710
8. RUBEN, A. W. FRENKEL AND Id. D. =MEN
SOME EXPERIMENTS ON CHLOROPHYLL AND PHOTOSYNTHESIS USING RADIOACTIVE TRACERS 8. RUBEN Chemical Laboratory, University of California, Berkeley, California
A. W. FRENKEL Department of Botany, University of California, Berkeley, California AND
M. D. KAMEN
Radiation Laboratory, University of California, Berkeley, California Received January 7 , 104.8
Wilstiitter and Stoll (13) observed that in acid solution the magnesium of chlorophyll is displaced by two hydrogen ions. Recently, Mackinney and Joslyn (7) have reported that Mg++ in chlorophyll a dissolved in aqueous acetone is replaced at a rate some eight to nine times greater than is the case for chlorophyll b. The reason for this difference in reaction rates is a t present unknown. According to Fischer and others (31,chlorophyll b differs structurally from chlorophyll a only in that the CH8 group in position 3 is replaced by a CHO group. The difference in rate of magnesium displacement might be clue to greater lability of magnesium in chlorophyll a. This suggestion may be tested by exchmge experiments with radioactive M P . In addition, if the rate of exchange with Mg++ were markedly different for the two chlorophylls, it would lead to a condition in which chlorophyll a was labeled with radioactive magnesium. Such an event would prove very useful in a study of the r6le of chlorophyll in photosynthesis. We have in mind the interesting suggestion of Dixon and Ball (2) that, during photosynthesis by green plants', the two chlorophylls are involved in a reversible oxidation-reduction cycle. The cycle has been formulated as follows: 1 nlhv RCHs COZ-+ RCHO ;(H&O), (1)
+
%hv
+
+
+ RCHO + Hz0 -+
RCHI
+ 02
(2)
the net reaction beiig (nl
+ m)hv + COZ+ HzO
-+
0 1
+
(HzCO),
(3)
where RCH8 and RCHO are chlorophylls a and b, respectively. Evidence (4,8,9,11), both indirect and direct, has been recently presented which indicates that such a cycle, if it occurs at all, does not consist of reactions 1 and 2 as such. Nevertheless the idea of a reversible oxidation-reduction cycle in1 While the great majority of plants contain the two chlorophyll components, in a few groups, notably among brown algae, i t has been reported that chlorophyll b could not be detected.
RADIOACTIVE TRACERS IN PHOTOSYNTEEBIS
711
volving the two chlorophylls can be formulated in other ways; moreover, it is not unreasonable when considered in the light of recent work on biocatalysta (enzymes, etc.). If one of the chlorophylls could be labeled in some manner, my with radioactive magnesium, a direct study of the interconversion cycle would be poasible. However, there is one serious experimental difficulty: namely, the short halflife (10.2 min.) of Mg2', which at the time these experiments were started was the only known radioactive isotope of magnesium. A search for MgZ3,which could be produced by the nuclear reaction NaZ3(42n) Mgz3,waa made in the hope that it would have a longer half-life. Bombardment of metallic sodium with 16 MeV. deuterons for 100 microampere hours in the 60-in. cyclotron of the Radiation Laboratory failed to produce a detectable long-lived Mg activity. Shortly afterward, White et d. (12) reported the production of Mgz3 with a half-life of 11.6 sec. However, it wm considered worthwhile to attempt the chlorophyll experiments using Mgs7as a tracer. EXPERIMENTAL
Metallic magnesium waa bombarded with 50 for 10 min. The nuclear reaction is
ID2
+ MgZB
--t
Mgz7
p
amperes of 8 MeV. deuterons
+ IH'
The magnesium waa dissolved in concentrated hydrochloric acid, and excess sodium hydroxide was added to precipitate magnesium hydroxide. The precipitate was filtered, washed, and dissolved and reprecipitated three times in order to remove the last traces of radioactive sodium (NaZ4), which is also produced by the deuteron bombardment. The final magnesium hydroxide, rid of NaZ4, was converted into aqueous magnesium chloride and used in this form for the experiments described below.
