VOL. 21 (1956) BIOCHIMICA ET BIOPHYSICA ACTA 245

P H O T O S Y N T H E S I S IN F L A S H I N G L I G H T

by

BESSEL KOK

T.N.O. Solar Energy Pro#ct*, Laboratory o/Plant Physiological Research, Agricultural University Wageningen** (The Netherlands) and Carnegie Institution o/ Washington,

Department o/Plant Biology, Stan[ord, Cali[. (U.S.A.)

In a previous article 1 we described a few measurements of photosynthesis in inter- mittent light of intensity about equal to that of bright sunlight. The purpose of this study was to investigate the intermittency patterns eventually required for increasing the yield of outdoor algal mass cultures by means of inducing turbulence in the medium.

In order to collect additional kinetic information concerning the rate-limiting photosynthetic reactions, this work was extended with the aid of further improved techniques of illumination, intermittency and volumetry. Our versatile arrangement for obtaining intermittency made it possible to corroborate most of the often apparently conflicting results of earlier workers. It appeared, however, that these results are largely open to reinterpretation and that the phenomena to be observed in flashing light are of complex character. The use of flash periods of the order of milli- seconds introduced complications. After a description of the technique we shall therefore discuss data obtained with the shortest flashes available. Then the influence of flash period will be described and finally a few kinetic observations in continuous light will be dealt with.

EXPERIMENTAL TECHNIQUE

Cellular material and volumetry

Chlorella cells suspended in o.2M carbonate buffer pH 8.8, saturated with air con- taining 2 % CO s, were used. The cell density was adjusted on the basis of a chlorophyll determination made on each batch of algae. A density of 5 V chlorophyll per cm 2 irradiated area was generally used.

Gas exchange was measured with our recording microvolumeter*; the small irradiated area (0.02-0.8 cm 2) of the reaction vessels allowed the concentration of high light intensities on the algae. Since an exposure of 2- 3 minutes sufficed for a single determination, the measurement of large numbers of rates, was possible, e.g. more than IOO per day. Ideally, all data to be mutually compared were collected using one and

* Work in Wageningen was aided by a grant from the Charles F. Kettering Foundation, Yellow Springs, U.S.A.

** Communication No. i4o of the Laboratory of Plant Physiological Research, Agricultural University, Wageningen, The Netherlands; 49th Communication on Photosynthesis. Re]erences p. 258.

246 B. KOV: VOL. 21 (1956)

the same sample of cells. This was often possible, since most of the posit ive rates were

small and to a certain ex ten t balanced by the dark readings tha t were f requent ly

taken. In this way, the to ta l pressure change in the vessel in the course of an experi-

ment could be kept small ( < I0 %).

In most exper iments we a t t e m p t e d to compute the sa tura t ion rate in given

pat terns of i l lumination. In such cases a series of gradual ly increasing intensit ies was

applied unti l the slope of the trace on the recorder increased no further. A severe

difficulty encountered was tha t even short exposures of the cells to bright l ight

- - e i t h e r cont inuous or f l a s h i n g - - m a y damage their photosynthet ic apparatus. Since

blue light is most injurious in this respect, a yellow filter (Schott GG 14) was usually

inser ted in the path of light. The rates in weak light are the first to be affected by this

in jury (@3) and frequent checks were made in the course of each experiment . If the

rate in a given low in tens i ty differed substant ia l ly from tha t observed immedia te ly

after the sample was first used, a fresh al iquot of algae was taken. In this way an

a t t emp t was made to obtain mutua l ly consistent da ta in each experiment . With every

batch of cells used an in tens i ty vs. assimilat ion curve was measured in continuous

light.

In the course of our investigations (c/. also 3) the increasing demands for high intensities brought about the use of smaller reaction-vessels. At present, spherical vessels o ~ 5 ram, volume ~-~4 o/,1, fluid phase~3 o/~1, are more or less standard. Before each filling the vessel is carefully dried with the aid of acetone and by subsequent evacuation. The main difficulty is the transfer of the sample into the reaction vessel after it has been pipetted on top of the opening of the index capillary (c[.2 Fig. 3). The evacuation required for this transfer has to be performed cautiously so that splashing of the liquid onto the walls of the compensating vessel is avoided.

In Table I we collected the values of the rate of oxygen evolution as observed in a weak and a saturating intensity (corrected for the dark exchanges recorded alternately as usual). These data served as checkpoints in the course of an experiment (lasting 7 hours in total), in which seven analogous samples of algae were used. (This experiment was one of the series used for Fig. 9.) The indicated mean error comprises all experimental limitations involved.

TABLE I

RATES OF P H O T O S Y N T H E S I S OBSERVED W I T H P A R A L L E L SAMPLES (3 ° / l l ) oI," AN ALGAE SUSPENSION (mm RECORDER/MXNUTE)

No. of sample • 2 3 4 5 6 7 % error

Rate in weak light 12.5 13 i1 12-5 13..5 13 16.5 7-4 Rate in saturating light 69 73 65 64 73,5 62 66 5

Illumination and intermittency

Fig. I shows the ar rangement used to i l luminate the react ion vessel with ei ther cont inuous or flashing light. The light source H, the in tens i ty of which could be

decreased by insert ing neutra l wire screens W, is focussed in the plane of the double ro ta t ing disk D1D2, with the aid of lens L 2. Lens L 1 imaged lens L= upon the react ion

vessel R, placed in a the rmos ta t ba th T. Mirror M 2 mounted behind the react ion vessel, served to increase fur ther the in tens i ty in the cell suspension. Mirror M 1 could be swung in to the posit ion shown in Fig. i and thus directed the l ight via lens L2 to a large area thermopi le Th; in this way the in tegra ted intensit ies could be measured.

As a light source we used water-cooled 500 V, 2 A, d.c., high pressure mercury arc lamps (Philips S.P. IOOO \V 8o/2} run on a stabilizer. The very high brilliance (45 Ksb), the favourable dimensions

Re/erences p. 258.

VOL. 2 1 (1956) PHOTOSYNTHESIS IN FLASHING LIGHT 247

of t he arc ( length 2o m m , wid th ~ I m m ) and t he prac t ica l ly con t i nuous l ight emiss ion (the rec t i fy ing a p p a r a t u s h a d a vol tage ripple ~ 5 %), m a d e these l amps ideally su i tab le for f lashing l ight work.

