662
JOHS J. MCBRADY .1SD ROBERT LIVISGSTOX
RET-ERSIRLE PHOTOBLEA4CHISG OF CHLOROPHYLL1 JOHS J. LIcBR.SDT*
4x1)
School of C h e m i s t i y , I i l s t z t i ~ t esf l ' c c h n d o g y ,
RORLRT LIT ISG>?'OS n i c e ~ i l yof AIzrinesota, A\fllznneapolis 14,
Mzriiiesuta IzEcelLed A l r g l t s l 13, 19::
The reversible photobleaching of chlorophyll 17 as diwovered by Porret and Rabinon itch in 1937 ( 7 ) . Its existence T\ as confirmed aiid it was further studied by Livingston in 1941 (4). Since the experimentq, both photometric and chemical, are difficult, both the earlier and the present researches are more or less exploratory in character. The present resultq, \\ liile confirming some of the earlier findings, necessitate some modification of the mechanism suggested previously (1, 4). EXPERIMESTAL METHODS A S D 3IATERIAL
The photometer The bleaching was measured \I-ith a differential photometer. It v-as so arranged that one of the absorption cells could be illuminated from the side, n-ith a beam of intense light, without disturbing the photometric measurements. This apparatus is similar t o but is an improvement on that described elsen-here by 3IcBrady and LiT-ingston (6). To att'ain the precision desired for the present work, it n-as found necessary t o stabilize the mechanical system aiid to improve the amplifier.3 Figure 1 is a circuit diagram of the amplifier. The apparatus is relatively free from drifts and fluct'uations, and is capable of measuring relative changes of transmission of 0.01 per cent, reliably and reproducibly. The method of calibrating the photometer n-as similar t o that described by Liringston (4). The source of actinic light vas a 1000-n-att, projection lamp, Tvhich was housed in a double-n-alled \T-ater-cooled cylindrical jacket (figure 2 ) . The cooling lvater flon-ed between the glass mdls of the n-indon- of the lamp house, and thereby removed much of the heat radiation from the light beam. AAfilter system, consisting of a cupric sulfate cell and a Corning glass filter S o . 348, transmitted the red end of the spectrum but absorbed the blue and ultraviolet as xell as the greater part of the infrared. -4 spherical condensing lens, follon-ed by a cylindrical lens, served t o concentrate the beam fairly uniformly over the length of the reaction cell. The lamp \vas run on D.c., hand controlled t o 8 amp. -1lternating current could not be used, since the response of the photocell and amplifier to the fluctuat,ingfluorescent light is not negligible. The use of the reduced voltage increased the life of the lamp, n-ithout greatly reducing the intensity of the red light. * This ~ v o r k\vas made possible t)y t h e support of the Office o f S a v a l Research (Contract S6ori-212, Task Order 1 I . I t vas also supported i n part by a grant-in-aid froin thc Graduate School of the I'niversity of lIinnesota. 2 Present address : &search Lahoratorics, C'elancse C,'orpur;it ion of America. Suinmit , SF\\-Jersey. 3 lye arc indebted t o l l r . George DelVitt of t h e Dcpartlucnt of Physics of t h e L-niversity of lIinncsota for valuable assistance in redcsigning and testing this circuit.
REVERSIBLE PHOTOBLE-ICHIKG O F CHLOROPHYLL
663
The filter combination used on the scanning light (4) isolated a narrow band with its maximum a t approximately 6550 A.
