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The Major Carotenoid Pigment of a Psychrotrophic Micrococcus roseus Strain: Fluorescence Properties of the Pigment and Its Binding to Membranes
JOBNAME: BBRC 220#3 PAGE: 1 SESS: 7 OUTPUT: Thu May 2 01:14:00 1996 /xypage/worksmart/tsp000/69829b/37
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 724–728 (1996)
0471
The Major Carotenoid Pigment of a Psychrotrophic Micrococcus roseus Strain: Fluorescence Properties of the Pigment and Its Binding to Membranes M. V. Jagannadham, M. K. Chattopadhyay, and S. Shivaji1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received February 5, 1996 The fluorescence excitation and emission spectra are reported for P-3 (bis-dehydro-B-carotene-2-carboxylic acid), the major carotenoid pigment of psychrotrophic M. roseus. The excitation spectrum and the absorption spectrum showed good agreement with respect to the position of their peak maxima. The study also demonstrates that P-3 binds to liposomes prepared from synthetic lipids (PC, DOPG, or CL) or the total lipids of a mutant colourless M. roseus. Binding of P-3 to the membranes was accompanied by a decrease in the fluorescence emission intensity and a blue shift in lem maximum by 15 to 20 nm. The quantum yield of P-3 was observed to be low (1.7 × 10−5). © 1996 Academic Press, Inc.
Carotenoids are a group of naturally occurring pigments present in a wide variety of bacteria, algae, fungi and plants (1). It is hypothesised that in prokaryotes carotenoids interact with the membrane and regulate the fluidity and thus influence membrane function (2,3). This interaction of carotenoids with membranes could be monitored by using standard spectrophotometric or spectrofluoremetric methods. In the latter method one could use external fluorophores (4) or monitor the fluorescent properties of the carotenoid under study provided it is fluorescent. However, studies on the fluorescent properties of carotenoids of prokaryotes (5) have been limited due to the low fluorescence quantum yield (6) and due to the difficulties involved in purifying carotenoids. Previously we had established that a good proportion of psychrotrophic bacteria from Antarctica were pigmented (7–11), the pigments were of the carotenoid type and could interact and stabilise model membranes (4,12). However, nothing is known about the fluorescent properties of any of the carotenoid pigments present in the psychrotrophic bacteria from Antarctica. In the present study, for the first time, the fluorescent properties of P-3 (4), the major carotenoid pigment of a psychrotrophic bacterium M. roseus, is established. The interaction of the pigment with synthetic model membranes and membranes prepared from the lipids of a pigment less mutant of M. roseus is also demonstrated. MATERIALS AND METHODS Chemicals and reagents. All chemicals used for bacterial cultures including HEPES were obtained from Loba (Bombay, India). Methanol, chloroform and carbon disulphide were of HPLC or spectroscopic grade and were obtained from Spectrochem (Bombay, India). B-carotene, Phosphatidylcholine (PC) and Dioleoylphosphatidyl glycerol (DOPG) were obtained from Sigma Chemical Co., St. Louis, MO. Cardiolipin, from bovine heart was from Avanti Polar Lipids (Birmingham, AL). Bacterial strains and growth conditions. The red pigmented psychrotrophic bacterium identified as M. roseus (MTCC 678; IMTECH, Chandigarh, India) (7) and a colourless mutant of M. roseus (Shivaji et al., unpublished) were maintained in a medium containing peptone (0.5 w/v), yeast extract (0.2 w/v) and soil extract (0.5 w/v). Pigment purification and fluorescence of P-3. The major carotenoid pigment present in psychrotrophic M. roseus was purified as described earlier (4) and used immediately or stored protected from light at −70°C. The purity of the sample was monitored by HPLC before and after the end of each experiment. 1
Corresponding author: Fax: 00-91-40-671195; E-mail: shivaji%[email protected]. 724
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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All fluorescence measurements were recorded on a Hitachi spectrofluorimeter (F 4010) at 25°C. The fluorescence excitation and emission spectra of the pigment in an appropriate solvent or in the presence of liposomes were recorded at concentrations at which the absorbance of the pigment was <0.1 at the excitation wavelength. The slit widths of excitation and emission were 10 nm respectively. Interferences from Raman scattering due to the solvent were corrected for by subtracting a solvent blank under identical conditions. Fluorescence quantum yield of the pigment was measured using B-carotene as a standard and whose quantum yield was reported to be 6 × 10−5 (5). Interaction of the pigment with small unilamellar vesicles. Small unilamellar vesicles (SUVs) were prepared by sonication (Branson B-50 sonifier) of an aqueous solution of PC, DOPG, CL or the total lipids of a mutant of M. roseus which lacked the pigment in HEPES buffer to clarity. Total lipids from the mutant cells were extracted (13) and lipids were estimated according to the method described by Stewart (14). Binding of the pigment with these vesicles was studied by adding an ethanolic solution of the pigment (such that the final concentration of ethanol did not exceed 1% and the absorption was <0.1) and by subsequently monitoring the intrinsic fluorescence properties of P-3. The pigment was excited at 465 nm and the emission spectra were recorded between 550 to 650 nm.