If imaged i : i on t he disc, sli t w i d t h s as smal l as 1.8 m m ( length 20 ram) could be used, and th i s al lowed one to ob ta in r ea sonab ly shor t f lashes w i t h o u t necess i t a t ing the use of large discs or h igh speeds of revolut ion.

The two discs D 1 and D 2, shown in Fig. I, h a d a rad ius of 134 m m a t the cen t re of t he open ing O. T h e y were m o u n t e d closely t oge t he r (space of 2 m m be tween them) and to t he two fixed d i a p h r a g m s S, which res t r ic ted undes i red l ight trails. The two discs were m o u n t e d coaxia l ly and were dr iven v ia a th i rd axle (As) wi th the aid of easi ly in t e rchangeab le gears (G). Axle A s could be dr iven a t a n y desired speed by a mo t o r v ia a var iable speed dr ive V and a s y s t e m of bel t and pul leys P.

By proper choice of the var ious gears (G), the two discs could be t u r n e d a t speeds differing m u t u a l l y by a factor of 2, 4, 8, or 16. Disc D2, p rov ided wi th a re la t ive ly large opening, was a lways the s lowest one and, depend ing upon t he gear a r r a n g e m e n t , only t r a n s m i t t e d eve ry 2nd, 4th, 8 th or 16th flash t r a n s m i t t e d by t he fas t disc D 1. In th i s way shor t flash per iods (de te rmined by speed and sli t w id th of D1) could be a l t e rna ted wi th long da rk per iods (de te rmined by t he even tua l use and t he speed of D2).

Fig. I. E x p e r i m e n t a l a r r a n g e m e n t . T, t h e r m o s t a t ; R, reac t ion vessel; M1M v mir rors ; L1L~L3, lenses ; Th, t he rmop i l e ; S, d i a p h r a g m s al igned wi th disc open ings O; V~', wire screen; H, l ight source; D1D2, co- axial ly dr iven discs. Axle A 1 en- closes axle A2, bo th are d r iven v ia change gears G by m e a n s of axle A 3. P, pul leys ; V, var iable speed drive; M, motor . To t he r ight , two e x a m p l e s of the discs used are shown. The radia l d imens ions are ind ica ted in t he uppe r one; only one slit is shown. DaD 4 consis ts of two blades, each p rov ided wi th 9o ° cu t -ou t s ; by m u t u a l d i sp l acemen t a n y rat io tf/td be tween i / i o o and

i / i could be chosen.

L,,C s L2.w .

M 1

/04

On the o the r hand , a series of sho r t da rk per iods (for a g iven flash period) could be realized by provid ing D 1 with 16 sli ts a n d e v e n t u a l l y cover ing up every second, four th , etc., wi th t he aid of a l u m i n u m foil and adhes ive tape.

If, for example , a t a speed of 300o r .p .m, all I6 slits of 8 m m each in D 1 were left open, the f lashes las ted 0.32 msec and were a t in te rva ls of 1.25 msee and the da rk per iods a m o u n t e d to 1.25 0.32 -- 0.93 msec. B y success ive ly cover ing 8, 12, 14, and 15 slits, t h e da rk period was a b o u t doubled each t i me u n t i l - - w i t h on ly one slit o p e n - - i t a m o u n t e d to 20 msec. By then , us ing D 2 wi th decreas ing coincidence factors , t he da rk period for t he s ame flash period could be increased s tepwise to 320 msec.

I n o the r e x p e r i m e n t s i n t e r m i t t e n c y reg imes were given, in which the flash period was of the s ame order of m a g n i t u d e as the da rk period. For th is purpose we used ins tead of D 1 and D s t he double disc DzDa, m o u n t e d on axle A s (c[. Fig. 2). For ins tance , a n u m b e r of i n t e r m i t t e n c y reg imes were s tud ied wi th v a r y i n g flash per iods and a c o n s t a n t da rk period of 400 mil l iseconds. F i rs t the two slits be tween D 3 and D~ were a d j u s t e d to 3.2 m m and t he disc was t u r n e d a t 74 r .p .m. (tf~,J 4, td ~ 4 °0 msec) ; t h e n the sli ts were a d j u s t e d to 6. 4 m m and t he disc t u r n e d a t 74 r .p .m. (11,',~ 8, t d ~ 400 msee) ; n e x t the slits were I2.5 m m and the speed 72 r .p .m. ( t f ~ 16, tdt~.~ 400 msec). This g radua l increase in sli t size and decrease in speed was con t inued ; finally wi th sli ts of 127 m m and a speed of 48 r .p .m, a flash period of 256 and a da rk period of 400 msec was ad jus ted . If, as in th i s case, ve ry slow i n t e r m i t t e n c y was applied, t he n u m b e r of f lashes g iven du r ing the period of m e a s u r e m e n t was coun ted photoelectr ical ly .

B y a l i g n i n g t h e s l i t s o f D1, S 1 a n d S , , t h e t i m e c o u r s e o f t h e f l a s h e s c o u l d b e

p r o p e r l y a d j u s t e d . I n F i g . 2 t h e i n t e n s i t y f a l l i n g u p o n T h is p l o t t e d a s d e p e n d e n t o n

t h e p o s i t i o n O f t h e m o v i n g s l i t . T h e t i m e c o u r s e o f t h e f l a s h is s h o w n if a s l i t o f 8 m m

Re[erences p. 258.

248 B. KOK VOL. 21 (1956)

1 0 0

i .° 60

40

20

10

0 , I * I I , I ,

B 8 4 2

\ i I i I i , i

2 4 6 0 1 0

m i l l i m e t e r c i r c u m f e r e n c e

Fig. 2. Examples of the t ime course of the flash intensity. Relative intensi ty is plotted vs. displacement of disc circumference (96o mm), for a few different openings in disc D 1 (O) and the fixed d iaphragms (S). 0 : O = 8, S = 4 ; × : O = 1.8, S = 1.8 (mm); A : flash shape for O = 4 m m when the microvessel and a microscope condensing lens (L1, Fig. ~) were used. L 1 was mounted very close to disc D 1 and the fixed slits ad- j usted for best flash shape. For this measure- ment the thermopile was located at the

position of the reaction vessel.

in D 1 was used, combined with slits of 4 mm width in S 1 and S 2 (dots). In this case the flash occurred within 15.5 mm displace- ment of the disc circumference and if Dx was turned at 3,000 r.p.m, the total flash period including the rise and fall of the intensity was 0.32 msec.

