Action spectra for fluorescence excitation of pyridine nucleotide in photosynthetic bacteria and algae

14

N. BENIIAMOU, M. DAUNE, M. JACOB, A. LUZZATI, G. WEILL

Nous dCsirons remercier tout particuli~rement Monsieur le Professeur BENOiT qui au cours de cette 6tude nous a donn6 de prCcieux conseils, et Madame G. POUYET pour son amical concours lors des mesures de sCdimentation. BIBLIOGRAPHIE

Biochim. Biophys. Acta, 24 (1957) 548. 2 N. BENHAMOU, J. Chim. Phys., 53 (1956) 32. 3 A. NICOLAIEFF, rCsultats non publics. 4 M. DAUNE, L. FREUND ET GI SCHEIBLING, J~ Chim. Phys., 54 (1957) 924. s M. DAUNE ET L. FREUND, J. Polymer Sci., 23 (1957) 115. 8 M. CHAMPAGNE, A. M. JUNG ET M. DAUNE, J. Chim. Phys., 54 (1957) 149. BOLTZMANN, Wied. A~n., 53 (1894) 959. s E.' O. FIELD ET J. R. P. O'BRIEN, Biochem. J., 60 (1955) 656. 9 T. SVEDBERG ET K. O. PEDERSEN, The Ultracentri]uge, Oxford, 194 o. 10 V. N. SCHUMAKER ET H. K. SCHACHMAN, Biochim. Biophys. Acta, 23 (1957) 628. 11 W. D. HUTCHINSON ET J. R. VINOGRAD, Report o] the Year I956-::957, Calif. Inst. of Technol. 12 E. O. F1ELD E T A . G. OGSTON, Biochem. J., 60 (1955) 661. 13 V. HASSERODT ET J. R. VINOGRAD, Communication pr~sentde au z32~me Congr~s de Am. Chem. Soc., N e w York, N.Y. (Sept. 1957). 14 V. M. INGRAM, Nature, 178 (1956) 792. is M. F. PERUTZ, A. M. LIQUORI ET F. HEIRICH, Nature, 167 (1951) 929. 1, A. C. ALLISON ET M. P. TOMBS, Biochem. J., 67 (1957) 256. 17 H. G. KUNKEL ET Q. WALLENIUS, Science, 122 (1955) 288. 18 M. MORRISON ET J. L. COOK, Science, 122 (I95#) 920. 19 C. J. GUTTER, H. A. SOBER ET E. A. PETERSON, Arch. Biochem. Biophys., 62 (1956) 427 . 1 N . BENHAMOU ET G. WEILL,

Biochim. Biophys. Acta, 37 (196o) 1-14

ACTION SPECTRA FOR FLUORESCENCE PYRIDINE

NUCLEOTIDE

EXCITATION OF

IN PHOTOSYNTHETIC

BACTERIA AND ALGAE J O H N M. O L S O N * AND J A N AMESZ

Biophysical Laboratory**, State University, Leiden (The Netherlands) (Received March 23rd, 1959)

SUMMARY

An apparatus is described for the determination of u.v. action spectra of fluorescence excitation. Excitation spectra for the blue fluorescence increase caused b y photosynthetic illumination of photosynthetic bacteria and algae have been found to be very similar to the excitation spectrum of D P N H bound to yeast alcohol dehydrogenase. The results support previous proposals based on fluorescence spectra that illumination causes the reduction of intracellular pyridine nucleotide bound to protein constituents. * Postdoctoral Research Fellow of the U.S. Public H e a l t h Service. P r e s e n t address: G r a d u a t e D e p a r t m e n t of Biochemistry, Brandeis University, W a l t h a m 54, Mass. (U.S.A.). ** Postal address: Nieuwsteeg. 18, Leiden, The Netherlands.

