308 IN THE DIATOM NZTZSCHIA CLOXTERIUM'

Recent experiments (4) indicate that carotenoid-sensitized photosynthesis occurs in the marine diatom Nikschia closterium. These experiments also show...
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308

n.

J. DUTTON,

w.

Y. M . ~ N I X G ,AND B. M. DUGGAR

GAFFRON, H.: Am. J. Botany87, 273 (1940). GAFFRON, H.: J. Gen. Physiol. 26, 195 (1942). H., AND WOWL,K.: Naturwissenschaften 24, 87, 103 (1936). GAFFRON, RIEKE,F. F.: Quantum efficiencies-to be published. M. D., HASSID,W,Z , AXD D E ~ A U LDT., C.: Science 90,570 (1939); RUBEN,S., KAMEN, J. Am. Chem. Soc. 62, 3447 (1940). (9) WELLER,S., AND FRANCK, J.: J. Phys. Chem. 46, 1359 (1941). (4) (5) (6) (7) (8)

CHLOROPHYLL FLUORESCENCE BND EXERGY TRANSFER IN T H E DIATOM NZTZSCHIA CLOXTERIUM' HERBERT J. DUTTOK,~WINSTOX hi. R.I.AXNING,~AND B. M. DUGGAR Departments of Botany and Limnology, University of Wisconsin, Madison, Wisconsin Received December 4 , 1942

Recent experiments (4) indicate that carotenoid-sensitized photosynthesis occurs in the marine diatom Nikschia closterium. These experiments also showed that the same enzyme system is probably involved in the thermal reactions of photosynthesis, whether the effective light is absorbed by carotenoids or by chlorophyll. Emerson and Lewis (5, 6) have obtained evidence for partial carotenoid participation in photosynthesis in Chroococcus, a blue-green alga, and in Chlorella, a green alga. In Chroococcus, their evidence for phycocyaninsensitized photosynthesis was much more conclusive than the evidence for carotenoid-sensitized photosynthesis. Sone of these results showed whether energy absorbed by carotenoid molecules was transferred directly to subsequent reactions in the photos2.nthetic process or transferred to chlorophyll with subsequent reactions the same as though the energy had been originally absorbed by the chlorophyll. The fluorescence experiments described below were designed to give information regarding the method of utilization of energy absorbed by carotenoids. Earlier studies with the green alga Chlorella have shown that the quantum yield (and also the wave-length distribution) of fluorescence for light absorbed only by chlorophyll is nearly constant over a considerable range of wave lengths (10). For an organism rich in carotenoid pigments, e.g., Nitzschia, a constant quantum yield for chlorophyll fluorescence a t various wave lengths, despite wide variations in the proportion of exciting light absorbed by the various pigments, xould be good evidence for an efficient transfer of absorbed energy from other pigments to chlorophyll. If there Tvere no transfer of energy, the yield of chlorophyll fluorThis work vias supported by grants from the Wisconsin Alumni Research Foundation. Present address: Western Regional Research Laboratory, Bureau of Agricultural Chemistry and Engineering, C. S. Department of Agriculture, Albany, California. 3 Present address: Division of Plant Biology, Carnegie Institution of Washington, Stanford University, California. 1

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ENERGY TRANSFER IN NITZSCHIA CLOSTERIUM

309

escence should vary in proportion to that fraction of the absorbed light which is absorbed by chlorophyll. EXPERIMENTAL PROCEDURE

