- Email: [email protected]
Bleaching of Chlorophyll by Digitoxin
Department of Botany, University of Minnesota, St. Paul, Minnesota 66108, USA
Bleaching of Chlorophyll by Digitoxin HERBERT joNAS
With 6 figures Received May 14, 1975 · Accepted July 16, 1975
Summary Digitoxin has been found to bleach chlorophylls a and b when administered in an alcoholic solution to the dry powder of freshly frozen leaves of Digitalis purpurea. The cardenolide causes the formation of a phase separation with distinct color renditions within the final chloroform-ethanol and chloroform-ethyl ether extracts. A detailed analysis of light absorption bands from the green to the red shows almost complete disappearance of the 526 and 661 nm peaks, the latter indicating a probable aggregation of a colloidal chlorophyll-adigitoxin complex by coordination of the Mg atom of chlorophyll with the Cn-carbonyl group of digitoxin with absorption in the 670 to 680 nm range and with a free energy of reaction of - 9.4 kcal. Chlorophyll-b reacted with digitoxin by an apparent increase of concentration at a low dose of 10 mg of digitoxin per 25 g of powder and by its destruction at a higher dose of 30 mg with a free energy release of - 9.6 kcal as shown by the behavior of its 644 nm absorption peak. The reactions are composed of three concentration dependent phases with distinct halfdose values. Some thoughts are stated about the ability of the plant to control its production of autotoxic secondary plant products, specifically in regard to chlorophyll. Its implications on the status of plant communities are pointed out.
Key words: Chlorophyll, bleaching, digitoxin, autotoxicity.
Zusammenfassung Die Chlorophylle a und b sind durch Digitoxin gebleicht worden, wenn dieses in einer alkoholischen Losung mit dem trockenen Pulver von frisch gefrorenen Blattern von Digitalis purpurea vermischt worden war. Die Kardenolide verursachten die Entstehung einer Phasentrennung mit bestimmten Farbtonungen in den endgiiltigen Chloroform-Athanol- und Chloroform-Athylather-Extrakten. Die Absorptionsmaxima 526 und 661 nm sind beinahe ganz verschwunden, was auf eine Aggregation von Chlorophyll a mit Digitoxin via Mg und C 2 ~ mit einer freien Reaktionsenergie von -9.4 kcal schlieflen Iaiit. Chlorophyll b ist mit einer freien Reaktionsenergie von -9.6 kcal gebleicht worden. Diese Reaktionen bestehen aus drei Phasen, welche von den Digitoxinkonzentrationen abhangen. Diese Befunde haben Bedeutung fur Probleme der Autotoxizitat und der Pflanzengemeinschaften. Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
43
Introduction The phytotoxic properties of extracts of Digitalis purpurea cause bleaching and necrosis of the leaves of the originating plants and of others as reported herein (JoNAs, 1969, 1972). This striking autoxicity to unsaturated lactones of steroid glycosides (cardenolides) is similar to that reported already by MACHT and KRANTZ in 1927 for seed germination. Such toxic effects represent a situation, in which the originating plant of a toxic metabolite possesses a control mechanism for limiting the in vivo concentration to a subtoxic level as far as its «normaL> development is concerned. In order to illustrate the particular case of the autotoxic effect of digitoxin on chlorophyll the following observations are presented.
Methods The procedure of this study was in part influenced by a concurrent series of experiments for developing a new approach to the extraction, purification and determination of Digitalis cardenolides (JoNAS, 1974). The experimental methods for the removal of pigments from these plant extracts was applied to the present investigation, which made use of the pigment extracts only. The aquous filtrates and the secondary extracts with 1 : 1 v /v of ethyl-ether and chloroform or with 2: 1 v/v of chloroform-ethanol were subjected to spectrophotometric analyses to determine the quantity and quality of the plant pigments. Spectrophotometry was performed by using the optics of a Beckman model DU spectrophotometer with a Keithley model 410 amplifier and a Heath model EU-20 B recorder for manual and visual readouts. The power-supply for the Hamamatsu 1 P 28 P.M. T. and for the RCA 918 gas photodiode detectors is a Keithley model 240 A power supply. The light sources are a standard automotive tungsten light and a Hellma D-102 deuterium lamp. Intensive optical and electrical calibrations enabled the determinations of up to five optical density units. However, the particular design of the monochromator does not permit a linear or logarithmic strip chart readout of the absorption spectra. It was found that a three-segment linear relationship exists between 1/}. of the interval J. 2-J. 1 and 3 X Jd, in which dis a time interval on the strip chart. Therefore an absorption spectrum obtained from a linear or logarithmic strip chart would present distorted information without further digital data processing. A presentation of the absorption peaks by abstracted diagrams has been chosen instead. All absorbancy values are normalized for sample dilution in order to represent concentrations in the original undiluted extracts. Where indicated, digitoxin U. S. P. was added to samples of dried leaf powder in order to observe its influences on the release of the pigments from the powder and on their destruction. Digitoxin was added as a 1 Ofo solution in 95 Ofo ethanol. Aliquots of 10, 20 and 30 mg (1, 2 and 3 ml) were diluted to 50 ml with 95 Ofo ethanol and then slurried with 25 g of leaf powder on a Vortex mixer. The ethanol was evaporated on a 40 °C waterbath. The dried powder was extracted as described. The supplemental digitoxin, being essentially insoluble in water, can enter reactions with the powdered leaf fragments during slurrying with the ethanolic solution. The water-ethanol gradient extraction permitted digitoxin to react with the surfaces of tissue fragments during the initial aquous stage, which was followed by reactions within them during the later ethanolic stage. Eluted digitoxin, both from native and added sources, was free to interact with the macerated tissue during the Soxhlet extraction stage and with the constituents of the subsequently recovered solutions.
