JEANETTE S. BROWN
From the Carnegie Institution of Washington, Stanford, California 94305
ABSTRACT Density-gradient centrifugation of disintegrated cells from a variety of plants gave two kinds of chlorophyll particles from all except the blue-green algae. As in previous procedures using detergents, the lighter Fraction 1 particles usually had greater absorption at longer wavelengths; they always had a lower ratio of short to long wavelength fluorescence at low temperature, and a lower fluorescence yield per chlorophyll than the denser Fraction 2 particles. Although only one kind of particle fraction was found in each blue-green alga, the fine structure of the chlorophyll a absorption band differed significantly among the three species measured.
The absorption and fluorescence spectra of chlorophyll lipoprotein fractions prepared from a wide variety of plants have been measured, and a few examples are reported in this paper. The detection and characterization of the various forms of chlorophyll a by means of absorption spectroscopy have often been discussed (1-10). It is still not known if the various forms are identical as to peak position and width in different species; nor is it clear how the forms recognized in absorption spectra are related to the components of fluorescence spectra. The absorption anomalies which can be present in spectra of pigmented particles measured in scattering samples have beenreviewed by Butler (11). Possible artifacts in fluorescence measurements are even more numerous than in absorption spectrophotometry (12, 13). For the following reasons it may be unprofitable to draw conclusions from comparisons of the shapes and relative peak heights of fluorescence spectra measured in different laboratories. (a) The most common source of trouble is reabsorption of fluorescent light within the sample. Neither the absorption nor fluorescence spectra of whole algae or of normally green chloroplasts can represent the sums of the individual spectra of the component pigments because of reabsorption within single chloroplasts. Such distortion varies with the chloroplast size and pigment content (14). With disintegrated chloroplast particles that are themselves small enough to avoid distortion by internal reabsorption, it is possible to measure reliable fluorescence spectra if the concentration and sample thickness are small enough. If the sample in the measuring chamber is visibly green, we assume the fluorescence spectra will be erroneous. Only spectra that are proved to be free of reabsorption distortion by having identical shapes at concentrations or thicknesses varied by a factor of two, can be relied upon. (b) Fluorescence spectra are variously presented on the basis of either quanta or energy per unit of fre- quency, or of wavelength, i.e., Q/Av, Q/AX, E/Av, or E/AX. (c) The 680-685 nm band is so steep that the monochromator slit width is of critical importance in determining the shape of the curve. (d) Some published spectra are uncorrected for the wavelength variation of photomultiplier sensitivity and the monochromator transmission. One purpose of this work was to provide comparative data by measuring spectra for particles of similar sizes prepared from a variety of plants, using a spectropho- tometer designed to minimize errors due to light scattering. The second purpose was to correlate absorption and emission bands, recording both spectra from aliquots of the same sample of finely disintegrated particles. Although a correlation between long and short wavelength absorption and emission in fractions from the same species was observed, the data offered no satisfactory explanation for the different positions of the long wavelength emission maxima in different plants. In addition to the fluorescence peak of chlorophyll a in vivo near 680 nm, most plants at low temperature have a long wavelength emission band (15) and a few show a small band near 700 nm (16). Although some of the forms of chlorophyll a seen in absorption spectra may be weakly or not at all fluorescent, presumably one of them corresponds to each emission band. A third purpose of this study was to test density-gradient fractionation of dis- integrated cells without using a detergent on a wide variety of algae and higher plants. Michel and Michel-Wolwertz (17) found that after breaking chloroplasts, suspended in a KCI-Tricine buffer, in a French press and after centrifugation in a sucrose density gradient, three particle-fractions could be recovered from green bands in the gradient. In terms of chlorophyll a to b ratio, fluorescence spectrum and yield, and photochemical activity, the lighter fraction and the two heavier fractions corresponded respectively to the lighter 144,000 g particles (system 1) and to the 10,000g particles (system 2) isolated with the digitonin fractionation procedure of Boardman and Anderson (18). Two chlorophyll-containing particle fractions, differing in absorbance and fluo- rescence spectra, were isolated from several higher plants, a liverwort, several green algae, a red alga, and a diatom. No different chlorophyll fractions were ap- parent from the three species of blue-green algae, although the phycocyanin rated almost completely from the chlorophyll particles. Similar fractionation experiments with spinach, barley, Marchantia and Euglena were presented in a previous report (19).