Two unicellular algae were used in this study. Cultures of Nitzschia closterium, a diatom rich in yellow pigments (especially fucoxanthin), and of Chlorella pyrenoidosa, a green alga, were maintained by a procedure previously described for Nikschia (4), except that ChEoreEla was grown in a modified Warburg nutrient solution. A 2000-watt tungsten-filament spotlight lamp and a 1000-watt water-cooled high-pressure mercury arc (G. E. type H-6) were employed as light sources. Liquid filtzrs were used to isolate four spectral regions: namely, the 4358 .&. and the 5780 A. lines from th,e mercury arc an$ the regions 6600-5500 d. (average 6000 A.) and 5400-4000 A. (average 4700 A.) from the tungsten-filament lamp. Incident light intensities for most of the experiments were between 6000 and 25,000 ergs per cm.2 per second. For measurement of absorbed and fluoresced light, the algal suspensions (or the extracted pigments dissolved in acetone) were placed in a vessel having a capacity of 65 ml. and a distance of 0.5 cm. between the front and rear windows. A thermopile-galvanometer system was used for the measurement of intensities of incident and transmitted light. After the absorbed light was thus evaluated, the thermopile was removed from its position behind the algal suspension and replaced with a Weston photronic cell connected directly to a sensitive galvanometer. This more sensitive system was used for the measurement of the intensity of the fluorescent light. Two glass filters (Corning Nos. 597 and 241) placed between the algal suspension and the photocell absorbed practically all of the remaining incident li4ht and transmitted only the fluoresced light of wave lengths longer than 6800 A. In order to minimize the influence of physiological variation, measurements a t the wave lengths to be compared were made a t nearly the same time, using identical vessels and similar preparations from a single sample of plant material. A mixture of 5 per cent carbon dioxide in air was bubbled slowly through the algal suspensions during an experiment. This provided stirring and tended to maintain constant oxygen and carbon dioxide tensions. Measurements of fluorescence were made only after several minutes of illumination, since fluorescent intensities fluctuated over a wide range during the first minute or two of irradiation (cf. 7 and included references). The measurement of absolute quantum yields of fluorescence is made very difficult by the reabsorption of fluoresced light in an algal suspension or pigment solution. The same difficulty is encountered to a lesser extent in the measurement of relative quantum yields a t different wave lengths, but the magnitude of the error can be minimized by employing low concentrations, with a consequent low rate of absorption of fluoresced light. However, very low concentrations could not be used in the experiments described here, since the fluorescent intensities then became too small to measure. The usual practice was to use moderate

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H. J. DUTTON, 1%’. Y. MAXKING, AND B. M. DUGGAR

concentrations and to adjust either the concentration or the incident intensity until the total energy absorbed per unit time was approximately equal a t the two wave lengths for which relative fluorescent yi:lds mere to be determined. Calculations indicated that under these conditions errors due to reabsorption of fluorescence were relatively small. RESULTS

Table 1 summarizes the results of the fluorescence measurements, both for the suspensions of photosynthesizing algae and for the pigments dissolved in acetone. The figures in the t’hird column of the table, giving the calculated values for chlorophyll absorption, were obtained by methods described in an earlier paper (4). The calculations involve the assumption that, except for a shift in wave length, the pigment absorption in acetone is the same as the absorption in the living plant. All of the chlorophyll absorption in Nitzschia and in acetone extracts therefrom was calculated as being due to chlorophyll a. Subsequent experiments (9) indicate that another chlorophyll pigment, chlorofucine, is present as a natural constituent of diatoms, but it turns out that the calculated chlorophyll absorption in the wave-length regions employed here, as well as in the earlier photosynthetic studies (4), is not substantially altered when the chlorofuci2e spectrum is taken into consideration. The two wave lengths 5780 and GOO0 A. have been treated as equivalent in the presentation of results, since the difference in proportion of light absorbed by chlorophyll a t these wave lengths is too small to be significant for the experiments reported here. Table 2 shows the data and calculations for a typical experiment with Nikschia. Table 1 shows that for the living cells, both of N. closterium and of C:. pyrenoidosa, the observed ratios of fluorescent yields are 1.O within experimental error. The result is m p t striking in the case of Nitzschia, where the yield for incident light of 4700 A. is as high as the yiel? for red light, despite the small absorption calculated for chlorophyll at 4700 A. The results appear to constitute good evidence for an efficient transfer of absorbed energy from carotenoid molecules to chlorophyll molecules in the plastids of ~Vitzschia. The results for ChloreEla are in agreement with those for Nitzschia but are less conclusive because of the smaller number of experiments and the relatively low absorption by yellow pigments in Chlorella. On the other hand, the experiments with Chlorella may also be regarded as control experiments with an organism for which chlorophyll absorption predominates a t most wave lengths. From this point of view, the results for Chlo,elZa constitute a check on the results for Nitzscizia. The results shown in table 1 for the acetone extracts of N . closterium indicate that little energy is transferred from yellow pigments to chlorophyll in acetone solution. This difference betiveen in vivo and in vitro behavior suggests that the condition of the pigments in the plastids of Nitzschia is much more favorable for efficient transfer of energy than is the condition existing in acetone solution. The results for chlorophyll (American Chlorophyll Co. grade 5X) in acetone solution show equal yields a t 4358 and 5780 ,&. and constitute a further check on the assumptions involved in the experimental procedure.