RESULTS Fluorescence Properties of P-3 Figure 1 depicts the absorbance, fluorescence excitation and emission spectra of P-3 in carbon disulphide recorded at room temperature. The excitation spectrum exhibited bands at 507, 528 and 568 nm when the emission maximum was fixed at 610 nm and bands at 504, 534 and 568 nm respectively when the emission maxima was fixed at 620 nm. The emission spectrum exhibited maxima at 540, 586 and 615 nm when the excitation maxima was fixed at 505 nm (Table 1). A good agreement was observed between the absorption maxima and the peaks of the excitation spectra and an obvious overlap was visible between the excitation and the emission spectra Fig. 1). Further both the excitation and the emission spectra of P-3 varied depending on the solvent (Table 1). The quantum yield of the fluorescence of P-3 in carbon disulphide was 1.7 × 10−5. Interaction of the Pigment with Membranes The absorption spectra of P-3 in buffer, and in MLVs of PC is shown in Fig. 2. In aqueous buffer it showed an absorption band at z443 nm and weak bands at approximately 482, 517 and 545 nm
FIG. 1. Fluorescence excitation (– – –), emission (–?–), and absorption (—) spectra of P-3 from M. roseus in carbon disulphide. The lem and the lex were fixed at 610 nm and 500 nm, respectively. 725
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TABLE 1 Fluorescence Properties of the Major Carotenoid Pigment (P-3) of Psychrotropic M. roseus in Various Solvents
Solvent Methanol
Chloroform
Carbon disulphide
Wavelength of excitationa (nm) 465 490 525 470 505 540 505 525 570
Emission spectrum em max (nm)
Wavelength of emission (nm)
Excitation spectrum ex max (nm)
502, 521, 565 534, 565 563 512, 546, 583 550, 581 579 540, 580, 615 574, 613 615
570 590
465, 506, 535 465, 490, 520
570 580
475, 506, 537 477, 508, 541
610 620
507, 528, 568 504, 534, 568
a The three wavelengths of excitation chosen for each solvent correspond to the three absorption maxima observed for P-3 in that particular solvent.
where as the pigment in lipid vesicles exhibited absorption bands at 476, 507 and 545 nm similar to the absorption spectra of P-3 in chloroform (with absorption bands at 478, 505 and 538 nm respectively) indicating that the pigment in the presence of a suspension of MLVs is associated with the MLVs. This is further confirmed by fluorescence studies. The fluorescence emission spectrum of the pigment in buffer and in the presence of various liposomes is shown in Fig. 3. In aqueous buffer the pigment exhibited an emission maximum of z615 nm. But, in the presence of the lipids from the mutant M. roseus or synthetic lipids such as PC, DOPG and CL there was a decrease in the fluorescence emission intensity and a blue shift in the emission maximum by about 15 to 20 nm. In these experiments the fluorescence intensity of the emission peak of the pigment was monitored as a function of lipid concentration and only the final spectra when maximum change was observed is presented in Fig. 3. Maximum change in em max was observed with 25 mg of mutant lipid, 42 mM of PC, 65 mM of DOPG and 50 mM of CL.
FIG. 2. The absorption spectra of P-3 from M. roseus in MLVs prepared from PC (a) and in HEPES buffer (b). 726
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FIG. 3. Emission spectra of P-3 of M. roseus in HEPES buffer (a) and in the presence of SUVs prepared with the total lipids of a mutant M. roseus (b), PC (c), DOPG (d), or CL (e).
