and 78 000 which are n o t linked by disulfide bridges. The visible absorption peaks of ovoverdin at 460 and 640 nm are shown to arise from two astaxanthin molecules each b o u n d at a different site. The spectral characteristics of the 460 nm site suggest a rigid h y d r o p h o b i c environment for astaxanthin, in which no charge-ligand interactions occur. The mechanism of the spectral shift in the 640 nm site is the same as in the crustacyanins, i.e. a charge-polarisation effect. Resonance Raman spectra of ovoverdin and the crustacyanins could be obtained in situ; they were identical to the spectra of the purified proteins showing that the carotenoid sites were unperturbed by protein isolation.
* To whom
corresPondence should be addressed.
177 Introduction
While the biological role of many carotenoids remains enigmatic [1] there is considerable current interest in the mechanism of the spectral shifts observed for carotenoids in protein [2] and photosynthetic membrane [3] environments and during the visual process [4]. The carotenoid astaxanthin is responsible for the color of several carotenoproteins isolated from the lobster Homarus americanus. However depending on the protein, astaxanthin undergoes .widely different spectral shifts upon binding. The object of t h e present study was to elucidate the mechanisms for these different spectral shifts and thereby provide a basis for a more general understanding of carotenoid spectra in terms of biological sites. In the crustacyanins, a group of proteins isolated from the carapace of the lobster (H. americanus or Hormarus gammarus) astaxanthin undergoes a large red shift in its )'max from the 480 nm value for free astaxanthin to 580--640 nm in the protein-bound form. In contrast, in the 'yellow protein' isolated from the same source [5] the astaxanthin absorbance is narrowed and markedly blue shifted to 410 nm. This protein has recently been identified in the carapace in vivo [6] and it was shown that the spectral properties resulted from electronic (exciton) interactions between proximal astaxanthin molecules. A third protein, ovoverdin, which occurs in lobster eggs, has two absorption peaks, one near 480 n m essentially unshifted from free astaxanthin and the other around 640 n m resembling the absorption found in the crustacyanins. There is some disagreement as to the number of astaxanthin molecules per mol of ovoverdin, a lipoprotein of molecular weight 360 000 [7]. Stern and Salomon [8] favored one while Zagalsky [9] and Ceccaldi et al. [10] presented evidence for two astaxanthins per mol of protein. Furthermore, it was not known if the two absorption bands in ovoverdin arose from one protein-bound astaxanthin species or two and the origin of the multiple peaks in the 440--480 nm band presented a further puzzle. The new results reported herein rely heavily on the use of resonance Raman spectroscopy. This technique provides detailed vibrational spectra of chromophores in the concentration range 10-4--10 -7 M. In contrast to the other optical techniques used to study carotenoproteins, e.g. absorption, CD and ORD which are sensitive to effects in both the ground and excited electronic states, the position of features in the resonance Raman spectrum is a property solely of the ground state. This provides an opportunity to elucidate with some certainty the mechanisms of spectral shifts in carotenoproteins. A further advantage of the resonance Raman technique is that spectra can frequently be obtained from chromophores in situ, i.e. without purification. Thus the spectra of astaxanthin in the crustacyanins in the shell of the live lobster and in ovoverdin in intact eggs were compared to those of the purified proteins. Thus, if artifacts arose during the preparation of the carotenoproteins they could be readily detected. Materials and Methods
Ovoverdin was extracted from undeveloped eggs of the lohster, H. arnericanus. Female mature lobsters were caught from Eastern Canadian Waters