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
44
H.
JoNAS
Results
The first indication of a specific action by digitoxin appears during the extraction process, when layering within some columns of extract can be observed (Table 1). Each layer possesses a distinct color and volume, which is dependent on the solvent and the dose of administered digitoxin, which may be termed an agent of phase separation.
Table 1: Phase separation and color rendition of 50 ml of leaf powder extracts. Type of extract
Volumes in ml and colors of each phase for each dose of digitoxin in mg/25 ml of sample 0 50 Bordeaux-*
10 50
20 50
30 50
Secondary, 1 : 1 chloroform-eth. ether
2 gold-yell. 48 dk. green
7 gold-yell. 43 dk. green
50 dk. green
50 dk. olive gr.
Secondary, 2: 1 chloroform-ethanol
1 red 49 gold-yell.
2 red 48 gold-yell.
T red 50 !.oak
T red 50 !.olive gr.
Primary, water after elimination of ethanol of the H20-EtOH gradient extraction
The aquous extract is uniformly Bordeaux red and shows an increasing visual density with an increasing dose of digitoxin. The transmission spectrum confirms this in the 625 nm range for the indicated doses to 20 mg/25 g of sample in the primary extracts. Absorbancy maxima (A = 10 /lx) exist at 495 nm for a 10 mg dose and also at 480 nm for a zero dose. In contrast, the secondary extracts exhibit phase separations to a varying degree. The chloroform-ethanol system produced a red layer superimposed over the bulk solution. As chloroform is immiscible with ethanol the layering must be due to a distribution of solutes. Increased doses of digitoxin result in a doubling of the red super-layer at the 10 mg dose and its disappearance at higher ones. The bulk solution turns from golden-yellow to a light olive green via light oak. The absorption spectrum (Figure 1) of the bulk solution shows a red shift from the 580 nm range for the 10 mg dose and less so progressively by the 20 and 30 mg applications and by the control. The increase of the digitoxin dose from 10 to 20 mg is accompanied by a color change from golden-yellow to light oak. The ether-chloroform bulk extract is dark green. Phase separation exists only in the zero and 10 mg doses. The ether-chloroform bulk extract contains more chlorophyll than the other extracts, which is desirable both from the points of view of purifying the digitoxin extract and of studying its interaction with the pigment. The multitude of distinct absorption peaks lends itself to the following analysis. Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
45
AI
....
/~ Water'~
10
500
600
Extracts A, nm 700
800
8 -
A 6
800
Fig. 1: Absorbancy maxima of extracts of Digitalis purpurea leaf powder with added digitoxin. Wavelengths A. in nm and absorbancy A corrected for sample dilution.
When plotting the maximum absorbancy of seven absorption bands of the bulk extract against the mean concentration of native plus the added dosage of digitoxin, all bands (Table 2, col. 4) exhibit a loss of absorption (Figure 2). Starting with the mean native digitoxin concentration in the leaf powder of 13.14 mg/25 g (JoNAS, 1974) all bands except the B-band 509-512 nm follow this trend. Bands A toE have lost all absorbancy at an extrapolated total digitoxin level of 45.7 mg, which is equivalent to 32.6 mg of added digitoxin or 2.6 mg in excess of the actual maximum addition. Shifts of absorbancy maxima occurred within each band in response to digitoxin. The C, G and B bands had blue shifts, while the D and A bands moved to the red. For purposes of elucidating in more detail changes in the absorption spectra of the chloroform-ether preparations the absorption bands require their identification in terms of specific forms of pigments. To accomplish this the experimental values are extrapolated to a) the theoretical absorbance (Figure 2 a) and the related peak waveZ. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
46
H. JoNAS
Table 2: Absorption bands and their assigned pigments of the chloroform-ethyl ether preparations. Band
Absorbancies in nm 2
3
4
5
Extrapolated to digitoxinfree leaves
Reported in the literature
Extent of bands
Pigments
A
526
527
HEATH, 1969
526-551
chlorophyll a
B
520
520
STRAIN and SvEc,
520-509
1966
644-627
phaeophytin b, (chlorophyll b?) chlorophyll b
c
644
643 642
D
661
663 662 660 661
E
517
HoLT, 1965; STRAIN, 1949 STRAIN and SvEc, 1966; HEATH, 1969 BROWN, 1972 FRENCH, 1960; FRENCH in BROWN, 1972 SRTAIN and SvEc, 1966; HEATH, 1969; STRAIN, 1949 STRAIN, 1949
517
F
G
865
661-681
chlorophyll a in vitro chlorophyll a aggregation chlorophyll a aggregation chlorophyll a artifact?
517
Iycopene
520s-522
phaeophytin b?