Biochim. Biophys. Acta, 37 (196o) i 4 - 2 4

FLUORESCENCE EXCITATION OF PYRIDINE NUCLEOTIDE

15

INTRODUCTION In previous studies x,~ of changes in blue fluorescence caused b y photosynthetic irradiation of photosynthetic bacteria and algae, the fluorescence changes have been attributed to intracellular changes in bound pyridine nucleotide on the basis of the spectra of these changes and their similarity to the fluorescence spectra of D P N H (reduced diphosphopyridine nucleotide) bound to various dehydrogenases. In the present investigation this interpretation has been tested by determining action spectra of fluorescence excitation. Excitation spectra have been obtained for the light-induced fluorescence increase in two species of photosynthetic bacteria and two species of algae. For purposes of comparison the fluorescence excitation spectra for the D P N H - y e a s t ADH (alcohol dehydrogenase) complex and free D P N H have also been determined. APPARATUS

The set-up for the determination o f action spectra (26o-4o~ mt~) of fluorescence excitation is shown schematically in Fig. I. Lens L 1 places a slightlY defocused image of the Xenon arc at the entrance slit of the monochromator. The exit slit of the monochromator is imaged on the sample cuvette b y I/2 L 1. A quartz plate (thickness I mm) in th.e excitation, beam directs about 8 % of the incident light to a phototube which monitors the intensity. The. fluorescing portion of the sample is imaged on the photocath0de of the photomultiplier b y L3. The tungsten lamp in the light condenser system illuminates the rectangular stop uniformly, and lens L 4 images the stop on the sample cuvette. The IOOO watt Xenon lamp (Osram XBO iooi) is operated on batteries. The quartz lerises L1 and Lz are the standard entrance slit optics of the Bausch & Lomb

/'~I~

,~

tungsten lamp

rectangular

system

~ 4 ~ , / ~ 11L4 circular

~ " ~ ~ \ 'j~

chopper

sampte~ll/~ u e,,e

~A Ufl A "MR{L,nu

p~hotoceli photomultlplier ~ ~_~

Fa

Fig. I. Apparatus for determining action spectra of fluorescence excitation. Monochromatic u.v. light excites fluorescence which is detected by the photomultiplier. The tungsten lamp irradiates photosynthetic organisms simultaneously with red light. Lenses are designated L, filter sets F, and mlrrors M. The figure is not exactly to scale. Biockim. Biopkys. Acta, 37 (196o) I4-24

16

I . M . OLSON, J. AMESZ

grating monochromator (//4-4, 500 mm focal length, 12oo lines/mm grating). The 50 c/sec hght chopper is a sectioned disk rotated by a synchronous motor. Filter F 1 is a Coming 9863 which reduces stray light from the monochromator. The monitor photocell (CsSb surface) operates as part of a Photovolt photometer, model 5oI-M. The sample cuvette is a I mm quartz cell placed perpendicular to the excitation beam to prevent reflected light from reaching the fluorescence detector. The glass lens system Ls is of high aperture,//I.2. Filter combination F~ consists of a wide band interference filter GAB K 2 and Schott filters BG 7, I mm, and GG 3, 2 ram. The transmission of this combination drops from a maximum value of 36 % at 450 m y to ~-- 9 % at 420 and 47 ° m~. Schott BG 23, 2 mm is added to F 2 when the sample is illuminated with 678 m~ light. An RCA I P 2 I photomultiplier serves as light detector. Filter F4 is water, 2 cm, to absorb heat radiation above 1.2 m/~. L4 is a glass lens, //2. 9. For illumination of photosynthetic bacteria, F3 is 2 × Schott RG 9, 2 mm, providing a broad transmission band with the maximum at 825 m~, and the light source is a 15o W projection bulb. The intensity available at the sample cuvette is 12 mW/cm ~. For illumination of algae, F3 is Schott RG 5, 2 mm and interference filter GAB 678, and the source is a 500 W bulb. In this case the intensity is 1.2 mW/cm ~. The light condenser system is that of the Aldis "Star" 5 × 5 cm slide projector minus heat filter. A rectangular stop, io × 30 mm, is placed in one slide of the slide transport, and a metal blank in the other. Illumination is turned on and off by means of the slide t~ansport. The electrical signal from the photomultiplier is amplified and rectified by a phase sensitive amplifier previously described s. The rectified signal is recorded by a Philips I second recorder. The recording apparatus responds only to the modulated fluorescence signal and rejects any signal due to scattered radiation from the photosynthetic illuminator. The filter set F 2 reduces the intensity of scattered light sufficiently to avoid noticeable increase in phototube shot noise when the cuvette is illuminated with lJhotosynthetic light. Over the wavelength interval 280 to 400 m~ stray light in the excitation beam is less than 1/2 %. For a typical case in which a 6 % (wet cell volume ratio) suspension of Rhodospirillum rubrum is excited to fluorescence, the signal from scattered stray light is estimated to be of the order of I o/ ,o of the signal from fluorescence of bacteria, suspension medium, and cuvette over the excitation wavelength interval 280 to 380 m/~. At 39 ° m~, scattered excitation light accounts for roughly IO % of the signal, but for 2 ~ 360 m y the signal from scattered excitation light is less than 1% of the fluorescence signal. The signal from fluorescence of the suspension medium and cuvette is estimated to be IO to 20 % of the signal from bacterial fluorescence for the 335 m/~ excitation maximum. Fluorescence from the medium is minimized by the use of concentrated cell suspensions. Fortunately, t}~,¢,, ct~m;~'~ ~n signal caused by photosynthetic illumination of the sample is independent of scattered lig.C and fluorescence of the medium and cuvette. Only changes in fluorescence are recorded, and these changes can be caused only by photochemical reactions within the cells. The intensity of the photosynthetically active illumination is determined by means of a thermopile in the sample cuvette position. Similarly the monitor phototube in the position shown in Fig. I is calibrated in terms of the energy intensity of Biochim. Biophys. Acta, 37 (~96o) 14-24