TABLE 1 Results of the fluorescence measurement&

P E E CENT OF ABSORBED LIGHT ABSORBED

WAVE LENGTBS COMPARED

BY CBLOBO. PEYLL

Nitzschia ctosterium

CALCULATED FLUORESCENT YIELD RATIOS

Assuming no Assumenergy Ing transfer comfrom plete carot- transfer enoid to of chloro- energy Dhvll

I 1 1

1

6

10.52 1.0 11.05 d= 0.041

2

4358 us. 51 5780 or 6000 95 or gg 0.53 ~

Chlorella pyrenoidosa. . , . . , . , . . ,

$

5780 4700 or 21s. 8ooo

5780 4358 or us. 6ooo

1

19

4700 us.

6000

li

4358 ws.

5780

I

1.0

1.1 d= 0.2

I

10.81 1.0 10.93

0.181

4

0.19

1.0 0.22 et 0.02

2

0.40

1.0

0.49 i 0.07

2

__-__

Acetone extract of N . closterium

NUMBER OF PAIRS OF EXPEBILIENTS

OBSERVED RATIOS (GEOMETRIC LIEANI STANDARD ERROB)

;t

-

Acetone solution of chlorophylls a + b .......................

* The proportion of light absorbed by these two chlorophyll components is nearly equal a t 4358 and 5780A. Hence the yield ratio should be approximately 1.9,despite differences in the fluorescence spectra of chlorophylls a and b. TABLE 2 Calculation of fluorescent yield ratio for a n experiment with Nitzschia closterium Wave length of incident light.

.,

,,

1

1

aoooI.

Incident energy (transmitted through water-filled cell). . . . . . . . . . . . . . . . . . 12.10

x

103 ergs/cm.*/ second

Energy transmitted through cell filled with diatom suspension.. . . . .

4.85 X 103 ergs/cm.a/ second Absorbed energy.. . . . . . . . . . . . . . . . . . . 7.25 X 103 ergs/cm.*/ second Fluorescence (galvanometer deflection) ............................. 0.36 cm.*



4700A. 11.51 X 103ergs/cm.2/ second 4.56 X lo3 ergs/cm?/ second 6.95 X lo3 ergs/cm.l/ second

0.32 cm.

0.32 X 7.35 X 6000 = 1.18 Ratioof fluorescencequantumyieldat4700A. toyieldat6000i. = 0.36X 6.95 X 4700

* After a correction of 0.04 em. for incident radiation 311

passing suspension and filters.

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H. J. DUTTON, W. M. UAXNING, A S D B. M. DUGGBR DISCUSSIOR'

In interpreting the results of the fluorescence measurements it has been assumed that the chlorophylls are the only pigments shoFving appreciable fluorescence at wave lengths longer than 6800 A. This is in accord with the results of Wilschke (11) and Dh6r6 et al. (1,2,3),who observed only the fluorescence bands of chlorophyll in the red region of the spectrum for the several species of green algae and diatoms n.hich were examined. From the results shown in table 1, it may be concluded that carotenoid-sensitized photosynthesis in N . closterium takes place through the transfer of absorbed energy from carotenoid molecules to chlorophyll molecules with subsequent reactions the same as though chlorophyll molecules were the primary absorbers. However, the results do not eliminate the possibility that a small fraction of the carotenoid-sensitized photosynthesis may proceed by ot,her pat>hswithout, the action of chlorophyll as an intermzdiate. The accuracy of the fluorescence measurements was not great enough to show whether or not all of the carotenoid pigments in Nifzschia are ablc to transfer energy to chlorophyll. Fucoxanthin is present in high goncentration and must transfer most of the energy it absorbs a t 4358 and 4700 A,, since failure to do so would materially decrease the fluorescent yield at these n-ave lengths. However, any yelloTv pigments occurring in very low concentrations could be inactive without, appreciably reducing the quantum yields of chlorophyll fluorescence or of photosynthesis, The mechanism of energy transfer from carotenoid t o chlorophyll cannot be determined from these fluorescence measurements. Possible mechanisms are: ( 1 ) transfer by collision ; ( 2 ) intramolecular transfer (implying that carotenoid and chlorophyll units are present together in larger molecules in the plastid) : (3) radiation by carotenoid and immediate reabsorption by chlorophyll, a process analogous t o the internal conversion of nuclear gamma rays (8). Mechanisms intermediate between these might also be possible. SUMUSRY