865-778s
length (Figure 2 b) for the state of complete absence of digitoxin, both native and added, and b) to the optical transmission at the extrapolated level of 45.7 mg of digitoxin. The postulated pigments with their absorption bands are given in Table 2. The absorption bands exhibit both changes in magnitude and peak wavelength. Bands A and D undergo almost complete destruction with a simultaneous red shift with increasing concentrations of digitoxin. While the significance of the A-shift remains obscure, that of band D can be construed as evidence for the formation of a chlorophyll-a aggregate in the 670 to 680 nm range (BROWN, 1972; FRENCH, 1960; BuTLER in BROWN, 1972; STRAIN and KATZ, 1969). In this case a digitoxin-chlorophyll-a aggregation might have taken place by a coordination between the Mg-atom of chlorophyll-a and the C23 -carbonyl group of digitoxin. In the absence of information about the site of cardenolide within or without the chloroplasts of Digitalis leaves, its coordination with Mg cannot be excluded in vivo (JoNAS, 1969). However the grinding of frozen and dried leaves and their subsequent water-ethanol gradient Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
47
extraction may create the proper conditions for the aggregation with the native cardenolide plus the added digitoxin in competition with the solvent. The red shift of the D-band appears to simulate or is even equivalent to the red shift observed during the formation of densely aggregated chlorophyll monolayers from 660 to 678 nm (TRURNIT and CoLMANO, 1959, figure 3) and from 659 to 680 nm (BELLAMY et al., 1963). Conversely, the deterioration of such monolayers is accompanied by a blue shift from the monolayer absorption maximum (KE and SPERLING, 1967, figure 4 replotted for time against log Amax. and log 6 A). The aggregate and a leuco-form of chlorophyll (Figure 2) at high digitoxin concentrations (DIJKMANS, 1973) apparently are sufficiently stable to survive the preparative stages leading to the final secondary extracts. The solubilization effect of the cardenolides could enhance the availability
9
7
6
<(
w'
u
z
~
~
4
<(
0
10
20
30
40
50
MG Of TOTAL DIGITOXIN IN 25 GRAMS OF DRY LEAF POWDER
Fig. 2 a (legend see page 48).
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
48
H.
}ONAS
--------0
800
•........................ G
~-----A
--------
-----------
.................................
SOO 0
10
20
30
40
srfOO
MG Of TOTAL DIGITOXIN IN 25 GRAMS OF DRY LEAF POWDER
Fig. 2b
Fig. 2: a) Maximum dilution-corrected absorbancy bands (A log ![) of ether-chloroform extracts as a function of total digitoxin content. Dotted sections refer to obscured experimental Amax. -values; dashed sections are extrapolations to values for leaf powder free of native and added digitoxin and for zero absorbance, where applicable. The inverted solid triangles at the upper margin designate from left to right the digitoxin content of the leaf powder of each treatment, 0, 10, 20, 30 mg, plus the native content of 13.140 ± 0.005 mg/ 25 g. - b) Shifts of the wavelengths of maximum absorbance with digitoxin concentration. Curve G refers to the 700 to 900 nm scale.
of chlorophyll and further the reaction. This hypothesis requires the acceptance of a loss of approximately 2 Ofo or less of digitoxin from the pigment-free final extract (Figure 5), which is being used for its assay. This aggregation might in turn possess colloidal properties, which also causes a red shift from the solution peak of 660 to
Z. P/lanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
49
1/G OF TOTAL DIGITOXIN IN 25 GM OF DRY LEAF POWDER
Fig. 3: Fraction of the remaining chlorophyll in the ether-chloroform extracts as a function of total digitoxin content as well as the corresponding ratio of chlorophyll a to b. Calculations are based on the absorption bands C and D (chlorophylls b and a, respectively). The molecular extinction coefficients are 86,300 for chlorophyll-a at 661 nm and 56,100 for form b at 643 nm.
663 nm to 670 nm (Figure 3 in lZAWA, 1969). However no Tyndall effect was observed visually in the final extract. A potentiation of photosystems I and II may also be considered (GoviNDJEE, 1971) as well as a situation similar to the reaction of chloroform-a with digitoxin in the 660 to 700 nm range (BAILEY et al., 1969; STRAIN and SvEc, 1966). In contrast, bands B and C respond to a low concentration of digitoxin by an increase of absorption and by a blue shift, both followed at higher concentrations by a strong elimination of light absorption with a stabilization of the lower Amax. Neither response can be assigned to a familiar chlorophyll-b reaction, though there may be a relation to a destruction of phaeophytin-b, which was probably formed by the 0.05 molal acidification. Bands F and G do not lend themselves to definite statements in the absence of available methods for greater spectral resolution. Based on these data the ratio of chlorophylls a: b is reduced by digitoxin, while their sum approaches zero as shown in Figure 3. Chlorophyll-b appears to be more stable, perhaps due to the 3-formyl group. Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
50
H.
JoNAS
__, __, >-
:X:
~
0
10
9:X:
__, __, >-
c
er::
:X: ~
u
0 er::
g
a/b ·-·---·
·--
:X:
u
1.0
0.1
O.O 0~--'--1'-0--'-...J20'-----L--'30_..,!___!40-..:L--l.50_
_L__6..LO_.L_...J70
TOTAL DIGITOXIN, mg /SAMPLE Fig. 4: Triphasic reaction (A, B, C) between digitoxin and chlorophyll as expressed by the logarithm of the fraction of unbleached chlorophylls a and b in the ether-chloroform extract as a function of the total amount of digitoxin in the leaf powder.
Discussion A triphasic relationship can be discerned between the total digitoxin concentration and the logarithm of the fraction of unbleached chlorophylls a and b remaining (Figure 3). The three phases A, B and C can best be characterized by their rates of the digitoxin effect and by its half-dose of effectiveness. These reactions increase in rate with increasing digitoxin concentrations, while the half-dose required to support them falls off. The apparent intermediate increase of the concentration of chlorophyll-b is quite evident, which is demonstrated when relating the mole-quantity of unbleached chlorophyll-b to the sample mass (Figure 5). This two component response of chlorophyll-b is even more evident, when the mole ratios of digitoxin to destroyed Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
+-0
..,
---
600
: b ---+ I
I
-,1
-'
0
\
X
UJ
-'
a.. ~
<{
Vl ~
Ol
0
9
"' ...!:
u
-- --
UJ
-'
0
~
I
0
0 w
400
\
\
\
''
10
UJ
:::cu "-,_
z
·~
200
30
0 ...... Ci
-'
<{
......