FLUORESCENCE EXCITATION OF PYRIDINE NUCLEOTIDE

17

the excitation b e a m incident at the cuvette over the entire useable wavelength i n t e r v a l 260 to 400 m/~. The calibration curve is converted into q u a n t u m i n t e n s i t y t e r m s b y the relation I O ~- I E / h v . All excitation spectra are d e t e r m i n e d as fluorescence per u n i t q u a n t u m i n t e n s i t y of excitation. The m o n o c h r o m a t o r slits are open to a w i d t h of 3.12 m m a n d stopped down to an average height of 15 ram. The h a l f - m a x i m u m - i n t e n s i t y b a n d w i d t h of the excitation b e a m is thus 5.0 m/~. The i n t e n s i t y of the exit slit image at the sample cuvette is a t t e n u a t e d b y m e a n s of a small circular stop between F 1 a n d lens 1/2 L 1 in order to minimize photochemical reactions in the sample due to excitation light. The m a x i m u m q u a n t u m i n t e n s i t y of the excitation b e a m is o b t a i n e d at a b o u t 365 m/z. A t this wavelength the energy i n t e n s i t y averaged over the entire slit image for one chopping cycle never exceeds o.I m W / c m 2, which is less t h a n i/IO the lowest energy i n t e n s i t y used for p h o t o s y n t h e t i c illumination. The relative increases in fluorescence u p o n illumination are found to be the same whether the i n t e n s i t y of the excitation b e a m is increased or decreased b y a factor of 2. The reliability of excitation spectra o b t a i n e d with the a p p a r a t u s was tested by comparing the excitation spectrum of D P N H , with the absorption (I-T) s p e c t r u m as d e t e r m i n e d b y m e a n s of a Zeiss PMQ I I spectrophotometer. The results summarized in Table I appeared satisfactory. TABLE I COMPARISON OF ABSORPTION AND FLUORESCENCE

Waveleneth {m/t)

31o 315 320 325 33° 335 34° 345 35° 355 360 365 37° 380

A~ A 340

0.69 0.76 0.84 0.92 0.96 I .oo I .oo o.98 0.92 0.83 o.72 o.62 o.51 o.23

EXCITATION FOR

DPNH

(1/I) 2 (l/l}a,o

0.68 0.75 0.85 0.93 0.98 t .02 i .oo i .oo 0.94 0.86 o.76 0.67 0.54 o.3t

4-5" IO-a M DPNH in 0.05 M phosphate buffer pH 7.7d = ~ mm; . 12 = 8.0 m/t: 3 34o = 47.5 °/o..4a is the absorption (t - - Ta). (]/I),l is the fluorescence per incident quantum. MATERIALS All organisms were grown in a light c a b i n e t u n d e r i l l u m i n a t i o n from t u n g s t e n bulbs. P h o t o s y n t h e t i c bacteria were grown anaerobically in glass-stoppered bottles. Algae were cultured in E r l e n m e y e r flasks partially filled with m e d i u m t h r o u g h which air was b u b b l e d continuously. Concentrated suspensions ( ~ 50/,o wet cell volume per volume suspension) of each organism were prepared for e x a m i n a t i o n . l~iochim. Biophys. Acta, 37 (196o) I4-24