When a microreaction vessel was used (cross section < 5 mm), the lens L 1 was a microscope condensing lens. The effective in- tensity attained in this way was more than a thousand times the photosynthetic "satu- ration intensity" (I s computed as illustrated in Fig. II). Flash saturation could be obtained in this case with a slit of 4 mm in D 1 and a speed of revolution of 4,000 r.p.m. The total flash period then amounted to o.I5 msec (half width 60 /,sec, c/. Fig. 2 triangles).

In studies concerning the influence of length of flash period, we encountered the difficulty of properly evaluating this mag- nitude. In all experiments unless otherwise stated, we indicate the total flash duration. It may be pointed out that as long as the disc is turned at equal speed the tails of the flashes

remain identical, regardless of the opening used. Therefore in a series of measurements in which the disc opening is varied, the increment of flash time is accurately known.

RESULTS

Before describing our experiments we must first discuss a few obvious points that are often neglected. The rate per flash (Rz) is computed by dividing the observed (integrated) rate of 02 evolution in intermittent light by the number of flashes applied. The flash yield therefore is the sum of the amounts of oxygen evolved during one flash and a subsequent dark period.

Suppose an experiment is made, in which (saturating) flashes of equal duration (t/) are alternated with dark periods of varying length (td). For the sake of simplicity we accept that the intensity rises and falls infinitely quickly at the beginning and the end of the flash, so that the duration of the flash can be easily evaluated. If the light is given continuously (td = o), photosynthesis will proceed at its maximum (saturation) rate: S ~1 02/see. During a period equal to the flash period t / a n amount of oxygen t fS will be evolved.

If the dark periods between the flashes are made shorter and shorter, the condition of continuous illumination is approached.

The first consequence of this is that if the flash yield is plotted vs. dark period, the curve will intercept the ordinate (t d = o) at a value R f = tlS. Re/erences p. 25X.

VOL. 21 (1956) PHOTOSYNTHESIS IN FLASHING LIGHT 249

Secondly, it is evident, that the initial slope of this plot (td -~ o) must be equal to the saturation rate in continuous light. This is illustrated in Fig. 3. The evaluation of the initial slope of the Rf vs. td plot therefore only requires a simple rate measurement in continuous strong light.

For an accurate determination of the height of the intercept, the value of tf has also to be estimated - - t h i s is often difficult since ideal flash shape cannot be realized in practice.

In view of the above considera- tions it may be stressed, that for a given time course of the dark re- action (e.g. of first order character as is often accepted), the values of

Rr

T

/ / /

/

< t f

---0

/ /

/ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . . .

tf. 8

> t d .

Fig. 3. See text.

Rf~ (the maximum yield per flash for td -~ oo) and the time constant are interdependent.

EXPERIMENTS WITH SHORT FLASHES

Fig. 4 shows a representative experiment made at two temperatures. The flashes lasted o.49 msec (including both tails) and were in all cases sufficiently intense to yield saturation. The dark period between the flashes varied between 1. 4 and 24 ° msec. From this type of experimentation we drew the following conclusions about maximum flash yield and the dark period time course:

The maximum l~ash yield is independent o/ temperature A number of exper iments of the type shown in Fig. 4 were made at various tempera tures with dark periods of up to a second. I t often proved somewhat difficult to evaluate properly the

X 10-4

7 7 / 3 0 o C 7 /S30oC. S 15 ° C.

• 15OC 6

m 5 ~ 5 i I I " 3 0 ° C

i I • /

4 I / ~ o

3 3 l/ ~ J 2 2 15 C

! I I

100 200 10 20 30 rn .sec , d a r k t i m e ~ m. sec.

Fig. 4. Relation between flash yield and dark period at 3 °0 C and 15 ° C, lr = 0-48 msec. The t ime scale is expanded Io-fold in the r ight-hand drawing (b). The dashed slopes indicate the satu- rat ion rates in cont inuous light. The dotted curves show the t ime course to be expected if the

decay had first order character (for 3 °0 C).

Re/erences p. 258.

250 B. KOK VOL. 21 (1956)

readings made at very long clark intervals. The rates of gas exchange in such cases are only small and close to or below the compensating point. Plots of integrated photosynthetic rate vs. integrated intensity either applied continuously or intermittently hardly ever extrapolate to zero rate (dark respiration) for zero light. The intercept with the ordinate may vary between dark uptake and half the compensation point (@4). We gained the impression, moreover, that the anomaly is often enhanced by slow intermittency of the light (t~t >o .2 sec).

Some experiments yielded more complicated curves than those shown in Fig. 4. An additional rise of the flash yield as well as a marked decrease of it have been observed in the range of (lark periods beyond o.l sec. Such deviations were often significant and obviously correlated with the pretreatment of the algae.

We restrict ourself to a discussion of the illustrated type of curve, which predominates in our experimental material available at present.

The two curves of Fig. 4 c lear ly t e n d to a p p r o a c h a c o m m o n final value. F r o m this we

conc lude t h a t the m a x i m u m flash y ie ld R I , , is i n d e p e n d e n t of t e m p e r a t u r e . The

abso lu t e v a l u e of Rf,, was one o x y g e n molecu le per 1,5oo to 4,ooo ch lo rophy l l mole-

cules p resen t in the r eac t ion vessel. This a p p r o x i m a t e l y th ree fo ld v a r i a t i o n of t he

m a x i m u m flash yie ld was co r re l a t ed w i t h t he p r e t r e a t m e n t of t he algae.