FIG.1. Diagram of t h e amplifier. The indicated resistances have t h e following values in megohms: Itl, 100; R?. 100; R:, 0.24; RA, 1.0; Ra, 0.005; Re, 2.0; R;. 0.5; Rs, 0.004; RIO. 1.6; R1i,0.01; R1?.0.5;Rls,0.0015;It14,0.001;RIj, 2.0. RBi s t h e g a i n control potentiometer, constructed of several xire-1%-oundresistors. Its total resistance is 1.5 niegohnis. RIGis a 0.01-megohm potentiometer, used to control t h e S o . 2 grid of 954. R1i is a 0.003-megohm Ayrton shunt which regulates t h e input t o t h e galvanometer. The indicated condensers have the following capacities in microfarads: CI, 0.0002; C?, 0.002; Car 4.0; C4, 0.05; CS, 0.002; Cc.4.0; C;. 0.1; C,, 0.002; Cy,4.0: Cl0,5.0; Cll,20; Clp,1.0; Cia, 1.0. L i s a f i l t e r c h o k e tuned by a suitable condenser for t h e 150-cycle signal. T is a coupling transformer, tuned for 150 cycles. Positive potentials are a s indicated, -B being grounded.
FIG.2 . Diagram of xater-cooled lamp house for 1000-xatt projection l a m p .\I.ITERI.\LS
The chlorophyll used in the majority of these experiments IT-as isolated from market spinach by a modification ( 5 ) of Zscheile's method (11). 3Iost of the experiments vere performed n-ith solutions of pure chlorophyll a , which n-ere checked (11) for pheophytin and chlorophyll b content with the Beckmann
664
JOHN J. 3lCBRADY ASD ROBERT LIVISGSTOS
spectrophotometer.4 -4felv esperinients iT-ere performed n-ith chlorophyll b solutions, which were prepared and checked by the same methods. For comparison with the earlier measurements (4) some measurements were made with an old solid sample (4) of a natural mixture of chlorophj-11s a and b. The methanol was prepared by treating a sample of commercial synthetic methanol n-ith a quantity of sodium which as estimated to be about, three times as much as was required t o react 11-ith the water present. It was then refluxed with dimethyl pht'halate to remove the sodium hydroxide,j after n-hich it was distilled through an efficient packed column. TKO samples of carbon tetrachloride were used. One ]vas of reagent grade and 11-as used without further purification. The other, originally of I-.S.P. grade, n-as saturated n-ith chlorine and allon-ed to remain in the daylit laboratory for about 48 hr. It Tvas then treated n-ith an excess of potassium iodide solution. Finally, it was ivashed exhaustively, first n-it'hsodium thiosulfate solution and then with distilled n-ater. It T T - ~ Ydried over calcium chloride and distilled. E S L R G l 3IEAsTIIE\IE1\ TS
To determine the inteiisity of the light absorbed by the chlorophyll, the chlorophyll-sensitized photooxidation of phenylhydrazine by methyl red ( 5 ) v a s used as an actinometer. The chief advantages of this actinometer are that it uses the same absorbing substance ichlorophj-11) at the same concentrations as Tvere used in the photobleaching experiment>,and that no changes in the lens or filter system are necessary. It has the further advantage that the extent of the actinometric reaction can be readily measured with the Beckmann spectrophotometer. Measurements n ith this actinometer indicate that, n hen the full intensity of the -11 in chlorophyll a, 2.3 x actinic light was used and the solution ivas 2 x 10l6quanta per cubic centimeter per second 11 ere sbqorbed. t-nder similar circumstances, chlorophyll b absorbed 1.6 X quanta per cubic centimeter per second. EXPER1IIEST.kL RESL-LTS
Figures 3 to 9 summarize the principal experimental results. With the esception of figure 5 , these plots illustrate the course of typical experiments. The duration of each esperiment, in seconds, is plotted as abscissa, and the decrease in molarity of chlorophyll (A(') as ordinate. Except where othern-ise indicated, the interval between measurements is 3 sec. The point at the end of each interval of illumination is indicated by an open circle: that follon-ing a dark interval, by a solid dot. I n computing the decrease in molarity of chlorophyll, it n-as assumed ('7) that the bleached form of chlorophyll does not absorb at all in the red end of the spectrum. If the bleaching reaction results only in a lowering of the average extinction coefficient, a, for red light, the reported changes in the concentrations of chlorophyll are too small, but are directly proportional t o the correct values. This instrunicnt n a s the property of the Bureau of Ordnance of the ITnited States S a v y and TTas kindly put a t our disposal by D r . Bryce L CraTTford. 5 We are indebted t o D r R .Irnold of the Division of Organic Chemistry. who suggested this procedure and placed the distilling column a t our disposal.