DISCUSSION Studies on the fluorescent properties of carotenoids have been hampered primarily due to their low quantum yield (5,6,15) and also due to the accompanying risk that the fluorescence observed is due to a contaminant. From the spectra of P-3 it is evident that P-3 from M. roseus has intrinsic fluorescent properties. To exclude the possibility of the presence of any impurity, P-3 was purified using at least two different methods namely by HPLC and by TLC (4) and each time the P-3 fraction obtained showed similar fluorescence spectral characteristics. Further the excitation spectra of P-3 were in good agreement with the absorption spectra and the emission maximum was independent of the excitation wavelength, thus it is very unlikely to have impurities with absorption spectral shift exactly similar to that of carotenoids. Based on similar reasoning the fluorescent properties of b-carotene, rhodopsin and spheroidenone were established (5). The present study also demonstrates that the quantum yield of P-3 like other carotenoids was low and was similar to that of b-carotene (6 × 10−5) and spheroidenone (3 × 10−5). Further the small Stokes’ shift (z300 cm−1) observed in Fig. 1 between the highest energy vibrionic transition of the ground state and the lowest energy vibrionic transition of the excited state is a characteristic feature of the carotenoids and was calculated as described earlier (16). Studies done earlier indicated that in larger carotenoids with more than 11 conjugated double bonds like P-3 (which has 13 conjugated bonds) the fluorescence emission is due to S2 → S0 (5,15,17–19) and in such carotenoids the 727
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origins of emission and absorption overlap as observed in case of P-3 in the present investigation. Table 1 also clearly indicates that the emission of P-3 is different in different solvents thus confirming a previous report that S2 → S0 emission is solvent dependent where as S1 → S0 emission is independent of the solvent used (20,21). Due to the low fluorescence quantum yield of carotenoids earlier studies on the interaction of carotenoids with membranes was studied either by monitoring the UV-visible spectral properties of the pigment (22), by using spin labels (2) or by using external fluorophores (4). The present study clearly demonstrates for the first time that binding of a carotenoid pigment could also be evaluated using the intrinsic fluorescence of the pigment. Binding of P-3 to membranes was accompanied by a decrease in the fluorescence emission intensity of P-3 and em maximum was blue shifted by 15–20 nm. This study also confirms our earlier observation that P-3 binds to membranes (4). Further, the present spectrophotometric studies also indicate that the visible spectrum of P-3 in MLVs is similar to that observed in organic solvents implying that P-3 is associated with the hydrophobic region of the MLVs. Though studies have highlighted that carotenoids stabilise membranes the physiological necessity of this change if any is still unknown. In psychrotrophic bacteria carotenoids may play an important role in regulating the fluidity of the membrane which is known to increase under low temperature conditions due to increase in the synthesis of unsaturated fatty acids (22–24). REFERENCES 1. Goodwin, T. W. (1980) The Biochemistry of Carotenoids, Vol. 1, Chapman & Hall, London. 