Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism

Nicholas Geacintov, F. Van Nostrand, Joseph F. Becker

Research output: Contribution to journalArticle

Abstract

1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10-13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes. 2. The FP ratio is typically in the range of 1.2-1.9 in Chlorella, 1.20-1.25 in Scenedesmus and 1.4-1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Qy transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented. 3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720-730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615-617). 4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01-0.06) determined by previous workers with unoriented cell suspensions. 5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered. 6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Qy transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane. 7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena. 8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.

Original languageEnglish (US)
Pages (from-to)443-463
Number of pages21
JournalBBA - Bioenergetics
Volume347
Issue number3
DOIs
StatePublished - Jun 28 1974

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Photosynthetic membranes
Fluorescence Polarization
Chloroplasts
Light polarization
Chlorella
Spectrum Analysis
Fluorescence
Spectroscopy
Euglena
Light
Membranes
Spinacia oleracea
Polarization
Chlorophyll
Scenedesmus
Wavelength
Euglena gracilis
Porphyrins
Magnetic Fields
Carotenoids

ASJC Scopus subject areas

  • Biophysics
  • Medicine(all)

Cite this

Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism. / Geacintov, Nicholas; Van Nostrand, F.; Becker, Joseph F.

In: BBA - Bioenergetics, Vol. 347, No. 3, 28.06.1974, p. 443-463.

Research output: Contribution to journalArticle

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N2 - 1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10-13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes. 2. The FP ratio is typically in the range of 1.2-1.9 in Chlorella, 1.20-1.25 in Scenedesmus and 1.4-1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Qy transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented. 3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720-730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615-617). 4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01-0.06) determined by previous workers with unoriented cell suspensions. 5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered. 6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Qy transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane. 7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena. 8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.

AB - 1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10-13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes. 2. The FP ratio is typically in the range of 1.2-1.9 in Chlorella, 1.20-1.25 in Scenedesmus and 1.4-1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Qy transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented. 3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720-730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615-617). 4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01-0.06) determined by previous workers with unoriented cell suspensions. 5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered. 6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Qy transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane. 7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena. 8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.

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