T i m e c o u r s e o~ the d a r k r e a c t i o ~

E x p e r i m e n t s were made , in wh ich the r a t e of o x y g e n e v o l u t i o n was m e a s u r e d as a

f unc t i on of the ( in tegra ted) l igh t i n t e n s i t y g iven e i the r c o n t i n u o u s l y or i n t e r m i t t e n t l y . 250

o

.5 _.= o

200

"~ 150

- -O O -

lOO

, °

4 0

2 0

1 0 ' i I . , I , . , . I , i i , I , , . J I

0.5 1.0 1.5 2..0

°/to I n t e n s i t y ( i n t l Q r l l t e d )

Fig. 5. Relation between integrated rate of oxygen evolution and integrated light inten- sity, 23 ° C. O : continuous light; []: inter- mittent light, If -- o.47, td = 1.43, × : inter-

mittent light, // -- o.47, I d - - 60 msec.

As long as suff ic ient ly shor t da rk

per iods were used, t he resul t s were e x a c t l y

iden t i ca l in the two cases. If the d a r k per iod

is inc reased b e y o n d a ce r t a in va lue (this

l imi t is lower the h igher the t e m p e r a t u r e ) ,

t he s a t u r a t i o n r a t e in i n t e r m i t t e n t l igh t

becomes smal le r t h a n the ra te obse rved

in con t inuous l ight . This is i l l u s t r a t ed in

Fig. 5. I n some e x p e r i m e n t s m a d e a t 3 o ° C

the use of da rk per iods as long as 5 msec

had no effect upon the obse rved p h o t o s y n -

t he t i c s a t u r a t i o n rate . This po in t cou ld be

checked wi th a n y a c c u r a c y des i red b y al-

t e r n a t e l y r eco rd ing exposures of the a lgae

to f lashing a n d to con t i nuous l ight . F r o m

th is i t fol lows t h a t p h o t o s y n t h e t i c 0 2 evo-

lu t ion con t inues for a while at full r a te a f t e r

the l igh t is r e m o v e d . C o n s e q u e n t l y the re la t ion b e t w e e n R(L and tf is l inear ove r a

r ange of shor t d a r k periods, i .e. the da rk r eac t ion has in i t i a l ly zero o rder cha rac te r .

This is i l l u s t r a t ed in Fig. 4b ; a t b o t h t e m p e r a t u r e s used the two curves are s t r a igh t in i t i a l ly and the i r s lopes are iden t i ca l w i t h t he s a t u r a t i o n ra te in con t inuous l ight (S). The l inear s t r e t ch is longer a t t he lower t e m p e r a t u r e . In the final s tage of the da rk

r eac t i on the cu rves b e n d o v e r and a p p r o a c h r a t h e r s lowly t he i r c o m m o n final va lue .

The dotted curves in Figs. 4a and 4b represent the time course to be expected, if the decay had first-order character (computed for 3 °° C and the observed values of initial slope and maximum flash yield). The deviation from the observed values is well beyond experimental error.

Re/erel~ces p. 25,~'.

VOL. 9.1 (1956) PHOTOSYNTHESIS IN FLASHING LIGHT 251

Shape o~ the intensity-rate curves in flashing light The relation between intensity and flash yield in intermittent light, in which the flash period is short and the dark period 10o long, is of special importance; it des- R r ~ . .

cribes the charging of the primary light 60 5 0

acceptor complex. These curves always ¢ 4o had exponential character (c/. Fig. 6). This indicates that formation of the pri-

2 0

mary photochemical product occurs in a one-quantum process. Absorption of each

1 0

8

6

individual quantum by an (not yet excit- ed) acceptor results in its stabilization.

Discussion

Most of the observations described above are not new. The same value and the temperature independence of the maxi- mum flash yield were found by EMERSON AND ARNOLD', 6 a n d DAMASCHKE AND

ROTHBt~HR 7. The shortest dark period available to the first authors was 2o msec,

• . I 50 tO0 1S0 ~ 0 0

Rel. ]nt~nstty-.--~:-

Fig, 6. Relation between intensi ty and rate per flash, t / = o . t 8 , t,, - - 80 msec.

which must have made it difficult to evaluate properly the dark reaction time course s,9. The zero order character of the dark decay was also observed by WELLER AND FRANCK 1°, whether only at low tem- perature or with cyanide inhibited cells. However, their available dark periods were multiples of 16.7 msec and therefore only a few points of the decay curve could be collected at higher temperatures. The first order character of the decay at high tem- perature is not confirmed by our data. We find the same character of dark reaction regardless of temperature, but with different time constants. The exponential shape of the intensity-rate curve is the same as that observed by NOUN n.

The findings described lead to the following conclusion: during the flash a long- living intermediate (U) between light quanta and oxygen is formed photochemically. I ts maximum concentration (R/m) corresponds to one molecule of oxygen precursor per 1,5oo to 4,000 molecules of chlorophyll or to one transformed light quantum per 15o to 400 chlorophyll molecules if a quantum number of IO is accepted. In our opinion the latter way of expressing RI, . per light quantum, possibly in terms of a "photosynthetic unit", is the one to be preferred.

The stable intermediate (U) is transferred to oxygen via a (temperature-depend- ent) dark reaction. As long as an excess of U is present, the reaction proceeds at constant velocity, determined e.g. by concentration and working time of the enzyme (E, c/. p. 255) involved. As soon as the average substrate concentration decreases too far, the course of the rection will change from zero order to a more complex character. Obviously flashing light data of the type discussed do not yield direct information about concentration and velocity constant of that rate-limiting enzyme system. A further analysis of the time course is required for this. All that Fig. 4 shows is the relative amount of photochemical product and the time required at various temperatures for its removal.

R e / e r e n c e s p . 258 .

252 B. I,:OK VOL. 21 (1956)

C L E N D E N N I N G A N D E H R M A N T R A U T 12 a n d E H R M A N T R A U T A N D RABINO~,VITCH 13 m a d e comparitive studies of photosynthesis and the Hill reaction in Chlorella. These authors found the quantum yield, the saturation rate in continuous light, the "dark reaction time" and the maximum yield in flashing light to be equal in both processes. This suggests that intermediate U and the removing enzyme system are identical in the two processes and therefore are to be located in the chain of reactions between the conversion of light quanta and the liberation of oxygen.

E X P E R I M E N T S W I T H L O N G F L A S H E S

In a number of experiments we studied the relation between flash yield and flash period depending on both light in tens i ty and dark period.

Fig. 7 a and 7b show part of the data collected in an experiment, in which a sample of algae was exposed to either cont inuous or flashing light of vary ing intensities. Fig. 7 a shows the assimilation in tens i ty curve in cont inuous light. The curve is

S . . . . . . . . . . . . / . . . . . . . . . . . . . . . ~ . 3 -xlO-2 /;!