2. Subczynski, W. K., Markowska, E., Gurszecki, W. I., and Sielewiesiuk, J. (1992) Biochim. Biophys. Acta 1105, 97–108. 3. Subczynski, W. K., Markowska, E., and Sielewiesiuk, J. (1991) Biochim. Biophys. Acta 1068, 68–72. 4. Jagannadham, M. V., Rao, V. J., and Shivaji, S. (1991) J. Bacteriol. 173, 7911–7917. 5. Gillbro, T., and Cogdell, R. J. (1989) Chem. Phys. Lett. 158, 312–316. 6. Wasielewski, M. R., and Kispert, L. D. (1986) Chem. Phys. Lett. 128, 238–243. 7. Shivaji, S., Rao, N. S., Saisree, L., Seth, V., Reddy, G. S. N., and Bhargava, P. M. (1988) J. Biosci. 13, 409–414. 8. Shivaji, S., Rao, N. S., Saisree, L., Seth, V., Reddy, G. S. N., and Bhargava, P. M. (1989a) Appl. Environ. Microbiol. 55, 767–771. 9. Shivaji, S., Rao, N. S., Saisree, L., Reddy, G. S. N., Kumar, G. S., and Bhargava, P. M. (1989b) Pol. Biol. 10, 225–229. 10. Shivaji, S., Ray, M. K., Seshu Kumar, G., Reddy, G. S. N., Saisree, L., and Wynn-Williams, D. D. (1991) Polar Biol. 110, 267–272. 11. Shivaji, S., Ray, M. K., Saisree, L., Jagannadham, M. V., Kumar, G. S., Reddy, G. S. N., and Bhargava, P. M. (1992) Int. J. Syst. Bacteriol. 42, 102–106. 12. Chauhan, S., and Shivaji, S. (1994) Polar Biol. 14, 31–36. 13. Kates, M. (1972) Techniques in Lipidoloy (Work, T. S., and Work, E., Eds.), p. 351, Elsevier, New York. 14. Stewart, J. C. M. (1980) Anal. Biochem. 104, 10–14. 15. De Coster, B., Christensen, R. L., Gebhard, R., Lugtenburg, J., Farhoosh, , and Frank, H. A. (1992) Biochim. Biophys. Acta 1102, 107–114. 16. Dalle, J. P., and Rosenberg, B. (1970) Photochem. Photobiol. 12, 151–167. 17. Shreve, A. P., Trautman, J. K., Frank, H. A., Owens, T. G., and Albreckt, A. C. (1991) Biochim. Biophys. Acta 1058, 280–288. 18. Bondarov, S. L., Bachilo, S. M., Dovrnikov, S. S., and Tikhomorov, S. A. (1989) J. Photochem. Photobiol. A: Chemistry 46, 315–322. 19. Cosgrove, S. A., Gruite, M. A., Burnell, T. B., and Christensen, R. L. (1990) J. Physiol. Chem. 94, 8118–8124. 20. Gillbro, T., Andersson, P. D., Liu, R. S. H., Asato, A. E., Takaishi, S., and Cogdell, R. J. (1993) Photochem. Photobiol. 57, 44–48. 21. D’Amico, K., Christopher, M., and Christensen, R. L. (1980) J. Am. Chem. Soc. 102, 1777–1782. 22. Yamamoto, H. Y., and Bangham, A. D. (1978) Biochim. Biophys. Acta 507, 119–127. 23. Shivaji, S., Chattopadhyay, M. K., and Ray, M. K. (1994) Proc. NIPR Symp. Polar Biol. 7, 173–184. 24. Wada, H., Gombos, Z., and Murata, N. (1994) Proc. Natl. Acad. Sci. USA 91, 4273–4277.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 724–728 (1996)
0471
The Major Carotenoid Pigment of a Psychrotrophic Micrococcus roseus Strain: Fluorescence Properties of the Pigment and Its Binding to Membranes M. V. Jagannadham, M. K. Chattopadhyay, and S. Shivaji1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received February 5, 1996 The fluorescence excitation and emission spectra are reported for P-3 (bis-dehydro-B-carotene-2-carboxylic acid), the major carotenoid pigment of psychrotrophic M. roseus. The excitation spectrum and the absorption spectrum showed good agreement with respect to the position of their peak maxima. The study also demonstrates that P-3 binds to liposomes prepared from synthetic lipids (PC, DOPG, or CL) or the total lipids of a mutant colourless M. roseus. Binding of P-3 to the membranes was accompanied by a decrease in the fluorescence emission intensity and a blue shift in lem maximum by 15 to 20 nm. The quantum yield of P-3 was observed to be low (1.7 × 10−5). © 1996 Academic Press, Inc.