18

J . M . OLSON, J. AMESZ

Chromatium, strain D, was grown in a non-sterile inorganic medium containing bicarbonate, sulfide, and thiosulfate as substrates (c/. NEWTON AND KAMEN4). Bacteria were resuspended in fresh medium for observation. Rhodospirillum rubrum, strain 4, was grown in sterile medium containing 1% peptone (Difco) and 1/2 To NaC1 in tap water. Since the peptone medium is highly fluorescent, bacteria were washed once and resuspended in a medium containing 15 m M Na butyrate, 1/2 To NaC1, and IO m M phosphate, pH 7.0. Samples were gassed with 5 % COs and 95 % Ns before examination. The blue alga Anacystis nidulans was grown in Medium C of KRATZ AND MYERS5 initially adjusted to pH 7,4- Suspensions were examined in culture liquid. Chlorella vulgaris was grown in an inorganic salt solution according to WARBURGe. The gas phase was enriched with COs (5 To). The algae were examined suspended in culture liquid. For the determination of excitation spectra for free and bound pyridine nucleotide, reduced diphosphopyridine nucleotide (Sigma) and yeast alcohol dehydrogenase (Boehringer) were used. EXPERIMENTS

The u.v. excitation spectrum for the increase in fluorescence (420-47 ° m/z) caused by photosynthetic irradiation of a bacterial or algal suspension is obtained by repetition of a fixed cycle of light and dark periods. The sequence of fluorescence changes is then in general reproducible. The wavelength and intensity of the photosynthetic light remain the same for each light period, while the wavelength of the radiation for excitation of fluorescence is varied from 39 ° to 26o m/~ in 5 m~ steps. The ratio of fluorescence signal to quantum intensity of excitation is proportional to the percentage of excitation light absorbed by fluorescent material only. Therefore the excitation spectrum indicates the absorption characteristics of the fluorescent material.

Chromatium The sequence of fluorescence changes in Chromatium during and after illumination have been described in a preceding paper ~. Upon illumination the fluorescence initially increases to a maximum and thereafter may either decrease or remain approximately constant. For determination of the excitation spectrum for the initial fluorescence increase a suspension was illuminated (12 mW/cm s) for 3 sec every 2 min. The light interval was long enough to permit the fluorescence to reach its maximum value, and the dark interval was sufficient to allow the return to the dark steady-state level before the next light interval. The excitation spectrum (Fig. 3 A) has maxima at 280 and 335 /z. The fluorescence excitation spectrum of bacteria in the dark steady-state shows similar maxima, even though this spectrum includes fluorescence from the medium and the cuvette in addition to a contribution from scattered excitation light for ~ 36o m~.

Rhodospirillum rubrum A typical sequence of fluorescence changes in anaerobic Rsp. rubrum is shown in Fig. 2A. Illumination (12 mW/cm s) resulted in an initial fluorescence increase followed Biochim. Biophys. Acta, 37 (196o) 14-24

FLUORESCENCE

EXCITATION

OF

PYRIDINE

z9

NUCLEOTIDE i

A. RHODOSPlRILLUM RUBRUM

ANACYSTIS NIDULANS

B

6

i

C.

4O

CHLORELLA

5

8

4

L 2

0

f

f

3

7

9/A

L

i

I 4

0

2

4

0

t in rain

t in m m

2

t In

rr

Fig. 2. Recordings of light-induced fluorescence changes in (A) anaerobic Rsp. rubrum, (B) aerobic Anacystis nidulans, a n d (C) aerobic Chlorella vulgaris.

!

O 260

I 280

I 300

I

I

I 320

I 340

I 360

I 380

]

I

~, in m F

I

I

I

RHODOSPI~ILLUM

[

I

Iq UBP, U M

(5 f B.

~O

i/io ',~ I

x~ "a

O 2(}0

I

I

I

I

I

t

:)80

300

320

340

360

380

k in mlJ.