/ ~ i I :

i i - 4 111 x ,1o 1

o / f:l 0 ~ 2 If=l,9 td=SO

1

, :o ' 2 0 ' . ; °/0 I n te .s l t y

I I

I ~ I s 2 3

°/o I n tens i ty

Fig. 7. (a) Relation between rate of oxygen evolution and intensity, measured on the sample of cells used also in the experiments shown in Fig. 71), 3 °o C. Inset: rate vs. intensity curve observed in inter-

mittent light, tf ~ 1.9, t,t = 5 ° msec. x ~0 -4

20

o ~.. 16

o~14

a: 12

l 0

M 8 aN.t.

6 I 4 --

i I I ! I I I I I b I I I

2 4 6 8 10 12 14 16 1,8 20 22 24 26

1: f ----~4~- m sec

(b) Relation between flash yield and flash period for o/ • various intensities: ~ , 2 -8°/~o," O, 5,o, +, 113"o; S,

saturating light, td = 5 ° msec.

characterized by two asymptotes. The ini t ia l slope observed in weak light represents the ma x i mum e/fi-

c i e n c y (;~) with which the used sam- ple of algae can convert light into oxygen. The second asymptote re- presents the ma x i mum rate (S." mol. O2/mol. chlorophyll/sec), with which these algae can evolve oxygen at the prevail ing temperature. (We define the "sa tura t ion in tens i ty" Is - - S / ;~ ).

Under no condit ion of in t e rmi t t en t or cont inuous i l luminat ion can these limits of efficiency and rate be sur- passed.

In Fig. 7 b the flash yields ob- served in in t e rmi t t en t light are plot ted vs. flash durat ion. In all in- t e rmi t tency experiments we used 3 intensi t ies: 2.8 %, 5 % and I I % re- spectively. If these intensi t ies had at all t imes been used with the m a x i m u m efficiency ;: (c/. Fig. 7a), the observed rates would have fitted the three indicated (lotted slopes. If, on the other hand, dur ing all flashes photosynthesis had run at ma x i mum rate, curves parallel to slope S (dashed) - - representing the sa tura t ion rate in cont inuous light - - would be expected.

Actually, Fig. 7b shows that very short flashes are used with m a x i m u m efficiency (dotted slopes),

Re/ere~ces p. 258.

VOL. 21 ( I956 ) PHOTOSYNTHESIS IN FLASHING LIGHT 253

but that during long flashes the limiting rate (dashed slope) is approached, i.e., the curves of Fig. 7 a and 7 b have indeed identical asymptotes.

M a x i m u m lash yield as dependent upon flash period

The final part of the curves--paral le l to S - - a p p e a r s to be shifted upwards over an amount of oxygen (expressed per unit chlorophyll and per flash). This amount is greater the higher the intensity is used (and the longer the dark period), but reaches a final value (for I - , oo, ta ~ oo) ; in all intermittency patterns shown, we increased the intensity beyond the three values discussed until no further increase in flash yield occurred (points indicated s). The final part of the curve so obtained extrapolates to a value 6.I-lO-* O2/mol. chlorophyll/flash (indicated M 2 in Fig. 7b). A point of considerable interest is that this value was more than twice as high as the maximum flash yield that we derived from parallel experiments with very short flashes. In con- nection with this it should be noted that the flash saturation yields at short flash periods do not fit the straight final slope but are lower. The inset of Fig. 7 a shows the intensity vs. rate curve as it was measured for obtaining the saturation value plotted in Fig. 7 b for tf ~ 1. 9 msec. I t is evident that saturation was closely approached.

I t may be noticed tha t in these measurements we did not increase the intensi ty beyond the value required for sa tura t ion (in this case 60°0 of the available maximum) . Photoinhibi t ion could be restricted in this way and all da ta collected wi th one sample of algae. This was helpful for obta ining the accuracy required for this type of measurement . Also, to achieve m a x i m u m accu- racy we used high temperatures , at which the dark reactions (c[. Fig. 4) are faster and shorter dark periods can be used, This yields higher rates of gas exchange, so tha t experimental errors and uncertaint ies in the dark respirat ion correction are restricted.

Use of dark periods, which are not long compared to the time required for completion of the dark reaction(s), will make the Rf vs. tf curves more difficult to interpret. The flashes in this case will hit an incompletely discharged system and after a few cycles of light-dark periods a steady state flash yield will be observed, which will decrease the shorter the dark period used.

Fig. 8 shows an experi- 9

ment, in which a much longer dark period was used (0.4 sec). 8 The results are qualitatively identical to those of Fig. 7 b.

Fig. 9 shows the effect of , 6 temperature. Flash saturation | values measured at 15 ° C and I e s 3o°C are plotted vs. flash g

4 period. The final slopes extra- polate to about the same in- 3 tercept (M2) on the ordinate. The "maximum yield in long 2 flashes', defined as the height of intercept M 2, is not greatly influenced by temperature. The a t ta inment of the steady state rate, however, occurs faster (the R l - - t I curve rises

Re/erences p. 258.

x l O -3 0 2

1 ~ . , ' " - " t d = 0.4 sec

1 - " m i l l i s e c o n d s

50 100 150 200 tf >

Fig. 8. Relation between m a x i m u m flash yield and flash period. 3 ° ° C, t d = 0. 4 sec.

254 B. t<oK v o L 2 1 (1956)

s t eepe r ) t h e h i g h e r t h e t e m p e r a t u r e used , i.e. t h e t i m e cour se is m o r e t e m p e r a t u r e -

X 10 -4 12 . ~ . . ~ O°C

~ ~o o

8 =': 6 y / ~ ° c

Ml--t~2"

I 0 20 30 if b re.see.

Fig. 9. Relation between flash yield (saturation values) and flash period. I d = o.15 sec; × : 3 °0 C; O : 15 ~' C. Both curves are arbitrari ly extrapolated in this drawing to a cominon intercept M 1 (dotted).

d e p e n d e n t .

The photosynthetic saturat ion rate is usually not very constant and may vary lO to 3 ° % in tile course of an experiment for no apparent reasons. This further re- stricted the accuracy with which we could determine the effect of temperature upon tile value of 31~.