178 in July, 1976, June 1977 and December 1977. The ovaries were used immediately or covered with 0.5 M NaC1/5 mM EDTA (pH 5) buffer and deep frozen. The extraction of crude ovoverdin was based on the procedure of Stern and Salomon [8]. The ovaries were ground with sand, extracted with cold 0.5 M NaC1/5 mM EDTA (pH 5) and precipitated with (NH4)2SO4. Purification was performed by dialysis against 45% {NH4}2SO4 solution followed by gel filtration [7] on an 8% agarose column (Bio-l~ad Laboratories Inc., l~ichmohd, Calif.), 2.5 × 90 cm, run in the above buffer. The determination of astaxanthin and protein content was carried out on samples purified by agarose gel filtration, and dialyzed against distilled water. For astaxanthin measurement, I ml of the solution was treated with an equal volume of acetone. The astaxanthin was extracted and dissolved in pyridine. The a m o u n t of astaxanthin was calculated using a molar extinction coefficient of 1.15 • l 0 s [11]. The protein content was determined b y s p e c t r o p h o t o m e t r y using the extinction coefficient A280nm 1~ = 0.85 [7]. The ~-, ~- and ~/-crustacyanins were purified from lobster shells as previously described [6]. Absorption spectra were measured in a Cary 14 spectrophotometer. Spectra of solutions above room temperature were taken with the aid of a circulating bath. The procedure for measuring Raman spectra has been described previously [12,13]. Excitation lines were provided b y the emission lines of a carbostyril 165 dye laser (430.0--460.0 nm), a Spectra Physics model 185 He-Cd laser (441.6 nm), Spectra-Physics 164 or Coherent Radiation CR-12 argon ion lasers {454.5, 457.9, 465.9, 472.6, 476.5, 488.0, 496.5, 501.7, 514.5 nm), Rhodamine 6G dye laser {590.0--640.0 nm), l~hodamine B dye laser ( 6 3 6 . 0 - ~ 7 8 . 0 nm) and a Spectra-Physics 164 Kr ÷ laser (647.1 nm). Resonance Raman excitation profiles were measured from solutions of ovoverdin in 0.05 M phosphate buffer (pH 7), 2 M sodium formate. The intensities of the Raman bands in the 1500 and 1158 cm -1 regions were measured relative to the formate band at 1354 cm -1. The spectra were taken using static cells. There was no evidence for variation of the Raman spectra with length of irradiation time, or changes in absorbance using 5--50 mW laser powers. Electrophoresis in the presence of sodium dodecyl sulfate was carried out by the m e t h o d of Weber and Osborn [14] using gels prepared from a stock acrylamide solution containing 16.7 g acrylamide and 0.225 g methylene bisacrylamide per 100 ml {i.e. a 7.52% gel with 'half cross-linker'). The gels were stained with Coomassie Brilliant Blue, and after removal of excess stain, the bands were quantitated b y photometric scanning on a Gilford 240 system. The proteins employed as standards were ~-galactosidase, phosphorylase a, bovine serum albumin, and a commercial cross-linked protein mixture containing species of molecular weights 53~000, 106 000 and 159 000 (B.D.H. Chemicals Ltd.). The reaction b e t w e e n astaxanthin and a strong base was studied using a 1 cm diameter glass tube provided with a side arm which permitted de-aeration of the reactants. Solutions of astaxanthin in tert-butanol and either tert-butanol saturated with KOH or 1"--3% potassium butoxide (prepared by reacting potassium metal with tert.butanol) were placed in separate compartments of the tube, thoroughly evacuated, sealed and mixed. Absorption measurements
179 on the sealed tube were made a b o u t half an h o u r or longer after mixing. Resonance Raman spectra were obtained b y backscattering. Results
Characterisation of ovoverdin Purification of ovoverdin by gel filtration on 8% agarose gels resulted in a single slightly asymmetric peak of protein in which the astaxanthin absorbance was skewed to the high molecular weight side of the peak. The ratio A640nm/ A46onm was constant across t h e peak, b u t the A 2 8 o n m / A 6 4 o n m increased. Fractions with A28onm/A48onm between 5.2 and 6.5 were combined for further characterisation. Rechromatography of this material gave a single peak with little change in the A28onm/A46onm ratio, On disc gel electrophoresis the green colour of the protein changed to orange and one protein band was observed (Fig. 1) which migrated with the orange colour. The ~ability of the blue (640 nm) chromophore to revert to the orange appearance of native astaxanthin without dissociation from the. protein is in accord with other denaturation experiments (see below). Gel electrophoresis in the presence of sodium dodecyl sulfate showed the protein contained t h r e e major polypeptide chains all of considerable size, as well as a minor c o m p o n e n t close to the third band. Comparison with the migration of standard proteins yielded molecular weights of 105 000, 95 000 and 78 000 for the major bands, and 83 000 for the minor band. There were no
Fig. 1. P o l y a c r y l a m i d e gel electrophoresis of o v o v e r d i n . I n s e t (a), disc gel electrophoresis of the purified p r o t e i n , 5% gel, p H 8.9; (b) e l e c t r o p h o r e s i s in 0.1% s o d i u m d o d e c y l sulfate; a P h o t o m e t r i c s c a n of t h i s gel is also s h o w n .