Carotenoids are a group of naturally occurring pigments present in a wide variety of bacteria, algae, fungi and plants (1). It is hypothesised that in prokaryotes carotenoids interact with the membrane and regulate the fluidity and thus influence membrane function (2,3). This interaction of carotenoids with membranes could be monitored by using standard spectrophotometric or spectrofluoremetric methods. In the latter method one could use external fluorophores (4) or monitor the fluorescent properties of the carotenoid under study provided it is fluorescent. However, studies on the fluorescent properties of carotenoids of prokaryotes (5) have been limited due to the low fluorescence quantum yield (6) and due to the difficulties involved in purifying carotenoids. Previously we had established that a good proportion of psychrotrophic bacteria from Antarctica were pigmented (7–11), the pigments were of the carotenoid type and could interact and stabilise model membranes (4,12). However, nothing is known about the fluorescent properties of any of the carotenoid pigments present in the psychrotrophic bacteria from Antarctica. In the present study, for the first time, the fluorescent properties of P-3 (4), the major carotenoid pigment of a psychrotrophic bacterium M. roseus, is established. The interaction of the pigment with synthetic model membranes and membranes prepared from the lipids of a pigment less mutant of M. roseus is also demonstrated. MATERIALS AND METHODS Chemicals and reagents. All chemicals used for bacterial cultures including HEPES were obtained from Loba (Bombay, India). Methanol, chloroform and carbon disulphide were of HPLC or spectroscopic grade and were obtained from Spectrochem (Bombay, India). B-carotene, Phosphatidylcholine (PC) and Dioleoylphosphatidyl glycerol (DOPG) were obtained from Sigma Chemical Co., St. Louis, MO. Cardiolipin, from bovine heart was from Avanti Polar Lipids (Birmingham, AL). Bacterial strains and growth conditions. The red pigmented psychrotrophic bacterium identified as M. roseus (MTCC 678; IMTECH, Chandigarh, India) (7) and a colourless mutant of M. roseus (Shivaji et al., unpublished) were maintained in a medium containing peptone (0.5 w/v), yeast extract (0.2 w/v) and soil extract (0.5 w/v). Pigment purification and fluorescence of P-3. The major carotenoid pigment present in psychrotrophic M. roseus was purified as described earlier (4) and used immediately or stored protected from light at −70°C. The purity of the sample was monitored by HPLC before and after the end of each experiment. 1
Corresponding author: Fax: 00-91-40-671195; E-mail: shivaji%[email protected]. 724
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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All fluorescence measurements were recorded on a Hitachi spectrofluorimeter (F 4010) at 25°C. The fluorescence excitation and emission spectra of the pigment in an appropriate solvent or in the presence of liposomes were recorded at concentrations at which the absorbance of the pigment was <0.1 at the excitation wavelength. The slit widths of excitation and emission were 10 nm respectively. Interferences from Raman scattering due to the solvent were corrected for by subtracting a solvent blank under identical conditions. Fluorescence quantum yield of the pigment was measured using B-carotene as a standard and whose quantum yield was reported to be 6 × 10−5 (5). Interaction of the pigment with small unilamellar vesicles. Small unilamellar vesicles (SUVs) were prepared by sonication (Branson B-50 sonifier) of an aqueous solution of PC, DOPG, CL or the total lipids of a mutant of M. roseus which lacked the pigment in HEPES buffer to clarity. Total lipids from the mutant cells were extracted (13) and lipids were estimated according to the method described by Stewart (14). Binding of the pigment with these vesicles was studied by adding an ethanolic solution of the pigment (such that the final concentration of ethanol did not exceed 1% and the absorption was <0.1) and by subsequently monitoring the intrinsic fluorescence properties of P-3. The pigment was excited at 465 nm and the emission spectra were recorded between 550 to 650 nm.
RESULTS Fluorescence Properties of P-3 Figure 1 depicts the absorbance, fluorescence excitation and emission spectra of P-3 in carbon disulphide recorded at room temperature. The excitation spectrum exhibited bands at 507, 528 and 568 nm when the emission maximum was fixed at 610 nm and bands at 504, 534 and 568 nm respectively when the emission maxima was fixed at 620 nm. The emission spectrum exhibited maxima at 540, 586 and 615 nm when the excitation maxima was fixed at 505 nm (Table 1). A good agreement was observed between the absorption maxima and the peaks of the excitation spectra and an obvious overlap was visible between the excitation and the emission spectra Fig. 1). Further both the excitation and the emission spectra of P-3 varied depending on the solvent (Table 1). The quantum yield of the fluorescence of P-3 in carbon disulphide was 1.7 × 10−5. Interaction of the Pigment with Membranes The absorption spectra of P-3 in buffer, and in MLVs of PC is shown in Fig. 2. In aqueous buffer it showed an absorption band at z443 nm and weak bands at approximately 482, 517 and 545 nm
FIG. 1. Fluorescence excitation (– – –), emission (–?–), and absorption (—) spectra of P-3 from M. roseus in carbon disulphide. The lem and the lex were fixed at 610 nm and 500 nm, respectively. 725
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TABLE 1 Fluorescence Properties of the Major Carotenoid Pigment (P-3) of Psychrotropic M. roseus in Various Solvents
Solvent Methanol
Chloroform
Carbon disulphide
Wavelength of excitationa (nm) 465 490 525 470 505 540 505 525 570
Emission spectrum em max (nm)
Wavelength of emission (nm)
Excitation spectrum ex max (nm)
502, 521, 565 534, 565 563 512, 546, 583 550, 581 579 540, 580, 615 574, 613 615
570 590
465, 506, 535 465, 490, 520
570 580
475, 506, 537 477, 508, 541
610 620
507, 528, 568 504, 534, 568
a The three wavelengths of excitation chosen for each solvent correspond to the three absorption maxima observed for P-3 in that particular solvent.