A g a i n i t m u s t be s t a t e d (c/. t h e p a r a -

g r a p h d e a l i n g w i t h s h o r t f lashes)

t h a t t h e r e su l t s p r e s e n t e d so f a r do

n o t c o v e r all o u r o b s e r v a t i o n s .

C u r v e s as s h o w n in Figs. 7 b - 9 a re

o n l y o b s e r v e d r e p r o d u c i b l y w i t h a

few t y p e s of cells. W i t h o t h e r , a n d

m a y b e e v e n t h e m a j o r i t y of, a lgae

we f o u n d o n l y t h e i n i t i a l p a r t of

t h e R / - - t / c u r v e to be i d e n t i c a l w i t h

t h e ones p r e s e n t e d in t h i s p a p e r .

O v e r a r a n g e of l ong f lashes t h e f lash y ie ld r i ses s t e e p e r t h a n c o r r e s p o n d s to t h e

s a t u r a t i o n r a t e in c o n t i n u o u s l igh t . T h i s t y p e of b e h a v i o u r is s t i l l t h e s u b j e c t of

s t u d y . W e r e s t r i c t ou r se l f a t p r e s e n t to t h e t y p e of c u r v e i l l u s t r a t e d , w h i c h is r e l a t i v e l y

s i m p l e to i n t e r p r e t . T h o u g h t h e r e s u l t i n g k i n e t i c p i c t u r e wil l s t i l l b e i n c o m p l e t e

( r e q u i r i n g t h e i n c l u s i o n of f u r t h e r s low d a r k s teps ) , i t m a y g ive a r a t h e r g e n e r a l

d e s c r i p t i o n of t h e fas t d a r k r e a c t i o n s .

The time course o~ photosynthesis during short flashes

W e r e a c h e d t h e c o n c l u s i o n t h a t , in t h e r eg ion of s h o r t f lashes , t h e m a x i m u m f lash

y ie ld is a f u n c t i o n of t h e f l ash d u r a t i o n . T h i s i m p l i e s t h a t t h e m a x i m u m c o n c e n t r a t i o n

of i n t e r m e d i a t e s b e t w e e n l i g h t a n d o x y g e n n e e d s a c e r t a i n p e r i o d of i l l u m i n a t i o n in

o r d e r to be b u i l t up. ( S t r i c t l y s p e a k i n g , of course , in o u r t y p e of e x p e r i m e n t s we can

o n l y d e t e c t i n t e r m e d i a t e s w i t h a l i f e t i m e s h o r t e r t h a n ~-~ I second . )

Experimental evidence will now be required to decide between two possible a priori mechanisms : (a) The maximum fash yield approaches zero if the flash period is decreased to an infinitely

small value (which might then require an infinite intensity to achieve saturation). This would imply tha t in the first moments of illumination the chloroplasts are unable to convert tile im- pinging light quanta. But a photochemical product, induced by these first quanta, might act ivate some enzyme system, so tha t it now acquires tile ability to convert excited pigment molecules into a stable form. In this type of autocatalytical reaction the total amount of the enzyme finally becomes active in turning over quanta into oxygen. If we accept tha t the enzyme is completely discharged (and deactivated) in each dark period between the flashes, the height of tile intercept :ll 2 on the ordinate of Fig. 7 b represents its concentration in continuous strong light.

(b) The maximum flash yiehl approaches a finite value for extremely short flashes. In this case two intercepts on the ordinate are to be expected in plots as given in Fig. 7 b 9: a t rue inter- cept (M1) at a relatively low value of R/ and the second one (M=), obtained by extrapolation from the final slope measured with tile aid of long flashes. One type of mechanism, which might fit such results, may be briefly described:

The magnitude of the true intercept, if unaffected by temperature, might indicate a concen- tration, for instance, of a photochemically formed and stable product (U) (as was discussed in the paragraph dealing with the influence of dark period).

The magnitude of the second intercept may also be correlated with a concentration. I t would

Re/ere~*ces p. A58.

VOL. 21 (I956) PHOTOSYNTHESIS IN FLASHING LIGHT 255

be most attractive to relate this second concentration (M2-M1), to an enzyme (E) acting upon substrate (U) :

hv + U kl [--> U* (U*)max ~ M1 (I)

U*+ E - k~ > E*+ U (E*)st. state ( : ) M 2 - - M 1 (2)

E* ka - ~ E + ~ ( O 2 ) (3)

In this reaction sequence not only U* but also E* is an intermediate, which in the dark period will continue to yield oxygen until depletion. Its concentration at the start of the flash is zero; after a certain period of illumination a stationary value (E*)s.s. is attained, determined by tempera- ture and intensity.

To decide whether the R f - - t f curve extrapola tes to zero or to a finite yield for zero

flash period, and to s tudy its t ime course, exper iments were made in which the flash

period var ied between o.2 and 5 msec.

To ensure tha t flash saturat ion was obtained in all cases, we used for each obser-

va t ion a series of intensities extending well beyond this requirement . Photoinhibi t ion

can hardly be avoided in this way and this necessi tated the use of a fresh sample of

algae for each determinat ion. Wi th the aid of measurements in weak and sa tura t ing

cont inuous light we checked the equal i ty of the samples and mutua l ly corrected the

da ta of each series.

Moreover, the absolute values of flash yield and sa tura t ion rate (expressed per

uni t chlorophyll) var ied with each batch of algae used. For each exper iment we

arbi t rar i ly put the yield observed at t l = o.5 msec (measured in all series) equal to

ioo. The data obta ined in this way are shown in Fig. io.

For the reasons discussed earlier in this paper, a short dark period (40 msec) was

used for these experiments. This 20O

could par t ly explain (@21) why the

ini t ial rise of the curve is so much _= ~80 -~ 160 faster than observed in the experi-

ments i l lustrated in Figs. 7b-9 . ~. ~4o

The R l v s . tf curve of Fig. IO i ,20 clearly tends to hit the ordinate at

10o a finite value ra ther than to extra- polate to zero. If e.g., both the final 80

s t raight par t of the curve and the 6o

ini t ial part are extrapolated, as 40

shown by the dot ted lines, we ob- 20

ta in two intercepts differing by a

factor of about two. The logar i thm

of the difference between the two

curves is proport ional to l I. Ev i - dence der ived from other exepri- ments supports this type of t ime course.