Fig. 3. E x c i t a t i o n s p e c t r a for dark fluorescence (//IO) a n d light-induced fluorescence increase (ZI~) in (A) Chromatium, a n d (B) Rsp. rubrum.

Biochim. Biophys. Acta, 37 ([96o) 14-24

20

j . M . OLSON, J. AMESZ

by a much slower decrease. (The intensity required for a half-maximal fluorescence increase was o.16 mW/cm2). Upon cessation of irradiation the fluorescence decreased abruptly. In order to obtain an excitation spectrum for the initial fluorescence increase upon illumination, an anaerobic suspension was illuminated for IO sec every I /1/ 2 rain, and the maximum fluorescence change recorded for each excitation wavelength. The spectrum as shown in Fig. 3]3 has maxima at ~-~ 280 m~ and at 335 m/~. The excitation spectrum for the Lotal fluorescence of the suspension in the dark also shows maxima in the same regions.

Anacystis nidulans The effect of illumination on the fluorescence of aerobic Anacystis nidulans is shown in Fig. 2B. Illumination caused an immediate increase in fluorescence to a new steady-state. Upon darkening the fluorescence returned to the previous dark steady-state level after one oscillation. In the determination of the excitation spectrum for the light-induced fluorescence increase (Fig. 4 A) the ratio of light period to dark period in the illumination cycle was made large enough to maintain aerobiosis throughout the experiment. The uncertainty in the measurement of the fluorescence change increased markedly at wavelengths below 30o mtz. Nevertheless, a maximum in the region of 280 m/~ is apparent as well as a rather broad maximum at ~ 33o m/z. In contrast to the results obtained for the photosynthetic bacteria, the excitation spectrum for Anacystis in the dark steady-state bears little resemblance to the spectrum of the light effect.

Chlorella vulgaris The time course of the light effect in aerobic Chlorella is shown in Fig. 2C. The intensity of illumination (1.2 mW/cm ~) was close to saturation. (The intensity required for the estimated half-maximum effect was o.16 mW/cm) 2. Upon illumination the fluorescence increased to a steady level, and upon darkening it undershot slihgtly before returning to the dark steady state. The difference between the illuminated steady-state level and the maximum point of undershoot upon darkening was quite reproducible and was therefore used for the determination of the excitation spectrum of the light effect (Fig. 4B). Although poorly defined below 29 ° m~, this spectrum appears to have a maximum or plateau in the 280 m/z region in addition to the maximum at 335 m/~. The spectrum for the total dark fluorescence shows only a broad maximum in the 325-340 m~ region.

Diphosphopyridine nucleotide Action spectra of fluorescence excitation for both free and bound D P N H were obtained by following the procedures used by DUYSENS AND KRONENBERG7 in determining the corresponding fluorescence emission spectra. All experiments were carried out at a temperat,n:e :~lightly abow~ o °. Each fluorescence measurement was corrected for the ttuorescence of cuvc~te and solvent. This correction became sizable only for excitations at wavelengths below 270 m/~. The excitation spectrum of the complex, D P N H and yeast ADH, is shown as curve I in Fig. 5A. Maxima occur at about 278 and 344 m#. Addition of excess acetaldehyde causes oxidation of nearly all D P N H and a decrease in fluorescence as shown by curve 2. ADH alone (curve 3) shows only a slight fluorescence in the wavelength Biochim. Biophys. Acta, 37 (I96O) 14-24

FLUORESCENCE EXCITATION OF PYRIDINE NUCLEOTIDE I

I

I

A. ~"

I

0

I

I

21

I

ANACYSTIS N IDUL ANS

t5

-

t0

~

~A_/,

&f

. '.d

- -'~a.z x

f/IO

0 260

I

t

I

I

t

i

280

300

320

340

360

380

1

i

). in ma

i,x B.