Plant experiments To a 0.1 M sodium bicarbonate solution containiing 3 cc. of young actively photosynthesizing unicellular green algae (ChloreZZa pyremidosa) waa added 10-3 mole of magnesium chloride having an activity of 2 X lo7counts per minute. The algae were illuminated and after 30 min. were centrifuged and washed once with distilled water. The chlorophyll was extracted with boiling methanol and acetone. Water waa added to the acetone-alcohol extract until it comprised N 20 per cent by volume; this solution was then extracted with petroleum ether. The petroleum ether layer which contained the major fraction of the chlorophyll was rigorously washed with a large volume of aqueous sodium chloride solution to remove water-soluble impurities. An aliquot of this chlorophyll solution was pipetted off and was found to contain radioactive magnesium. In control experiments it was demonstrated that magnesium salts are completely removed from petroleum ether by several washings with aqueous sodium chloride solutions. An attempt wm made to separate chlorophylls a and b and examine each for radiomagnesium. It waa hoped that, if the magnesium in chlorophyll
712
5. RUBEN, A . W. FRENKEL AND M. D. KAMEK
a was more labile ‘than in chlorophyll b, a dark control experiment should show the radio-Mg++ to be mainly in chlorophyll a, Le., the specific activity of chlorophyll a would be greater than that of chlorophyll b ; whereas in the light, if there were interconversion of the two chlorophylls, the specific activities should be equal. It was not feasible to use the WillstatterStoll method of separating the two chlorophylls from one another because of the length of time required. The chromatographic adsorption method, using talc, was tried. However, this proved unsuccessful because by the time an adequate separation was obtained and the pigments removed from the talc column, the activity of the MgZ7was too low for satisfactory measurement. Similar experiments were tried with young barley plants, the root system of which was vigorously aerated in order to increase the rate of salt absorption ( 5 ) . In addition, the Mg* was added as magnesium nitrate, since magnesium ion is most readily absorbed in this form. In these experiments, as observed with the unicellular algae, a chlorophyll solution containing radiomagnesium was obtained. Again, however, the activity was too low to enable a separation of the two chlorophylls. The specific activity of the chlorophyll (a 4- b ) was only 2 per cent of the value calculated for complete randomness of the Mg* between the chlorophyll and magneiium ions. Thif result could be due to any of the following reasons: ( I ) Slow diffusion of Mg*++ into the cells containing chlorophyll, coupled with the circumstance that only a small fraction of the chlorophyll may be in contact uith the aqueous solution, the remainder being “buried” below the surface of the chloroplast. To avoid this possible difficulty, it might be advisable to employ systems in which the pigments are in solution and therefore more accessible for exchange. (2) The magnesium of the native chlorophyll-protein complex may not exchange readily with magnesium ions. (3) The presence of Mg27 in the chlorophyll could be due to the formation, during extraction, of an associati’on complex between chlorophyll and magnesium ion (see below). Despite our lack of success using short-lived Mg2’, it would, appear that valuable information (which is sorely lacking) regarding the r6le of chlorophyll in photo5ynthesis could be obtained, provided a longer-lived tracer were available. When the separated stable isotopes of magnesium are available, this problem may be fruitfully studied.
-
Exchange rrperiments an vatro wa2h chlorophyll Although little is known regarding the state of chlorophyll tn vzvo, it is known to be combined nith protein (1, 10). Because of this fact, it is generally agreed that the chemical properties of native chlorophyll may be quite different from those of chlorophyll dissolved in organic solvents. Severtheless, a study of chlorophvll free of its protein is of some interest for its own sake. We have qtudied the exchange of various preparations of chlorophyll with Mg*++. Puri-
713
R4DI0.4CTIVE TRACERS IN PHOTOSYNTHESIS
fied crude mixtures of chlorophyll were obtained by treating fresh spinach leaves with 80 per cent acetone. The chlorophyll was extracted from the abueous acetone with petroleum ether. After careful prolonged washing of the petroleum ether layer with water, the chlorophyll precipitated and was filtered off. Samples obtained in this manner contain small quantities of the carotenoids, particularly xanthophyll. The exchange experiments consisted 200 mg. of this chlorophyll (a b ) dissolved in of shaking for 10 to 20 min. 80 per cent acetone with a Mg*Cl, solution (pH 7 to 8). The chlorophyll was extracted with petroleum ether, which mas rid of traces of magnesium ion by copious washings with aqueous sodium chloride. The resulting petroleum ether solution of chlorophyll was strongly radioactive. The results of a typical experiment are summarized in table 1
-
+
TABLE 1 "Ezchange" of Mg++wdh crude chlorophylls a and b RADIOACTIVITY'
UOLES OF
Mg++
1.65
x
1
Chloroph 11 (a
+by
Z X l O - Y !