where as the pigment in lipid vesicles exhibited absorption bands at 476, 507 and 545 nm similar to the absorption spectra of P-3 in chloroform (with absorption bands at 478, 505 and 538 nm respectively) indicating that the pigment in the presence of a suspension of MLVs is associated with the MLVs. This is further confirmed by fluorescence studies. The fluorescence emission spectrum of the pigment in buffer and in the presence of various liposomes is shown in Fig. 3. In aqueous buffer the pigment exhibited an emission maximum of z615 nm. But, in the presence of the lipids from the mutant M. roseus or synthetic lipids such as PC, DOPG and CL there was a decrease in the fluorescence emission intensity and a blue shift in the emission maximum by about 15 to 20 nm. In these experiments the fluorescence intensity of the emission peak of the pigment was monitored as a function of lipid concentration and only the final spectra when maximum change was observed is presented in Fig. 3. Maximum change in em max was observed with 25 mg of mutant lipid, 42 mM of PC, 65 mM of DOPG and 50 mM of CL.
FIG. 2. The absorption spectra of P-3 from M. roseus in MLVs prepared from PC (a) and in HEPES buffer (b). 726
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FIG. 3. Emission spectra of P-3 of M. roseus in HEPES buffer (a) and in the presence of SUVs prepared with the total lipids of a mutant M. roseus (b), PC (c), DOPG (d), or CL (e).
DISCUSSION Studies on the fluorescent properties of carotenoids have been hampered primarily due to their low quantum yield (5,6,15) and also due to the accompanying risk that the fluorescence observed is due to a contaminant. From the spectra of P-3 it is evident that P-3 from M. roseus has intrinsic fluorescent properties. To exclude the possibility of the presence of any impurity, P-3 was purified using at least two different methods namely by HPLC and by TLC (4) and each time the P-3 fraction obtained showed similar fluorescence spectral characteristics. Further the excitation spectra of P-3 were in good agreement with the absorption spectra and the emission maximum was independent of the excitation wavelength, thus it is very unlikely to have impurities with absorption spectral shift exactly similar to that of carotenoids. Based on similar reasoning the fluorescent properties of b-carotene, rhodopsin and spheroidenone were established (5). The present study also demonstrates that the quantum yield of P-3 like other carotenoids was low and was similar to that of b-carotene (6 × 10−5) and spheroidenone (3 × 10−5). Further the small Stokes’ shift (z300 cm−1) observed in Fig. 1 between the highest energy vibrionic transition of the ground state and the lowest energy vibrionic transition of the excited state is a characteristic feature of the carotenoids and was calculated as described earlier (16). Studies done earlier indicated that in larger carotenoids with more than 11 conjugated double bonds like P-3 (which has 13 conjugated bonds) the fluorescence emission is due to S2 → S0 (5,15,17–19) and in such carotenoids the 727
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Vol. 220, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