I t is thus possible to fit the

da ta with a finite intercept at t / - , o,

t i i I I I I I I 2 3 4 5

I I ~ r e . t e e .

Fig. IO. Relation between maximum flash yield and flash period in the region of short flashes, I d - - 4 ° msec. Data arise from 9 series of measurements, each in- cluding an observation at tf ~ 0.5 msec, which was used as a reference point and taken as IOO (cross). The circles drawn at l/ = 2.04 and tf ~ 4.16 represent the

average of the indicated data.

an exponent ia l ini t ial stage and a linear final stage (saturat ion rate).

Re/erences p. 258.

256 B. KOK VOL. 21 (1956)

Some uncer ta in ty still remains, owing to the lack of flash periods shorter than o.2 msec and the limited accuracy at tainable in these experiments . Two additional a rguments may therefore be given for the reali ty of the intercept M 1.

(i) In a few exper iments (unfor tunate ly not carried out expressly for the purpose of solving this problem) flash yields were measured with the aid of condenser discharges (45oo V o. 5/~F) th rough AH6 mercury lamps. The sa tura t ing flash yields observed were only slightly lower than those induced by shor t flashes obtained by the sector disc arrangement . The flash periods differed by a factor of about 5o (2o and iooo/ , sec resp.) in these two cases. This indeed indicates t ha t the @ vs. t/curve is discontinuous in the region of extremely shor t flashes.

(2) The second a rgument arises from exper iments on the influence of dark period in which we varied the temperature . The influence of t empera tu re upon Rfm (for t , ,--+ o0) appeared to decrease with decreasing flash periods; it was very small (io 2o%) for flashes shorter than o. 5 msec (c[. Fig. 4a), bu t rapidly increased if longer flashes were used. This is to be expected if a finite tempera ture- independent flash yield exists for t f - -~ o.

D i s c u s s i o n

Several flashing light studies, as those of WARBURG H, BRINGS ia, TAMI'ZA AND CI~IB.,\ TM, PHILLIPS AND MYERS 17, in which "long" flashes were used, yielded results differing qualitatively from those obtained with "short" flashes (c/. also the recent reviews by LUMRY el al. is, 19).

The data presented above demonstrate that a variety of results may be expected when "long" flashes are used for studying the dark decay. Relatively weak intensities may suffice for obtaining saturation, flash yields will depend upon temperature and length of dark period; the dark reaction time course will depend upon the flash period used. Many effects can be understood on the basis of a reaction sequence as discussed on p. I I (1)-(3), which is qualitatively similar to those proposed by ORNSTEIN 6t al. 2° and BRIG(;S 15. But a further analysis of additional slow dark steps is still required.

A more detailed analysis of the experimental material presented will be given in a forthcoming paper 2~.

SHAPE OF THE INTENSITY-RATE CURVES IN CONTINUOUS LIGHT

The shape of the intensity-assimilation curve in continuous light has been the subject of several experiments and discussions (c/. RABINOWITCH 22 Chapter 28). Accurate knowledge of this curve may make it possible to check eventual kinetic schemes concerning the rate-limiting photosynthetic reactions. We therefore thought it worth-while to measure such curves as accurately as possible with dilute suspensions of algae (3 7 chlorophyll per cm 2 irradiated area).

This type of exper iment requires only the recording of a series of al ternate exposures of the algae to darkness and various light intensities. But a few difficulties are encountered. The first is the ra ther unpredictable behaviour of the algae in very weak light, yielding rates below or close to the compensat ion point. The second is tha t photoinhibi t ion effects m a y be induced by higher intensities a, which will decrease the rates in weak light observed subsequent ly. The sequence in which the readings are taken may therefore influence the shape of the observed curve. The best procedure is to measure a series of gradually increasing intensities. For the same reason it is sometimes difficult to approach experimental ly the exact value for the sa tura t ion rate: photo- inhibition may simulate the a t t a inment of a final plateau or even a decrease of the rate in high intensities.

A representative experiment in which the rate extrapolated to zero for zero light intens i ty is shown in Fig. 11. The data appeared to fit quite well with an exponent ia l function R R,,~ (I--e-k~), illustrated in Fig. I I as a solid line (c/. BRACKETT 23 and ARNOLD24).

Re/erences p. 258.

VOL. 21 (I956) PHOTOSYNTHESIS IN FLASHING LIGHT 257

The dashed line represents a non- rec tangu la r hype rbo la (c/. RABINOWlTCH22), ca lcu la ted for a s l ight ly higher value of R,. The rec tangu la r hyperbola , shown as a do t t ed line in Fig. I I , is ru led out comple te ly as a possible ana- R s - - - - ~

ly t ica l represen ta t ion of the ra te 8o ......... ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - r - - - - vs. in tens i ty curve. 7o :: .

60 / : ..... ' I t appea red t h a t in m a n y cases the g: a s s imi la t ion curve in con t inuous l igh t ~ " : ~:i~__._ R l 50 . . . . , ~ - - , . . . . 7 ' ~

could be a d e q u a t e l y represen ted by ~ R = Rs( I - -e I/Is),inwhichR is the ra te of pho to syn the s i s induced by in- 3o ,' . . ', tensity I, Rsisthe saturation rate at 321 ~ the p reva i l ing t empera tu re , Is is the " s a t u r a t i o n i n t e n s i t y " der ived from a m e a s u r e m e n t of the ( t empera ture - in - ~o dependent ) in i t ia l slope C in the w a y shown in Fig. i i (I s = Rs/ ~). : 5 lo 1~ 20

A prac t i ca l a d v a n t a g e of th i s for- Is Ret. ln t .ns i ty - -~ muta t ion is t h a t the comple te curve can be defined by the m e a s u r e m e n t of Fig. i i. Re la t ion be tween ra te of oxygen evo lu t ion and on ly two ra tes : one in weak and one l igh t i n t e n s i t y (3 °° C, 3 Y chlorophyll /cm2) • • : ac tua l in s a t u r a t i n g l ight, obse rva t ions ; - - - - - - : exponen t i a l func t ion ; - : non-

r e c t a ngu l a r hype rbo l a ; . . . . . . : r e c t angu l a r hyperbola . The hor izonta l a s y m p t o t e s for the var ious curves are

Though curves resembling the i nd ica t ed (Rs). one shown in Fig. I I p redomina te in our exper imen ta l mater ia l , dev ia t ions of this shape are not rare. Such dev ia t ions are near ly a lways in the d i rec t ion of a s t i l l shor ter t r ans i t ion range. The l inear re la t ion be tween ra te and in t ens i ty m a y ex tend up to or beyond half the sa tura- t ion rate.