RET-ERSIBLE PHOTOBLC.ICHIXG
O F CHLOROPHYLL
665
Figure 3 is a plot typical of the reversible bleaching n-hich n-as observed in the present experiments when air-free methanol solutions of chlorophyll a or 2, n-ere used. The outstanding characteristic of these results is the speed of the reverse process. I n some experiments not shown in figure 3 i t was observed that the steady-state bleaching was reached in less than 3 sec. (the period of the galvanometer) and that the solution returned t o its original color too rapidly t o he follon-ed by our apparatus. B y comparison v i t h other results, it can be estimated that the half-life of the bleached chlorophyll in these methanol solutions
FIG.3. Reversible photobleaching: chlorophyll a ( 2 X 10-6 JI) in methanol
is less than 0.5 sec. Experiments in which the illumination was prolonged showed that the irreversible bleaching \vas less than 10 per cent of the reversible effect. The maximum bleaching obtained with these solutions varied with the samples of chlorophyll and of solvent used and with the previous treatment of the solution. Illumination of a solution, even in the absence of air, reduced its ability t o undergo reversible bleaching, and this effect appeared to be greater than the concurrent irreversible bleaching. Using the full intensity of the actinic light and 2 x 10-6 X solutions, the follou-ing range of reversible bleaching, espressed as per cent reduction in the chlorophyll concentration, was obtained: chlorphylla-4.6 t o 0.2 per cent; chlorophyll b-1.5 to 0.4 per cent; solid chlorophyll (a natural mixture of a and b containing about 30 per cent of pheophytin)0.2 per cent. Air-free solutions of chlorophyll b appear to be more resistant t o irreversible photobleaching than corresponding solutions of chlorophyll a.
666
JOHK J. McBRADY AKD ROBERT L I V N G S T O S
Dissolved oxygen completely suppresses the reversible bleaching and somewhat increases the irreversible bleaching. Figure 4 is a plot of the bleaching occurring in a methanol solution saturated v i t h air. The rate of thi. irreversible bleaching is less than one-tenth of the rate of the reversible bleaching attained in the absence of oxygen but othern-ise under the same conditions. I t should be noted, however, that the rate of the irreversible blenching is increaped by five- or ten-fold by the addition of air.
FIG. 4. Irreversible photobleaching. Experimental conditions similar to those represented by figure 6, except t h a t the solution was saturated with air.
Figure 3 is a plot of the steady-state bleaching against the square root of the intensity of the actinic light. I t is apparent that the steady-state (i.e., maximum) bleaching is a linear function of the square root of the intensity. The fact that the straight lines do not pass through the origin is due to the bleaching produced by the scanning (i.e , analytical) light, n-hich cannot be neglected in comparison t o the actinic light of reduced intensity. Thib correction is discussed quantitatively in the section on computations. The short life of the hleachrd form in “pure” methanol solutions observed in the present experiments re3embles the results obtained by Porret and €
150 l7me Sac.
-
200
250
31
FIG. 6 . Reversible photobleachiny. Experinicntal conditions siniilar t o those represented by figure 3, escept that the sample of chlorophyll was prepared by a modified method in which carbon tetrachloride was substituted for ether as a n extracting agent.
the path of the actinic light in the solution is short, the intensity of the absorbed light should be approximately proportional to the concentration of the chlorophyll. The observed increase in 1 C can be attributed entirely to the increase in the intensity of the absorbed light. While no great precision can be claimed for the present measurements, they do show that the ratio. 1 C I:;:, does not increase appreciably with the concentration of chlorophyll. h number of experiments were performed to determine if the presence of added substances changed the course of the bleaching. I n methanol solutions, neither allylthiourea nor phenylhydrazine, a t the relatively high concentration of 0.10 M, had an appreciable effect upon the bleaching. It was previously
50
0
I
Time
FIG. 7 . Reversible bleaching. acid (lo-< 31) had been added.