Ideally the homogenate should contain between 0.5 and 1 mg chlorophyll per ml, but with the algae which were difficult to break, homogenates were usually obtained with 100-200 ,ug per ml. Linear, 12.5-50%, or step, 10-30-50%, top to bottom, gradients of sucrose dissolved in 0.15 M KCl, 0.05 M Tricine buffer at pH 8, were prepared in 34 ml centrifuge tubes. Two to four ml of homogenate were layered on top of the sucrose and spun at 64,000 g in the swing- ing-bucket rotor of a Spinco model L centrifuge (Spinco Div., Beckman Instruments, Inc., Palo Alto, Calif.) for 30-60 min depending on the plant species. The green bands in the gradient were removed with a syringe and either dialyzed against dilute buffer overnight or analyzed immediately. All steps in the procedure were carried out near 4°C.
The light intensity on the sample was approximately 2 X 104 ergs/cm2/sec`. The half bandwidth of the analyzing monochromator was 4 nm and the plot produced is in terms of relative quanta per unit frequency interval, Q/Av. The response of the apparatus was checked frequently against the emission of a Corning 3387 ifiter. The gain could be adjusted to correct for small changes in the lamp intensity, thus allowing a valid comparison of the fluorescence yield of samples recorded on different days. For comparisons of curve shapes the photomultiplier voltage was set to give curves of about the same size regardless of yield.
From the Carnegie Institution of Washington, Stanford, California 94305
ABSTRACT Density-gradient centrifugation of disintegrated cells from a variety of plants gave two kinds of chlorophyll particles from all except the blue-green algae. As in previous procedures using detergents, the lighter Fraction 1 particles usually had greater absorption at longer wavelengths; they always had a lower ratio of short to long wavelength fluorescence at low temperature, and a lower fluorescence yield per chlorophyll than the denser Fraction 2 particles. Although only one kind of particle fraction was found in each blue-green alga, the fine structure of the chlorophyll a absorption band differed significantly among the three species measured.
INTRODUCTION
The absorption and fluorescence spectra of chlorophyll lipoprotein fractions prepared from a wide variety of plants have been measured, and a few examples are reported in this paper. The detection and characterization of the various forms of chlorophyll a by means of absorption spectroscopy have often been discussed (1-10). It is still not known if the various forms are identical as to peak position and width in different species; nor is it clear how the forms recognized in absorption spectra are related to the components of fluorescence spectra. The absorption anomalies which can be present in spectra of pigmented particles measured in scattering samples have beenreviewed by Butler (11). Possible artifacts in fluorescence measurements are even more numerous than in absorption spectrophotometry (12, 13). For the following reasons it may be unprofitable to draw conclusions from comparisons of the shapes and relative peak heights of fluorescence spectra measured in different laboratories. (a) The most common source of trouble is reabsorption of fluorescent light within the sample. Neither the absorption nor fluorescence spectra of whole algae or of normally green chloroplasts can represent the sums of the individual spectra of the component pigments because of reabsorption within single chloroplasts. Such distortion varies with the chloroplast size and pigment content (14). With disintegrated chloroplast particles that are themselves small enough to avoid distortion by internal reabsorption, it is possible to measure reliable fluorescence spectra if the concentration and sample thickness are small enough. If the sample in the measuring chamber is visibly green, we assume the fluorescence spectra will be erroneous. Only spectra that are proved to be free of reabsorption distortion by having identical shapes at concentrations or thicknesses varied by a factor of two, can be relied upon. (b) Fluorescence spectra are variously presented on the basis of either quanta or energy per unit of fre- quency, or of wavelength, i.e., Q/Av, Q/AX, E/Av, or E/AX. (c) The 680-685 nm band is so steep that the monochromator slit width is of critical importance in determining the shape of the curve. (d) Some published spectra are uncorrected for the wavelength variation of photomultiplier sensitivity and the monochromator transmission. One purpose of this work was to provide comparative data by measuring spectra for particles of similar sizes prepared from a variety of plants, using a spectropho- tometer designed to minimize errors due to light scattering. The second purpose was to correlate absorption and emission bands, recording both spectra from aliquots of the same sample of finely disintegrated particles. Although a correlation between long and short wavelength absorption and emission in fractions from the same species was observed, the data offered no satisfactory explanation for the different positions of the long wavelength emission maxima in different plants. In addition to the fluorescence peak of chlorophyll a in vivo near 680 nm, most plants at low temperature have a long wavelength emission band (15) and a few show a small band near 700 nm (16). Although some of the forms of chlorophyll a seen in absorption spectra may be weakly or not at all fluorescent, presumably one of them corresponds to each emission band. A third purpose of this study was to test density-gradient fractionation of dis- integrated cells without using a detergent on a wide variety of algae and higher plants. Michel and Michel-Wolwertz (17) found that after breaking chloroplasts, suspended in a KCI-Tricine buffer, in a French press and after centrifugation in a sucrose density gradient, three particle-fractions could be recovered from green bands in the gradient. In terms of chlorophyll a to b ratio, fluorescence spectrum and yield, and photochemical activity, the lighter fraction and the two heavier fractions corresponded respectively to the lighter 144,000 g particles (system 1) and to the 10,000g particles (system 2) isolated with the digitonin fractionation procedure of Boardman and Anderson (18). Two chlorophyll-containing particle fractions, differing in absorbance and fluo- rescence spectra, were isolated from several higher plants, a liverwort, several green algae, a red alga, and a diatom. No different chlorophyll fractions were ap- parent from the three species of blue-green algae, although the phycocyanin rated almost completely from the chlorophyll particles. Similar fractionation experiments with spinach, barley, Marchantia and Euglena were presented in a previous report (19).