180 major differences between mercaptoethanol-reduced and non-reduced samples, hence the chains are not linked by disulfide bridges. Quantitation of the relative amounts of the chains by p h o t o m e t r y of the gels (Fig. 1) gave ratios of 1 : 1.3 : 1, the minor band being included with the third component. Gels with higher load and longer running times gave ratios of 1 : 1.2 : 1.16. These data suggest the chains are present in equimolar amounts, which is borne o u t by their combined weight, 278 000 dalton, being close to the weight of polypeptide in the lipoprotein, calculated from the data of Wallace et al. [7] to be 260 000 dalton. Quantitative analyses for the astaxanthin content of ovoverdin consistently gave values over 1 mol of astaxanthin per mol of protein, the range being 1.05-1.27 for duplicate determinations on t w o samples. The content was determined b y denaturing the protein and extracting the chromophore with organic solvent, the protein concentration in the sample being obtained spectrophotometrically. Some of the variations between the absorbance ratios obtained here and those of other workers (Table I) may arise from species differences. The absorption spectrum of the purified ovoverdin is shown in Fig. 2. In the visible region, ~ m a x is at 460 nm, with additional maxima at 433 and 483 nm, and a weaker, broad absorption centred approximately at 637 nm. In the ultraviolet region there is an inflexion at 320 nm and a maximum at 278 nm. The protein seems to have a low t r y p t o p h a n content from the appearance of the ultraviolet region, which would be consistent with its low ultraviolet extinction coefficient (A280nm 1~ = 0.85 [7]}. The absorption spectrum was unchanged in the pH range 4.0--9.0. As a test for free binding sites on ovoverdin, 0.1 ml 2 - 1 0 - 4 M astaxanthin in acetone was added to 4 ml ovoverdin At60,m = 0.76 in 0.5 M NaC1/5 mM EDTA buffer, pH 5.0. No evidence for additional binding was obtained. Resonance Raman spectra Thin-layer chromatography of the chromophore extracted from ovoverdin showed that it consisted entirely of astaxanthin. However, two distinct resonance Raman spectra can be obtained by irradiating ovoverdin with a blue or red laser line. The spectrum obtained with blue excitation at 488.0 nm is shown in Fig. 3a.
TABLE I A B S O R B A N C E R A T I O S O F O V O V E R D I N B A N D S IN D I F F E R E N T P R E P A R A T I O N S A278nm/A 460nm
A 460nm/A640nm
Species
Reference
2.00
2.95 2.76 2.21
H. a m e r i c a n u s H. gamrnarus H. g a m m a r u s
8 21 9 a n d 24
1.54--2.28 5.15 4.59 2.46
1.48--2.81 2.95 2.45 2.55
H. H. H. H.
10 7 This w o r k * This w o r k * *
gammarus arnericanus arnericanus arnericanus
* P~epared b y repeated precipitation w i t h a m m o n i u m sulfate, t h e n dialysis [ 8 ] . * * Pu_~ified b y gel filtration o n 8% aga~ose.
181
8.0
0.8
'E
0.6
o
ua (_) ~
~ ~~ ~
~
6 0 _z z ,,~
~0
~ ~
~ • • ~ ~
~ ~0~