(COUNTS PER MINUTE)
-
Chlorophyll
Mg++
1.67X10'
(a
~
+ b)
OX10'
PEPCENTOFRANDOM DlSTPlBUIlON Or h.Igc* AITAINFD
47
* The figures are comparable, sincc they are corrected for Mgz' decay to the time of counting the chlorophyll. TABLE 2 Non-ezchange OJ M g + + with pure chlorophylls a and b
* See footnote t o table 1.
We have also studied the exchange with pure chromatographically separated (6) samples of chlorophylh a and b.2 The results are tabulated in table 2. I t is apparent from table 2 that no exchange has taken place in 10 min. This is indeed a surprising result, since the civde chlorophyll preparations did take up Mg*++. Two explanations for this anomaly suggest themselves: ( 1 ) Mg*++ has not replaced the magnesium in the center of the phorbin 1~1g but rather is held by some other coordinating group of the chlorophyll molecule or another compound present in the crude preparation. (2) Chlorophyll is "activated" by a substance present in the crude extract but absent in the highly purified a and b samples. Explanation 1 seems most likely on the following grounds: Only fresh prepara-
* Very kindly supplied by Professor C . hfackinney.
714
8. RUBEN, A. W. FRENKEL AND M. D. KAMEN
tions of chlorophyll (or those kept in v a n t o ) will take up Mg*++. The product formed will liberate the Mg- in dilute acid and also in dilute alkaline solutions. The latter behavior is markedly different from that of magnesium held in the center of the phorbin ring. The centrally located magnesium is not liberated in concentrated alkali (13). Fresh chlorophyll preparations will also associate radioactive cupric ions'. When the cupric addition complex is run through a Tswett adsorption column (containing talc, inulin, etc.), the radiocopper is found in the chlorophyll a band. Attempts to separate chlorophyll a from the cupric addition complex were unsuccessful. The chlorophyll b band waa almost devoid of radioactivity. If chlorophyll solutions were allowed to stand at mom temperature for a few days or adsorbed on the talc, the ability to associate magnesium ion or cupric ion was lost, indicating a chemical change had taken place. When fresh chlorophyll was added to chlorophyll a which had been taken up was the same as previously adsorbed on talc, the amount of Cuin the absence of the extra chlorophyll a. This indicates that fresh chlorophyll solutions do not contain material which catalyzes the formation of the addition complex. Furthermore, it is interesting to note that fresh preparations of It is known from the work of Willstiitter pheophytin will sssociate Mg-. and others that magnesium ion does not enter the phorbin ring under these conditions. SUMMARY
1. Using short-lived radioactive magnesium as a tracer, an unsuccessful attempt was made to detect the interconversion of chlorophylls a and b during photosynthesis. 2. Purified crude mixtures of chlorophyll a and b take up Mgand Cu-, but the separated highly purified components of chlorophyll do not exchange with magnesium ion in 80 per cent acetone. The authors wish to thank Professor G . Mackinney for hie interest and helpful advice. They are also grateful to Professor E. 0. Lawrence and members of the Radiation Laboratory for their coiiperation in making this work possible, REFERENCES (1) ANRON: Science W. 186 (1941). (2) DIXONAND BALL:Notes from Botanical School, Trinity College, Dublin S, 52 (19%). AND STERN:Die Chemie de8 PyrOl8, Vol. 11, p. 23. Akademische Verlags(3) FISCHER gesellschaft, Leipzig (1940). (4) FRANCK AND GAFFRON: Advances in Enzymology 1, 199 (1941). AND ARNON:Soil Sei. 61, No. 6 (1941). (5) HOAQLAND (6)MACKINNEY: J. Biol. Chem. 1s0, 91 (1940). (7) MACKINNEY AND JOSLYN: J. Am. Chem. SOC.6% 2530 (1941). (8) RUBEN,KAMEN, AND HASSID:J. Am. Chem. SOC.83,3443 (1940). (9) RUBEN,RANDALL, KAMEN, AND HYDE:J. Am. Cbem. SOC. 89,877 (1941). (10) SMITH:J. Gen. Physiol. 94, 665 (1941). (11) VANNIEL:Advances in Enzymology 1.273 (1941). (12) WHITE,DELSASSO, Fox, AND CREUTZ: Phys. Rev. 66,512 (1939). (13) WILLSTA~TER AND STOLL:Untersvehungen Gbet Chlorophyll. Springer, Berlin (1913). 8
We employed CUM,which has a half-life of 12.8 hr.