I 5

o

I

[

I

o

~

CHLORELLA

,o

VULGARIS

t\ \\

S

A~ & - a -z~-

0 260

I 280

I 300

I 320 Xin

o

.&_

I 340

I 360

I 380

m#

Fig. 4. E x c i t a t i o n spectra Ior dark fluorescence (//io) and light-induced fluorescence increase (A/) in (A) Anacystis nidulans, and (B) ChloreUa vulgaris.

region where the apparatus is sensitive. The shoulder at about 280 m~ in the excitation spectrum corresponds to the strong absorption peak of A D H between 275 and 280 m~. WEBER8 has found that the excitation spectrum of free D P N H has maxima at 260 and 34 ° m~. A comparison of curve I of Fig. 5A and curve 2 of Fig. 5B indicates that the fluorescence yield of free D P N H is considerably less than the yield of the D P N H - A D H complex for excitation in the 34 ° m/x region (c/. DUYSENS AND

KRONENBERGT). For comparison with the excitation spectra of fluorescence changes in photosynthetic organisms, the difference excitation spectrum, A D H - D P N H minus ADH-DPN, was obtained b y subtracting curve 2 from curve I in Fig. 5A. The resulting Biochim. Biol~hys. Acta, 37 (I96o) 14-24

22

J.M.

OLSON, J. AMESZ

A,

~5

/

oH DoN.

t0

2

t

5 1

260

280

300

320

340

300

380

360

380

X in m~

B.

15

ADH-DPNH 10

/

"~

minus ADH-DPN t

5 2 I

O

t 260

280

300

320

340

k in m~

F i g . 5. E x c i t a t i o n s p e c t r a o f f l u o r e s c e n c e . (A) C u r v e I : 1.8- i o ~i M D P N H , 6 . IO -8 M y e a s t A D H , 2 . lO 4 M s e m i c a r b a z i d e , a n d 2. 5 . lO -3 M p h o s p h a t e , p H = 7.9- C u r v e 2 : A f t e r a d d i t i o n o f e x c e s s a c e t a l d e h y d e (2. i o -2 M ) t o a b o v e s o l u t i o n . C u r v e 3 : 6 . IO -5 M y e a s t A D H . (B) C u r v e I : d i f f e r e n c e s p e c t r u m u p o n a d d i t i o n o f e x c e s s a c e t a l d e h y d e t o 1 . 8 . lO 4 M D P N H a n d 6 . IO -~ M y e a s t A D H . C u r v e 2 : 1 . 8 . i o -~ M D P N H .

spectrum (curve I, Fig. 5 B) is remarkably similar to the spectra obtained from the bacteria and algae, whereas the spectrum for free D P N H is distinctly different. These results are summarized in Table II. DISCUSSION

The use of fluorometry as a method of following light-induced changes of intracellular pyridine nucleotide in photosynthetic organisms requires that the observed fluorBiochim. Biophys. Acta, 37 (196o) 1 4 - 2 4

F L U O R E S C E N C E E X C I T A T I O N OF P Y R I D I N E TABLE

NUCLEOTIDE

23

II

CHARACTERISTICS OF EXCITATION SPECTRA

Excitation spectrum

Free DPNH Yeast ADH DPNH-ADH

minus DPN-ADH

Location o] maxima first (my) second ( m # ) 26o* ~ 28o , ~ 28o

34 ° none 345

~ ~ ,~s ~

335 335 325-335 33o-34o

Ratio first/second i.o* -1.3

L i g h t effect for

Chromstium, s t r . D Rhodospirillum rubrum Anacystis nidulans Chlorella vulgaris