0......
,,
' I
·---------:~.
x
6
..
20
0c.::
...... Vl
'
'
>-
0
'a---+ I \
0
~
·~
+--- b -----·
Vl
w
-'
0 ~ -....... w
!~
9
51
\I
1:::: \\
40
60
0
TOTAL DIGITOXIN, mg /25g OF SAMPLE
Fig. 5: Quantitative evaluation of the interaction between digitoxin and chlorophyll in the ether-chloroform extract by comparing the concentrations of digitoxin with a) the moles of unbleached chlorophylls a and b per 100 g of sample and b) the mole ratios of digitoxin to destroyed chlorophylls a and b.
chlorophylls are inspected. The abrupt change of the slope at the 20 mg dose signifies this. In order to illustrate the dynamics of these reactions the equilibria between phases A and B and between B and C must be considered. The fraction of bleached chlorophyll in relation to the total available chlorophyll and to the unit mass of sample is highest at the interphase B-C. The supply of the 16.5 available moles of digitoxin per mole of bleached chlorophyll-a compares with the 110 available moles for chlorophyll-b, a ratio of 1: 6.7. However a ratio of only 1 : 1.4 exists for the junction A-B. These observations for these two equilibria permit the calculation of the free energies of reaction between digitoxin and the chlorophylls (Figure 6). The free energies of- 4.22 and- 4.58 kcal, respectively, for chlorophylls a and b at the junction A-B compare with - 5.20 and - 5.04 kcal at the B-C junction, which shows that the spontaneity of these reactions increases with the Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
52
H.
]ONAS CHLOROPHYLL
K ,(bleach chi) (chi) (dig1t)
~u -2
0 u
-4
h. Fo
h.(h.Fo)
-6
( B-C )
a
b
(A-B)
a
b
(A- B) -(B-C)
a
b
Fig. 6: Free energies of reaction between digitoxin and chlorophylls a and b (a, b) at the transition points of their reaction phases. The reaction constant K = (Bleached chlorophyll)/ (Unbleached chlorophyll) (Total digitoxin in the sample). The value for chlorophyll-b at the intersection of phases A and B refers to an apparent increase of concentration.
dose of digitoxin, regardless of whether a pigment is bleached at each dose or like chlorophyll-b produces an apparent augmentation at lower levels. The total free energies for both equilibrium points are -9.42 and -9.62 kcal, respectively, for chlorophylls a and b. The interrelation between these in-vitro processes and the bleaching of healthy plants of Digitalis purpurea, Nicotiana tabacum and of leaves of Lycopersicum esculentum, Coleus Blumei, and of Pelargonium zonale (JoNAS, 1969) remains to be clarified. The earlier attempt to formulate an explanation of bleaching of intact leaves may have to be reexamined in the light of this report. The mechanism for controlling the concentration levels of cardenolides by the living plant within acceptable limits of chlorophyll maintenance poses a challenge for further investigation, which has implications for an understanding of the formation and stability of plant communities by means of selected secondary metabolites. References BAILEY, J. L., and W. KREUTZ: Progr. in Photos. Res. I, 149 (1969). BELLAMY, W. D., G. L. GAINES Jr., and A. G. TwEET: J. Chern. Phys. 39, 2528 (1963). BROWN, J. S.: Ann. Rev. Plant Physiol. 23, 73 (1972). BuTLER, W. L.: Cit. BROWN, J. S.
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
53
DIJKMANS, H.: Europ. ]. Biochem. 32, 233 (1973). FRENCH, C. S.: Handbuch d. Pflanzenphysiologie V, 1, cit. page 279 (1960). - Cit. BROWN, ]. s. GoviNDJEE, and G. PAPAGEORGiou: Photophysiology VI, Chapter 1 (1971). HEATH, 0. V. S.: The Physiological Aspects of Photosynthesis, Stanford University Press, Stanford, California (1969). HoLT, A. S.: In: T. W. GoODWIN (Ed.), Chemistry and Biochemistry of Plant Pigments, Chapter 1., Academic Press, New York, 1965. IzAwA, S.: Progr. in Photos. Res. 3, 1742 (1969). JoNAS, H.: Z. f. Pflanzenphysiol. 60, 359 (1969). - Pl. Physiol. 49 suppl., 38 (1972). - Prep. Biochem. 4, 411 (1974). KE, B., and W. SPERLING: Brookhaven Symposium in Biology No. 10, 319 (1967). MACHT, D. I., and D. C. KRANTZ Jr.:]. Pharm. Expt. Therap. 31, 11 (1927). STRAIN, H. H., and].]. KATZ: Progr. Photos. Res. VII, 539 (1969). STRAIN, H. H., and W. S. SVEc: In: L. P. VERNON and G. R. SEELY (Eds.), The Chlorophylls, Chapter 2. Academic Press, New York, 1966. STRAIN, H. H.: In: ]. FRANK and W. E. LoOMIS (Eds.), Photosynthesis in Plants, Chapter 6. Iowa State University Press, Ames, Iowa, 1949. TRURNIT, H.]., and G. COLMANO: Biochim. Biophys. Acta, 30, 434 (1958).
HERBERT JoNAS, Department of Botany, University of Minnesota, St. Paul, Minnesota 66108, USA.