The observed curves could in all cases be adequa t e ly descr ibed by a hyperbol ic funct ion, p rov ided the pa ramete r s were p roper ly chosen. This formula t ion therefore appears to hold genera l ly and will be discussed in a fur ther paper2L

ACKNOWLEDGEMENT

The au thor is g rea t ly indeb ted to Mr. A. J. LU~TINGH for his ass is tance in the experi- men ta l work.

S U M M A R Y

Kine t ic s tud ies of pho tosyn thes i s , m a d e in f lashing and con t inuous l ight, led to the fol lowing conclusions :

If short , suff icient ly b r i g h t flashes are a l t e r n a t e d wi th long d a r k periods, the flash y ie ld a t t a i n s a finite, t e m p e r a t u r e - i n d e p e n d e n t m a x i m u m va lue of one oxygen molecule per 1,5oo- 4,ooo ch lorophyl l molecules. F r o m m e a s u r e m e n t s in which bo th i n t e n s i t y and leng th of d a r k per iod were var ied we concluded t h a t du r ing a shor t flash a long- l iv ing i n t e r m e d i a t e is formed p h o t o c h e m i c a l l y in a o n e - q u a n t u m process.

E x t e n s i o n of the flash per iod in i t i a l ly y ie lds a marked , t e m p e r a t u r e - d e p e n d e n t increase of the flash yield, which ind ica tes the fo rma t ion of an add i t i ona l i n t e r m e d i a t e be tween l ight and oxygen. I t s concen t r a t i on was of the same order of m a g n i t u d e as t h a t of the p r i m a r y photo- chemica l product , formed in the very first m o m e n t of the flash.

If the flash per iod is increased beyond ~ o . o 3 sec, the flash yield increases wi th the ra te obse rved in s t rong con t inuous l ight.

Re/erences p. 258.

258 I3. KOK rOE. 21 (I956)

R E F E R E N C E S

1 B. KOK in J. S. BURLEW, Algal Culture, Carnegie Institution of Washington, Pub. 600, 1953 2 B. KOK, Biochim. Biophys. Aeta, 16 (1955) 35. a B. KOK, Bioehim. Biophys. A eta, (in the press). 4 B. KOK, Biochim. Biophys..4cta, 3 (1949) 625. 5 R. EMERSON AND W. ARNOLD, .]. Gen. Physiol., 15 (1932) 39t . 6 R. EMERSON AND W. ARNOLD, .]. Gen. Physiol., 16 (1932) I91. 7 K. DAMASCHKE AND L. ROTHBglHR, Biochem. Z., 327 (1955) 39. s W. ARNOLD, J. Gen. Physiol., 17 (1933) 145.

W. ARNOLD, Cold Spring Harbour Symposia Quant. Biol., 3 (1935) 124- 10 S. \VELLER AND J. FRANCK, J. Phys. Chem., 45 (I94I) 1359. 11 H. J. KOHN, Nature, 137 (1936) 7o6. 12 K. A. CLENDENNING AND H. C. EHRMANTRAUT, Arch. Biochem., 29 (195 o) 387 . 13 H. FHRMANTRAUT AND E. RABINOWlTCH, Arch. Biochem. Biophys., 38 (1952) 67. 14 O. WARBURG, Biochem. Z., IOO (1919) 230. 15 G. E. BRIGGS, Proc. Roy. Soc. London, B, ~3 o (194 I) 24. 16 H. TAMIYA AND Y. CH1BA, Studies Tokugawa Inst., 6 (1949) I. 17 j . N. I)HILIPS AND J. MYERS, Plant Phys., 29 (1954) 148. 18 R. LUMRY, J. D. SPIKES AND H. EYRING, Ann. Rev. Phys. Chem., 4 0953) 399- 1~ R. J. D. LUMRY, SPIKES AND H. EYRING, .4nn. Rev. Plant Physiol., 5 (I954) 271. 20 L. S. ORNSTEIN, E. C. \.VAsSINK, G. H. REMAN ANt) D. VERMEULEN, Enzymologia, 5 (1938) i i o . 21 B. KOK AND J. A. BUSlNGER (in preparation). 22 E. RABINOWITCH, Photosynthesis and Related Processes, 2, Pt. i, 1951. za F. S. BRACKETT, Cold Spring Harbour Symposia Quant. Biol., 3 (1935) 117. 24 \V. ARNOLD, personal communica t ion .

R e c e i v e d O c t o b e r I 2 t h , I 9 5 5

C O N S T I T U E N T S OF T H E E L E C T R I C O R G A N OF TORPEDO

I. CHROMATOGRAPHIC STUDIES

by

G. B. M A R I N I - B E T T O L O AND G. T R A B A C C H I

Laboratory o/ Therapeutical Chemistry, lstituto Superiore di Sanitg~, Rome (Italy)

INTRODUCTION

Investigations carried out on the electric organs of species of Torpedo (1". ocellata, T. marmorata and T. occidentalis) and of gymnotes (Electrophorus electricus) have made a considerable contribution to the study not only of the phenomena of electro- genesis but also of the metabolism of nervous tissue.

The whole problem of electrogenesis might be said to have taken on a new interest after DALE, FELDBERG AND VOGT'S 1 statement of their theory of the linkage between nerve impulse transmission to striped muscle and acetylcholine discharge at the motor end plate level.

FELDBERG AND FESSARD 2 used biological methods of estimation to show that the electric organ in Torpedo contains from 40 to IOO /xg of acetylcholine per g of fresh organ, that substances such as eserine, which are capable of inhibiting choline- sterase, make it possible to isolate acetylcholine in the effluent from the veins of Torpedo during perfusion and that arterial injection of acetylcholine has an electro-

Re[erences p. 264.