origins of emission and absorption overlap as observed in case of P-3 in the present investigation. Table 1 also clearly indicates that the emission of P-3 is different in different solvents thus confirming a previous report that S2 → S0 emission is solvent dependent where as S1 → S0 emission is independent of the solvent used (20,21). Due to the low fluorescence quantum yield of carotenoids earlier studies on the interaction of carotenoids with membranes was studied either by monitoring the UV-visible spectral properties of the pigment (22), by using spin labels (2) or by using external fluorophores (4). The present study clearly demonstrates for the first time that binding of a carotenoid pigment could also be evaluated using the intrinsic fluorescence of the pigment. Binding of P-3 to membranes was accompanied by a decrease in the fluorescence emission intensity of P-3 and em maximum was blue shifted by 15–20 nm. This study also confirms our earlier observation that P-3 binds to membranes (4). Further, the present spectrophotometric studies also indicate that the visible spectrum of P-3 in MLVs is similar to that observed in organic solvents implying that P-3 is associated with the hydrophobic region of the MLVs. Though studies have highlighted that carotenoids stabilise membranes the physiological necessity of this change if any is still unknown. In psychrotrophic bacteria carotenoids may play an important role in regulating the fluidity of the membrane which is known to increase under low temperature conditions due to increase in the synthesis of unsaturated fatty acids (22–24). REFERENCES 1. Goodwin, T. W. (1980) The Biochemistry of Carotenoids, Vol. 1, Chapman & Hall, London. 2. Subczynski, W. K., Markowska, E., Gurszecki, W. I., and Sielewiesiuk, J. (1992) Biochim. Biophys. Acta 1105, 97–108. 3. Subczynski, W. K., Markowska, E., and Sielewiesiuk, J. (1991) Biochim. Biophys. Acta 1068, 68–72. 4. Jagannadham, M. V., Rao, V. J., and Shivaji, S. (1991) J. Bacteriol. 173, 7911–7917. 5. Gillbro, T., and Cogdell, R. J. (1989) Chem. Phys. Lett. 158, 312–316. 6. Wasielewski, M. R., and Kispert, L. D. (1986) Chem. Phys. Lett. 128, 238–243. 7. Shivaji, S., Rao, N. S., Saisree, L., Seth, V., Reddy, G. S. N., and Bhargava, P. M. (1988) J. Biosci. 13, 409–414. 8. Shivaji, S., Rao, N. S., Saisree, L., Seth, V., Reddy, G. S. N., and Bhargava, P. M. (1989a) Appl. Environ. Microbiol. 55, 767–771. 9. Shivaji, S., Rao, N. S., Saisree, L., Reddy, G. S. N., Kumar, G. S., and Bhargava, P. M. (1989b) Pol. Biol. 10, 225–229. 10. Shivaji, S., Ray, M. K., Seshu Kumar, G., Reddy, G. S. N., Saisree, L., and Wynn-Williams, D. D. (1991) Polar Biol. 110, 267–272. 11. Shivaji, S., Ray, M. K., Saisree, L., Jagannadham, M. V., Kumar, G. S., Reddy, G. S. N., and Bhargava, P. M. (1992) Int. J. Syst. Bacteriol. 42, 102–106. 12. Chauhan, S., and Shivaji, S. (1994) Polar Biol. 14, 31–36. 13. Kates, M. (1972) Techniques in Lipidoloy (Work, T. S., and Work, E., Eds.), p. 351, Elsevier, New York. 14. Stewart, J. C. M. (1980) Anal. Biochem. 104, 10–14. 15. De Coster, B., Christensen, R. L., Gebhard, R., Lugtenburg, J., Farhoosh, , and Frank, H. A. (1992) Biochim. Biophys. Acta 1102, 107–114. 16. Dalle, J. P., and Rosenberg, B. (1970) Photochem. Photobiol. 12, 151–167. 17. Shreve, A. P., Trautman, J. K., Frank, H. A., Owens, T. G., and Albreckt, A. C. (1991) Biochim. Biophys. Acta 1058, 280–288. 18. Bondarov, S. L., Bachilo, S. M., Dovrnikov, S. S., and Tikhomorov, S. A. (1989) J. Photochem. Photobiol. A: Chemistry 46, 315–322. 19. Cosgrove, S. A., Gruite, M. A., Burnell, T. B., and Christensen, R. L. (1990) J. Physiol. Chem. 94, 8118–8124. 20. Gillbro, T., Andersson, P. D., Liu, R. S. H., Asato, A. E., Takaishi, S., and Cogdell, R. J. (1993) Photochem. Photobiol. 57, 44–48. 21. D’Amico, K., Christopher, M., and Christensen, R. L. (1980) J. Am. Chem. Soc. 102, 1777–1782. 22. Yamamoto, H. Y., and Bangham, A. D. (1978) Biochim. Biophys. Acta 507, 119–127. 23. Shivaji, S., Chattopadhyay, M. K., and Ray, M. K. (1994) Proc. NIPR Symp. Polar Biol. 7, 173–184. 24. Wada, H., Gombos, Z., and Murata, N. (1994) Proc. Natl. Acad. Sci. USA 91, 4273–4277.
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