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin HERBERT joNAS
With 6 figures Received May 14, 1975 · Accepted July 16, 1975
Summary Digitoxin has been found to bleach chlorophylls a and b when administered in an alcoholic solution to the dry powder of freshly frozen leaves of Digitalis purpurea. The cardenolide causes the formation of a phase separation with distinct color renditions within the final chloroform-ethanol and chloroform-ethyl ether extracts. A detailed analysis of light absorption bands from the green to the red shows almost complete disappearance of the 526 and 661 nm peaks, the latter indicating a probable aggregation of a colloidal chlorophyll-adigitoxin complex by coordination of the Mg atom of chlorophyll with the Cn-carbonyl group of digitoxin with absorption in the 670 to 680 nm range and with a free energy of reaction of - 9.4 kcal. Chlorophyll-b reacted with digitoxin by an apparent increase of concentration at a low dose of 10 mg of digitoxin per 25 g of powder and by its destruction at a higher dose of 30 mg with a free energy release of - 9.6 kcal as shown by the behavior of its 644 nm absorption peak. The reactions are composed of three concentration dependent phases with distinct halfdose values. Some thoughts are stated about the ability of the plant to control its production of autotoxic secondary plant products, specifically in regard to chlorophyll. Its implications on the status of plant communities are pointed out.
Key words: Chlorophyll, bleaching, digitoxin, autotoxicity.
Zusammenfassung Die Chlorophylle a und b sind durch Digitoxin gebleicht worden, wenn dieses in einer alkoholischen Losung mit dem trockenen Pulver von frisch gefrorenen Blattern von Digitalis purpurea vermischt worden war. Die Kardenolide verursachten die Entstehung einer Phasentrennung mit bestimmten Farbtonungen in den endgiiltigen Chloroform-Athanol- und Chloroform-Athylather-Extrakten. Die Absorptionsmaxima 526 und 661 nm sind beinahe ganz verschwunden, was auf eine Aggregation von Chlorophyll a mit Digitoxin via Mg und C 2 ~ mit einer freien Reaktionsenergie von -9.4 kcal schlieflen Iaiit. Chlorophyll b ist mit einer freien Reaktionsenergie von -9.6 kcal gebleicht worden. Diese Reaktionen bestehen aus drei Phasen, welche von den Digitoxinkonzentrationen abhangen. Diese Befunde haben Bedeutung fur Probleme der Autotoxizitat und der Pflanzengemeinschaften. Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
43
Introduction The phytotoxic properties of extracts of Digitalis purpurea cause bleaching and necrosis of the leaves of the originating plants and of others as reported herein (JoNAs, 1969, 1972). This striking autoxicity to unsaturated lactones of steroid glycosides (cardenolides) is similar to that reported already by MACHT and KRANTZ in 1927 for seed germination. Such toxic effects represent a situation, in which the originating plant of a toxic metabolite possesses a control mechanism for limiting the in vivo concentration to a subtoxic level as far as its «normaL> development is concerned. In order to illustrate the particular case of the autotoxic effect of digitoxin on chlorophyll the following observations are presented.
Methods The procedure of this study was in part influenced by a concurrent series of experiments for developing a new approach to the extraction, purification and determination of Digitalis cardenolides (JoNAS, 1974). The experimental methods for the removal of pigments from these plant extracts was applied to the present investigation, which made use of the pigment extracts only. The aquous filtrates and the secondary extracts with 1 : 1 v /v of ethyl-ether and chloroform or with 2: 1 v/v of chloroform-ethanol were subjected to spectrophotometric analyses to determine the quantity and quality of the plant pigments. Spectrophotometry was performed by using the optics of a Beckman model DU spectrophotometer with a Keithley model 410 amplifier and a Heath model EU-20 B recorder for manual and visual readouts. The power-supply for the Hamamatsu 1 P 28 P.M. T. and for the RCA 918 gas photodiode detectors is a Keithley model 240 A power supply. The light sources are a standard automotive tungsten light and a Hellma D-102 deuterium lamp. Intensive optical and electrical calibrations enabled the determinations of up to five optical density units. However, the particular design of the monochromator does not permit a linear or logarithmic strip chart readout of the absorption spectra. It was found that a three-segment linear relationship exists between 1/}. of the interval J. 2-J. 1 and 3 X Jd, in which dis a time interval on the strip chart. Therefore an absorption spectrum obtained from a linear or logarithmic strip chart would present distorted information without further digital data processing. A presentation of the absorption peaks by abstracted diagrams has been chosen instead. All absorbancy values are normalized for sample dilution in order to represent concentrations in the original undiluted extracts. Where indicated, digitoxin U. S. P. was added to samples of dried leaf powder in order to observe its influences on the release of the pigments from the powder and on their destruction. Digitoxin was added as a 1 Ofo solution in 95 Ofo ethanol. Aliquots of 10, 20 and 30 mg (1, 2 and 3 ml) were diluted to 50 ml with 95 Ofo ethanol and then slurried with 25 g of leaf powder on a Vortex mixer. The ethanol was evaporated on a 40 °C waterbath. The dried powder was extracted as described. The supplemental digitoxin, being essentially insoluble in water, can enter reactions with the powdered leaf fragments during slurrying with the ethanolic solution. The water-ethanol gradient extraction permitted digitoxin to react with the surfaces of tissue fragments during the initial aquous stage, which was followed by reactions within them during the later ethanolic stage. Eluted digitoxin, both from native and added sources, was free to interact with the macerated tissue during the Soxhlet extraction stage and with the constituents of the subsequently recovered solutions.