The quantum yield of chlorophyll fluorescence in Nitzschia closterium was found to be constant, within rather large limitsoof experimental error, for exciting light of ware length 6000, 5780, 4700, or 4358 .4. Light absorbed by yelloir- pigments in Nitzschia can therefore reappear as chlorophyll fluorescence. This leads to the conclusion that the previously obsewed carotenoid-sensitized photosynthesis in N. closterium takes place principally through the transfer of absorbed energy from carotenoid to chlorophyll molecules with subsequent reactions the same as though chlorophyll molecules 1%-erethe primary absorbers. In acetone extracts of 21'. closterium light absorbed by the yellox pigments did not contribute appreciably to chlorophyll fluorescence, indicating that little or no energy \vas transferred between the pigments in acetone solution. REFERENCES ( 1 ) BACHRACB, E., AND DHERB,C.: Compt. rend. soc. biol. 108,385 (1931). (2) D H ~ R C.,~ AKD , FOXTAIBE, M.: Aiin. de l'inst. oc6anographique 10, 248 (1931).

KINETICS OF CHEMICAL REACTIONS

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(3) D H ~ RC.,~ AND , RAFFY,A , : Compt. rend. soc. biol. 119, 232 (1935). (4) DUTTON, H.J . , AND MANNING, W . M . : Am. J. Botany 26,516 (1941). (5) EMERSON, R . , AND LEWIS,C. M . : J. Gen. Physiol. 26, 579 (1942). (6) EMERSON, R., AND LEWIS,C. M . : In press. J., FRENCH, C. S., AND PUCK,T.T.:J. Phys. Chem. 46,1268 (1941). (7) FRANCK, (8)OPPENHEIMER, J. R . : Phys. Rev. 60, 158 (1941). (9) STRAIN,H . H., AND MANNING, W. M . : J. Biol. Chem. 144,625 (1942). D., WASSINK,E . C., AND REMAN,G. H.:Enzymologia 4, 254 (1937) (10) VERMEULEN, (11) WILSCHKE, A . : 2. wiss. Mikroskop. 31, 338 (1914).

A METHOD O F STUDY OF THE KINETICS OF CHEMICAL REACTIONS FRANCOIS OLMER' Laboratory oj Chemistry, &ole Nationale Supdrieure des Mines de P a r i s , France Received J u l y $4, 1848

The author has developed a new method for the study of chemical reactions. This method consists in raising the temperature of a chemical system linearly (that is, proportionally to the time) and observing the variations of some physical property of the system, for instance, the mass of one of the reagents. This method was first described by Guichard (1) and was applied to the study of the dehydration of certain hydrates by Vallet ( 5 ) , but has not yet, to the author's knowledge, been employed for the study of the kinetics of a pure chemical reaction. HOMOGENEOUS REACTIONS OF THE FIRST ORDER

In the case of a reaction in which two reagents, M and N, are both either liquid or gaseous and in which one of them, M, is in excess, the speed of the reaction a t a given temperature depends only on the mass x of the reagent N: dx = -kx dt

IC being the van't Hoff constant determined by the temperature 8: K = Aa"' where A, a, and CY are constants. If the temperature of the system increases uniformly with the time t,

e

= gt

the speed of the reaction may be written

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Present address: Diamond Alkali Co., Painesville, Ohio.