EXPERIMENTAL
Fractionation Procedure Algae or chloroplasts were sedimented by centrifugation at 3000 g for 10 min, washed once with distilled water, and resuspended in 0.3 M KCI, 0.05 M Tricinel adjusted to pH 8. The suspension was forced through a needle valve by approximately 12,000 psi (8300 NI cm2) pressure at least three successive times. Some algae required more passes in order to break a sufficient number of cells. The broken mixture was centrifuged as before to remove larger particles yielding the suspended homogenate.
Ideally the homogenate should contain between 0.5 and 1 mg chlorophyll per ml, but with the algae which were difficult to break, homogenates were usually obtained with 100-200 ,ug per ml. Linear, 12.5-50%, or step, 10-30-50%, top to bottom, gradients of sucrose dissolved in 0.15 M KCl, 0.05 M Tricine buffer at pH 8, were prepared in 34 ml centrifuge tubes. Two to four ml of homogenate were layered on top of the sucrose and spun at 64,000 g in the swing- ing-bucket rotor of a Spinco model L centrifuge (Spinco Div., Beckman Instruments, Inc., Palo Alto, Calif.) for 30-60 min depending on the plant species. The green bands in the gradient were removed with a syringe and either dialyzed against dilute buffer overnight or analyzed immediately. All steps in the procedure were carried out near 4°C.
Spectral Measurements
The absorption spectrophotometer and special cuvettes have been described (20). Both were designed to measure a representative fraction of the scattered light. A metal finger of the cuvette was immersed in liquid nitrogen to cool the sample to -196°C. The samples were diluted with buffer to give an absorbance of less than 0.5 at 675 nm. The spectrofluorimeter (21) automatically corrected the emission spectra for the spectral sensitivity of the appartus. Excitation at 435 nm, half-width 4.8 nm, was provided by a xenon lamp used with a Bausch & Lomb "high intensity" monochromator (Baush & Lomb Incor- porated, Rochester, N.Y.) and a Corning 9782 (4-96) filter (Corning Glass Works, Corning, N.Y.) to remove traces of stray light in the 650-770 nm region.
The light intensity on the sample was approximately 2 X 104 ergs/cm2/sec`. The half bandwidth of the analyzing monochromator was 4 nm and the plot produced is in terms of relative quanta per unit frequency interval, Q/Av. The response of the apparatus was checked frequently against the emission of a Corning 3387 ifiter. The gain could be adjusted to correct for small changes in the lamp intensity, thus allowing a valid comparison of the fluorescence yield of samples recorded on different days. For comparisons of curve shapes the photomultiplier voltage was set to give curves of about the same size regardless of yield.
CONCLUSIONS
One reason that the physical-chemical nature of the absorbing forms of chlorophyll a has remained obscure is because of the difficulty in separating them without de- struction. The procedure described above effects a partial separation and is appli- cable to a wide variety of plants. These very small chlorophyll-lipoprotein particles which differ in their chlorophyll absorption should be excellent material for t1w ther study to determine the aggregation and lipid or protein binding of chlorophyll in its native functional state. Since the same fractionation procedure produced two corresponding chlorophyll particles from so many different plants, the basic arrangment of the chlorophyll- lipid-protein molecules is probably similar in these plants.
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