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
44
H.
JoNAS
Results
The first indication of a specific action by digitoxin appears during the extraction process, when layering within some columns of extract can be observed (Table 1). Each layer possesses a distinct color and volume, which is dependent on the solvent and the dose of administered digitoxin, which may be termed an agent of phase separation.
Table 1: Phase separation and color rendition of 50 ml of leaf powder extracts. Type of extract
Volumes in ml and colors of each phase for each dose of digitoxin in mg/25 ml of sample 0 50 Bordeaux-*
10 50
20 50
30 50
Secondary, 1 : 1 chloroform-eth. ether
2 gold-yell. 48 dk. green
7 gold-yell. 43 dk. green
50 dk. green
50 dk. olive gr.
Secondary, 2: 1 chloroform-ethanol
1 red 49 gold-yell.
2 red 48 gold-yell.
T red 50 !.oak
T red 50 !.olive gr.
Primary, water after elimination of ethanol of the H20-EtOH gradient extraction
The aquous extract is uniformly Bordeaux red and shows an increasing visual density with an increasing dose of digitoxin. The transmission spectrum confirms this in the 625 nm range for the indicated doses to 20 mg/25 g of sample in the primary extracts. Absorbancy maxima (A = 10 /lx) exist at 495 nm for a 10 mg dose and also at 480 nm for a zero dose. In contrast, the secondary extracts exhibit phase separations to a varying degree. The chloroform-ethanol system produced a red layer superimposed over the bulk solution. As chloroform is immiscible with ethanol the layering must be due to a distribution of solutes. Increased doses of digitoxin result in a doubling of the red super-layer at the 10 mg dose and its disappearance at higher ones. The bulk solution turns from golden-yellow to a light olive green via light oak. The absorption spectrum (Figure 1) of the bulk solution shows a red shift from the 580 nm range for the 10 mg dose and less so progressively by the 20 and 30 mg applications and by the control. The increase of the digitoxin dose from 10 to 20 mg is accompanied by a color change from golden-yellow to light oak. The ether-chloroform bulk extract is dark green. Phase separation exists only in the zero and 10 mg doses. The ether-chloroform bulk extract contains more chlorophyll than the other extracts, which is desirable both from the points of view of purifying the digitoxin extract and of studying its interaction with the pigment. The multitude of distinct absorption peaks lends itself to the following analysis. Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
45
AI
....
/~ Water'~
10
500
600
Extracts A, nm 700
800
8 -
A 6
800
Fig. 1: Absorbancy maxima of extracts of Digitalis purpurea leaf powder with added digitoxin. Wavelengths A. in nm and absorbancy A corrected for sample dilution.
When plotting the maximum absorbancy of seven absorption bands of the bulk extract against the mean concentration of native plus the added dosage of digitoxin, all bands (Table 2, col. 4) exhibit a loss of absorption (Figure 2). Starting with the mean native digitoxin concentration in the leaf powder of 13.14 mg/25 g (JoNAS, 1974) all bands except the B-band 509-512 nm follow this trend. Bands A toE have lost all absorbancy at an extrapolated total digitoxin level of 45.7 mg, which is equivalent to 32.6 mg of added digitoxin or 2.6 mg in excess of the actual maximum addition. Shifts of absorbancy maxima occurred within each band in response to digitoxin. The C, G and B bands had blue shifts, while the D and A bands moved to the red. For purposes of elucidating in more detail changes in the absorption spectra of the chloroform-ether preparations the absorption bands require their identification in terms of specific forms of pigments. To accomplish this the experimental values are extrapolated to a) the theoretical absorbance (Figure 2 a) and the related peak waveZ. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
46
H. JoNAS
Table 2: Absorption bands and their assigned pigments of the chloroform-ethyl ether preparations. Band
Absorbancies in nm 2
3
4
5
Extrapolated to digitoxinfree leaves
Reported in the literature
Extent of bands
Pigments
A
526
527
HEATH, 1969
526-551
chlorophyll a
B
520
520
STRAIN and SvEc,
520-509
1966
644-627
phaeophytin b, (chlorophyll b?) chlorophyll b
c
644
643 642
D
661
663 662 660 661
E
517
HoLT, 1965; STRAIN, 1949 STRAIN and SvEc, 1966; HEATH, 1969 BROWN, 1972 FRENCH, 1960; FRENCH in BROWN, 1972 SRTAIN and SvEc, 1966; HEATH, 1969; STRAIN, 1949 STRAIN, 1949
517
F
G
865
661-681
chlorophyll a in vitro chlorophyll a aggregation chlorophyll a aggregation chlorophyll a artifact?
517
Iycopene
520s-522
phaeophytin b?