280 280 280 28o

~ ~ ~ ~

1.6 1.2 1.6 1.7

* WEBER8

escence changes be identified specifically as arising from changes in pyridine nucleotide without substantial contribution from changes in other fluorescent compounds. Previous studies 1, ~ have shown that the spectra of light-induced fluorescence changes are very similar to the fluorescence spectra of a number of D P N H - e n z y m e complexes. In the present study the excitation spectra for these light-induced changes have been shown to be very similar to the excitation spectrum for a typical DPNH--enzyme complex, D P N H bound to yeast ADH. Both fluorescence spectra and excitation spectra indicate that photosynthetic illumination causes an initial increase in the concentration of bound P N H (reduced pyridine nucleotide) rather than free P N H . The PNH-binding enzyme m a y be related to the photoreductase obtained b y SAN PIETRO AND LANG9 from spinach leaves. As yet no data have been obtained concerning a possible differentiation between D P N H and T P N H (reduced triphosphopyridine nucleotide) on the basis of fluorescence characteristics. I t has been tacitly assumed that the observed fluorescence changes in whole cells m a y reflect changes in either or both of the pyridine nucleotides. In the photosynthetic bacteria, Chromatium, strain D, and Rhodospirillum rubrum the spectra of the light-induced fluorescence increases are almost identical (c[. D U Y S E N S A N D S W E E P 1 a n d O L S O N , D U Y S E N S A N D K R O N E N B E R G 2 ) , a s a r e also t h e excitation spectra. For both organisms the excitation spectrum of fluorescence in non-illuminated cells indicates the presence of bound PNH. The transient increase in bound P N H upon illumination is about 20 % for Chromatium and roughly 50 % for Rsp. rubrum. For Chlorella the increase appears to be 20 to 30%. In Anacystis nidulans the spectrum of the light-induced fluorescence increase differs slightly from the spectra of the bacteria in having its m a x i m u m at 460-47 o m/z instead of at 44o m y 1. The excitation spectrum for the light effect in algae is basically similar to the spectrum for bacteria, but the 33o-34o m/z peak is somewhat broader in the former case. This slight difference is not unexpected in view of the great differences in size and internal structure between the two types of organisms. The excitation spectrum of fluorescence in non-illuminated Chlorella suggests the presence of P N H as does also the fluorescence spectrum determined b y DOYSENS AND AMESZ1°. For Anacystis, on the other hand, the corresponding excitation spectrum differs markedly from the pyridine nucleotide spectrum. This difference m a y be Biochim. Biophys. Acta, 37 (196o) 1 4 - 2 4

24

j.M.

OLSON, J. AMESZ

attributed to the considerable amount (up to o.I % dry weight) of other blue fluorescent material in Anacystis. FORREST, VAN ]3AALEN AND MYERS11,13 have identified the principle fluorescent substances as pteridines. The presence of a strong peak at 280 m/~ in the fluorescence excitation spectrum of the yeast A D H - D P N H complex is evidence for the transfer of quanta absorbed by aromatic amino acid residues in the protein moiety to the pyridine ring of the nucleotide moiety. Recent studies of dehydrogenases by SHIFRIN AND KAPLAN13 indicate that pyridine nucleotides and their analogues quench protein fluorescence (2 ~-~ 35o m/,) in proportion to the extent of binding. Thus it appears that quanta normally appearing as protein fluorescence are transferred to nucleotide upon the formation of a protein-nucleotide complex. ACKNOWLEDGEMENTS

The authors wish to express their appreciation for the advise and encouragement of Dr. L. N. M. DUYSENS. Dr. J. ]3. THOMAS and the Biophysical Research Group at the State University at Utrecht very kindly provided laboratory space and biological material for this work. This investigation was supported by the Netherlands Organization for Pure Scientific Research (Z.W.O.). REFERENCES 1 L. N. M. DUYSENS AND G. SWEEP, Biochim. Biophys. Acta, 25 (1957) 13. " J. M. OLSON, L. N. M. DUVSENS AND G. H. M. KRONENBERG, Biochim. Biophys. Acta, 36 (1959) 125. a L. N. M. DUYSENS, in H. GAFFRON, Research in Photosynthesis, Interscience Publishers, N e w York, 1957, p. 59. 4 j . \ v . NEWTON AND M. D. KAMEN, Biochim. Biophys. Acta, 2I (I956) 71. 5 W. A. KRATZ AND J. MYERS, Am. J. Botany, 42 (1955) 282. 6 0 . "WARBURG, Biochem. Z., IOO (1919) 230. 7 L. N. M. DUYSENS AND G. H. M. KRONENBERG, Biochim. Biophys. Acta, 26 (1957) 4378 G. YVEBER, Nature, 18o (1957) 14o9. 9 A. SAN PIETRO AND H. M. LANG, J. Biol. Chem., 23i (1958) 2I I. 10 L. •. M. DUYSENS AND J. AMESZ, Biochim. Biophys. Acta, 24 (1957) 19. 11 H. S. FORREST, C. VAN BAALEN AND J. MYERS, Science, 125 (1957) 699. 12 C. VAN ]~AALEN, H. S. FORREST AND J. MYERS, Proc. Natl. Acad. Sci. U.S., 43 (1957) 7oi13 S. SHIFRIN AND N. O. KAPLAN, Proc. Natl. Acad. Sci. U.S., 44 (1958) 177.

Biochim. Biophys. Acta, 37 (196o) 14-24