0
50
- Sec
Chlorophyll b (2 X 10-6 -11)in methanol t o whichIoxalic
IO0 Time
-
150 Sec.
200
25 0
FIG.S. Reversible blenching. Chlorophyll a 12 X 10-6 M) in methanol t o which iodine .If)had been added. 660
670
JOHK J. 11CBR.kDT A S D ROBERT L I V I S G S T O S
S~OTn I
(4) that hydroquinone and i>oamylamine are equally TI ithout effect. Both Porret and Rabinon itch ( 7 ) and Livingston (4)reported that formic acid increases the bleaching. This result \vas not confirmed by the present experi31 formic acid had only a slight effect upon ments, in which it m s found that the bleaching. This inarked discrepancy suggests that the obseryed effect of formic acid was due t o some impurity present in the samples used. I n contrast, oxalic acid (10V .I[) incremed the steady-state bleaching by about threefold and
FIG.9. Reversible bleaching: cl~loropllyllr~ ( 2 X
.l11 in carbon tetrachloride
the half-life of the bleaclied form by more than tenfold. Figure 7 shows the course of the bleaching in the presence of oxalic acid. The addition of allylthiourea t o methanol solutions containing carbon tetrnchloride decreases the steady-state bleaching and shortens tlie half-life of the bleached form from 3 or 10 sec. t o less than 0.3 sec.; in other Ti-ords, tlie allylthiourea neutralizes the effect of the carbon tetrachloride upon the methanol solution. These result3 strongly suggest that the effect of carbon tetrachloride is due to the presence in it of an osidizing impurity. Since iodine had been present during the purification of the carbon tetrachloride, it was suspected t h a t
RE\-ERJIDLE PHOTOB1,EACHISG O F CHLOII0PHYI.L
G'il
this might be the active impurity. This IT-as quickly verified. A methanol 21) had been added exhibited the photobleaching solution t o which iodine illustrated by figure 8. The addition of the iodine increased the steady-state bleaching from about 0.2 t o 26 per cent and the half-life of the bleached form from less than 0.5 see. t o about 20 see. I n addition, the irreversible bleaching was completely suppressed. Ainumber of experiments v e r e performed with solutions in carbon tetrachloride. -4typical experiment is illustrated by iigure 9. These reactions exhibit two nen- effects. The irreversible reaction is considerably increased, and the bleaching continues in the dark for a short time after the light is cut off. This after-bleaching amounts t o about 20 per cent of the photobleaching occurring in a 5-see. interval. It n-as never observed in methanol solutions or in mixtures of methanol and carbon tetrachloride. COllPKT.ITIOSS
I n att'empting a quantitative analysis of the steady-state bleaching, it is necessary t o allon- for the efi'ect of the sranning light. Khile the incident, intensity of the scanning light is much lees than that of the actinic light, this difference is partially offset' by the difference in the length;: of the ubsorption paths. -1 direct actinometric. determination, using the phen!.lhydrazine-meth~l red reaction ( 3 ) sensitized 1,- 2 x 1 0 P .ll chlorophyll cr, gave a value for thc ratio of the intensity of the absor1)ed scanning light t o tli:tt of the ac*tiniclight of about 0.012. 11-lien clilorophyll b nxs used, the ratio T Y W ;Itiout 0.017. The olmrved bleaching, lh,is the tlii'frrence bet\\-een tlic bleaching produced by t h e scanning and 11)- tlic actinic lights. If the hlcarliing is proportional t o the sqiiare root of tlie intensity of the a1~sorl)ctiliglit, lye mal- w i t e : 6 Ab
==
=
+ +
K ( I Z t C ISL,)1'? - I