865-778s
length (Figure 2 b) for the state of complete absence of digitoxin, both native and added, and b) to the optical transmission at the extrapolated level of 45.7 mg of digitoxin. The postulated pigments with their absorption bands are given in Table 2. The absorption bands exhibit both changes in magnitude and peak wavelength. Bands A and D undergo almost complete destruction with a simultaneous red shift with increasing concentrations of digitoxin. While the significance of the A-shift remains obscure, that of band D can be construed as evidence for the formation of a chlorophyll-a aggregate in the 670 to 680 nm range (BROWN, 1972; FRENCH, 1960; BuTLER in BROWN, 1972; STRAIN and KATZ, 1969). In this case a digitoxin-chlorophyll-a aggregation might have taken place by a coordination between the Mg-atom of chlorophyll-a and the C23 -carbonyl group of digitoxin. In the absence of information about the site of cardenolide within or without the chloroplasts of Digitalis leaves, its coordination with Mg cannot be excluded in vivo (JoNAS, 1969). However the grinding of frozen and dried leaves and their subsequent water-ethanol gradient Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
47
extraction may create the proper conditions for the aggregation with the native cardenolide plus the added digitoxin in competition with the solvent. The red shift of the D-band appears to simulate or is even equivalent to the red shift observed during the formation of densely aggregated chlorophyll monolayers from 660 to 678 nm (TRURNIT and CoLMANO, 1959, figure 3) and from 659 to 680 nm (BELLAMY et al., 1963). Conversely, the deterioration of such monolayers is accompanied by a blue shift from the monolayer absorption maximum (KE and SPERLING, 1967, figure 4 replotted for time against log Amax. and log 6 A). The aggregate and a leuco-form of chlorophyll (Figure 2) at high digitoxin concentrations (DIJKMANS, 1973) apparently are sufficiently stable to survive the preparative stages leading to the final secondary extracts. The solubilization effect of the cardenolides could enhance the availability
9
7
6
<(
w'
u
z
~
~
4
<(
0
10
20
30
40
50
MG Of TOTAL DIGITOXIN IN 25 GRAMS OF DRY LEAF POWDER
Fig. 2 a (legend see page 48).
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
48
H.
}ONAS
--------0
800
•........................ G
~-----A
--------
-----------
.................................
SOO 0
10
20
30
40
srfOO
MG Of TOTAL DIGITOXIN IN 25 GRAMS OF DRY LEAF POWDER
Fig. 2b
Fig. 2: a) Maximum dilution-corrected absorbancy bands (A log ![) of ether-chloroform extracts as a function of total digitoxin content. Dotted sections refer to obscured experimental Amax. -values; dashed sections are extrapolations to values for leaf powder free of native and added digitoxin and for zero absorbance, where applicable. The inverted solid triangles at the upper margin designate from left to right the digitoxin content of the leaf powder of each treatment, 0, 10, 20, 30 mg, plus the native content of 13.140 ± 0.005 mg/ 25 g. - b) Shifts of the wavelengths of maximum absorbance with digitoxin concentration. Curve G refers to the 700 to 900 nm scale.
of chlorophyll and further the reaction. This hypothesis requires the acceptance of a loss of approximately 2 Ofo or less of digitoxin from the pigment-free final extract (Figure 5), which is being used for its assay. This aggregation might in turn possess colloidal properties, which also causes a red shift from the solution peak of 660 to
Z. P/lanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
49
1/G OF TOTAL DIGITOXIN IN 25 GM OF DRY LEAF POWDER
Fig. 3: Fraction of the remaining chlorophyll in the ether-chloroform extracts as a function of total digitoxin content as well as the corresponding ratio of chlorophyll a to b. Calculations are based on the absorption bands C and D (chlorophylls b and a, respectively). The molecular extinction coefficients are 86,300 for chlorophyll-a at 661 nm and 56,100 for form b at 643 nm.
663 nm to 670 nm (Figure 3 in lZAWA, 1969). However no Tyndall effect was observed visually in the final extract. A potentiation of photosystems I and II may also be considered (GoviNDJEE, 1971) as well as a situation similar to the reaction of chloroform-a with digitoxin in the 660 to 700 nm range (BAILEY et al., 1969; STRAIN and SvEc, 1966). In contrast, bands B and C respond to a low concentration of digitoxin by an increase of absorption and by a blue shift, both followed at higher concentrations by a strong elimination of light absorption with a stabilization of the lower Amax. Neither response can be assigned to a familiar chlorophyll-b reaction, though there may be a relation to a destruction of phaeophytin-b, which was probably formed by the 0.05 molal acidification. Bands F and G do not lend themselves to definite statements in the absence of available methods for greater spectral resolution. Based on these data the ratio of chlorophylls a: b is reduced by digitoxin, while their sum approaches zero as shown in Figure 3. Chlorophyll-b appears to be more stable, perhaps due to the 3-formyl group. Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
50
H.
JoNAS
__, __, >-
:X:
~
0
10
9:X:
__, __, >-
c
er::
:X: ~
u
0 er::
g
a/b ·-·---·
·--
:X:
u
1.0
0.1
O.O 0~--'--1'-0--'-...J20'-----L--'30_..,!___!40-..:L--l.50_
_L__6..LO_.L_...J70
TOTAL DIGITOXIN, mg /SAMPLE Fig. 4: Triphasic reaction (A, B, C) between digitoxin and chlorophyll as expressed by the logarithm of the fraction of unbleached chlorophylls a and b in the ether-chloroform extract as a function of the total amount of digitoxin in the leaf powder.
Discussion A triphasic relationship can be discerned between the total digitoxin concentration and the logarithm of the fraction of unbleached chlorophylls a and b remaining (Figure 3). The three phases A, B and C can best be characterized by their rates of the digitoxin effect and by its half-dose of effectiveness. These reactions increase in rate with increasing digitoxin concentrations, while the half-dose required to support them falls off. The apparent intermediate increase of the concentration of chlorophyll-b is quite evident, which is demonstrated when relating the mole-quantity of unbleached chlorophyll-b to the sample mass (Figure 5). This two component response of chlorophyll-b is even more evident, when the mole ratios of digitoxin to destroyed Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
+-0
..,
---
600
: b ---+ I
I
-,1
-'
0
\
X
UJ
-'
a.. ~
<{
Vl ~
Ol
0
9
"' ...!:
u
-- --
UJ
-'
0
~
I
0
0 w
400
\
\
\
''
10
UJ
:::cu "-,_
z
·~
200
30
0 ...... Ci
-'
<{
......
0......
,,
' I
·---------:~.
x
6
..
20
0c.::
...... Vl
'
'
>-
0
'a---+ I \
0
~
·~
+--- b -----·
Vl
w
-'
0 ~ -....... w
!~
9
51
\I
1:::: \\
40
60
0
TOTAL DIGITOXIN, mg /25g OF SAMPLE
Fig. 5: Quantitative evaluation of the interaction between digitoxin and chlorophyll in the ether-chloroform extract by comparing the concentrations of digitoxin with a) the moles of unbleached chlorophylls a and b per 100 g of sample and b) the mole ratios of digitoxin to destroyed chlorophylls a and b.
chlorophylls are inspected. The abrupt change of the slope at the 20 mg dose signifies this. In order to illustrate the dynamics of these reactions the equilibria between phases A and B and between B and C must be considered. The fraction of bleached chlorophyll in relation to the total available chlorophyll and to the unit mass of sample is highest at the interphase B-C. The supply of the 16.5 available moles of digitoxin per mole of bleached chlorophyll-a compares with the 110 available moles for chlorophyll-b, a ratio of 1: 6.7. However a ratio of only 1 : 1.4 exists for the junction A-B. These observations for these two equilibria permit the calculation of the free energies of reaction between digitoxin and the chlorophylls (Figure 6). The free energies of- 4.22 and- 4.58 kcal, respectively, for chlorophylls a and b at the junction A-B compare with - 5.20 and - 5.04 kcal at the B-C junction, which shows that the spontaneity of these reactions increases with the Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
52
H.
]ONAS CHLOROPHYLL
K ,(bleach chi) (chi) (dig1t)
~u -2
0 u
-4
h. Fo
h.(h.Fo)
-6
( B-C )
a
b
(A-B)
a
b
(A- B) -(B-C)
a
b
Fig. 6: Free energies of reaction between digitoxin and chlorophylls a and b (a, b) at the transition points of their reaction phases. The reaction constant K = (Bleached chlorophyll)/ (Unbleached chlorophyll) (Total digitoxin in the sample). The value for chlorophyll-b at the intersection of phases A and B refers to an apparent increase of concentration.
dose of digitoxin, regardless of whether a pigment is bleached at each dose or like chlorophyll-b produces an apparent augmentation at lower levels. The total free energies for both equilibrium points are -9.42 and -9.62 kcal, respectively, for chlorophylls a and b. The interrelation between these in-vitro processes and the bleaching of healthy plants of Digitalis purpurea, Nicotiana tabacum and of leaves of Lycopersicum esculentum, Coleus Blumei, and of Pelargonium zonale (JoNAS, 1969) remains to be clarified. The earlier attempt to formulate an explanation of bleaching of intact leaves may have to be reexamined in the light of this report. The mechanism for controlling the concentration levels of cardenolides by the living plant within acceptable limits of chlorophyll maintenance poses a challenge for further investigation, which has implications for an understanding of the formation and stability of plant communities by means of selected secondary metabolites. References BAILEY, J. L., and W. KREUTZ: Progr. in Photos. Res. I, 149 (1969). BELLAMY, W. D., G. L. GAINES Jr., and A. G. TwEET: J. Chern. Phys. 39, 2528 (1963). BROWN, J. S.: Ann. Rev. Plant Physiol. 23, 73 (1972). BuTLER, W. L.: Cit. BROWN, J. S.
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.
Bleaching of Chlorophyll by Digitoxin
53
DIJKMANS, H.: Europ. ]. Biochem. 32, 233 (1973). FRENCH, C. S.: Handbuch d. Pflanzenphysiologie V, 1, cit. page 279 (1960). - Cit. BROWN, ]. s. GoviNDJEE, and G. PAPAGEORGiou: Photophysiology VI, Chapter 1 (1971). HEATH, 0. V. S.: The Physiological Aspects of Photosynthesis, Stanford University Press, Stanford, California (1969). HoLT, A. S.: In: T. W. GoODWIN (Ed.), Chemistry and Biochemistry of Plant Pigments, Chapter 1., Academic Press, New York, 1965. IzAwA, S.: Progr. in Photos. Res. 3, 1742 (1969). JoNAS, H.: Z. f. Pflanzenphysiol. 60, 359 (1969). - Pl. Physiol. 49 suppl., 38 (1972). - Prep. Biochem. 4, 411 (1974). KE, B., and W. SPERLING: Brookhaven Symposium in Biology No. 10, 319 (1967). MACHT, D. I., and D. C. KRANTZ Jr.:]. Pharm. Expt. Therap. 31, 11 (1927). STRAIN, H. H., and].]. KATZ: Progr. Photos. Res. VII, 539 (1969). STRAIN, H. H., and W. S. SVEc: In: L. P. VERNON and G. R. SEELY (Eds.), The Chlorophylls, Chapter 2. Academic Press, New York, 1966. STRAIN, H. H.: In: ]. FRANK and W. E. LoOMIS (Eds.), Photosynthesis in Plants, Chapter 6. Iowa State University Press, Ames, Iowa, 1949. TRURNIT, H.]., and G. COLMANO: Biochim. Biophys. Acta, 30, 434 (1958).
HERBERT JoNAS, Department of Botany, University of Minnesota, St. Paul, Minnesota 66108, USA.
Z. Pflanzenphysiol. Bd. 77. S. 42-53. 1975.