SPECTRA OF CHLOROPHYLLIN a
April 5 , 1964
1309
The sulfur trioxide may be formed by 0
(1)
+ so2 +SO8
The small amount of molecular oxygen probably arises by attack of 0 on SO3* according to 0
+ so3 --+- On + so2
The alternate explanation that the O2 is formed by recombination of 0 atoms in the spurs (analogous to H H = Hz in irradiated water) can be ruled out by the following argument: for gas phase reactions, the 0 M = 0 2 4- M is specific rate constant for 0 9.8 x lo8 1.* mole-* sec.-',I1 whereas for 0 SOz Lf = SOa M it amounts to 3 x 10101.2mole-zsec.-'.l2 In the condensed phase, in analogy to the events in irradiated water, the 0 atoms are probably clustered a few iingstrom units apart within isolated volume elements, each 0 atom being surrounded by SO2 molecules. Therefore, no significant amounts of 02 arising from recombination of 0 atoms can be expected. The reaction 0 SO3 + SOz Ozis competitive with 0 SOz --t so3. Applying the usual equations for such cases,15 the following relations are obtained among G(S03), G(O2),Go (the primary yield of oxygen atoms), the specific rate constants k of 0 SO3 = SO, Ozand 0 SOz = SOa,respectively, and the concentrations of SO2 and so3
+
+ +
+
+
+
[O,l =
+
+
(11) R . R Reeves, G Manella, a n d P. Harteck, J . Chem P h y s . , 32, 632 (1960). (12) F . K a u f m a n , Proc. R o y . SOL.(London), 8 2 4 7 , 123 (1958) (13) A 0 Allen. " T h e Radiation Chemistry of Water and Aqueous Solutions," D . Van Kostrand Co., I n c . , Princeton, N . J . , 1961, p. 7 . (14) C J . Hochanadel, "Comparative Effects of Radiation," John Wiley and Sons, I n c . , New York. N . Y . , 1960, Chapter V I I I . (15) See ref. 13, p . 29.
[CONTRIBUTION FROM
THE
(G(S03) X dose)2 ko +so2
(3)
2[SOZl 7
+
+
+
+
Equations 1 and 2 show that a t low doses ([SO3] = 0) G(S03) = Go and G(O2) = 0. With increasing accumulation of SO3, the rate of oxygen production increases whereas the rate of sulfur trioxide formation decreases. Equation 2 is integrated to give the concentration of oxygen molecules as a function of the dose. Noting that G(02) = d[Ozj/d dose, [SOz] = constant, (ko+so2/ko+~~3)([SO~I/[SO~I) >> 1, GO = G(SOs),and [SO31 = G(SO3) X dose, one obtains
RO SO^
where concentrations are in number of molecules per ~ m and . ~dose is in units of 100 e.v. ern.?. Figure 2 shows the curve according to eq. 3 with ko + so1/ ko + so3 = 0.1. From eq. 1 follows Go = 1.35. According to the proposed mechanism, Go equals Gso; therefore, the net decomposition of pure liquid sulfur dioxide proceeds with a primary G-value of 1.35. Acknowledgments.-The author wishes to thank Dr. W. J. Burlant for many fruitful discussions and his continued interest in this work, J. C. Neerman for the mass spectrometric analyses, and M. Valukonis for help with the experiments.
DEPARTMENT OF CHEMISTRY, POLYTECHNIC INSTITUTE OF BROOKLYN, BROOKLYN 1, N. Y , ]
Spectral Properties of Chlorophyllin u1 BY GERALD OSTER,SUSEB. BROYDE,AND
JUDITH
S.BELL IN^
RECEIVED JULY3, 1963 The infrared and visible absorption spectra and the fluorescence spectrum of chlorophyllin a were determined and compared with those of chlorophyll a. The infrared spectrum showed the absence of a carbonyl group except when chlorophyllin b was present as an impurity. This provides the basis of a convenient method of analysis of the chlorophyll b content of a given chlorophyll preparation. Since t h e cyclopentanone ring is absent in chlorophyllin, the observed infrared bands help t o clarify those observed in the case of chlorophyll. The visible absorption spectrum of chlorophyllin a has features in common with t h a t of chlorophyll a. In particular, the formation of colloidal aggregates is shown b y the appearance of an absorption band a t i 3 5 mp just a s is observed for microcrystals of chlorophyll a. The appearance of a pronounced absorption maximum a t 688 xnp attributed t o dimer formation is analogous t o t h a t observed by difference spectra in the case of concentrated solutions of chlorophyll a. In stretched polymer films chlorophyllin a and chlorophyll a exhibit a positive dichroism which is greater for the blue absorption maximum than for the red. The fluorescence spectrum of chlorophyllin a is markedly dependent on the wave length of excitation.
Introduction Because of the oleophilic character of chlorophyll, photochemical and spectral studies of this pigment have of necessity been carried out in organic solvents. In the chloroplast, however, some of the chlorophyll (1) ( a ) Supported b y t h e United S t a t e s Air Force through t h e Air Force Cambridge Research 1,aboratories under Contract No. AF19(628).475 a n d b y t h e National Institutes of Health under Research G r a n t No. C6351. ( b ) Taken in p a r t from t h e dissertation of Suse B. Broyde, submitted June, 1963, t o t h e Faculty of t h e Polytechnic I n s t i t u t e of Brooklyn in partial fulfillment of t h e requirements for t h e degree of Doctor of Philosophy. ( 2 ) Public Health Service Research Career Awardee.
molecules are in contact with an aqueous medium. We have therefore undertaken a study of the watersoluble chlorophyll derivative chlorophyllin, in aqueous solution. Chlorophyllin, the product of saponificaOf chlorophyll in has been known for more than 50 years,3 yet the photochemical and spectral properties of this interesting pigment have not been studied extensively. A preliminary account of our (3) (a) R. Willstiitter and A. Stoll, "Untersuchungen iiher Chlorophyll," Springer-Verlag, Berlin, 1913; (b) S. Aronoff in "Handbuch der Pflanzenphysiologie," Vol. 5, P a r t 1, W. Ruhland, Ed., Springer-Verlag. Berlin, 1960.
1
ereiice heairih tvitli their direction of vibration vertical. Since it fotintl that both beams are btrorigly polai-ized, the sxlnple UYIS ititerpoied lxt\veeii t h e polarizer arid the detector. :\ti\i)rptiori ipectrn ivere taken with t h e direction of stretch of the ~ ~ i p:it l r variiiui angles with respect t o t h e orientation of the po1:irizc.r. 'The diclirriic saiiiples $vert. prepared conipletcly i n the dark l)y atitling a concentr:i tell water s~)lutionof chlorophyllin (I t o a water ~iilutionr i f pol>.viri)1 alcohol and itirriiig for L' llr. with :I iiiagnetic 5tirrer. T h e reiultiiig liornogeneoiir viieciub ?i~lution W:LS caht o i i ;I \ilicone-trcated g l a i s pl:tte and allowed t o i i r y . The stripped-riff f i l i r i \ w s held firInly a t both ends, tlicri warmeti to abuut 75' b y holding it inoirientcirily over ;I Iiot plate. 'I'hcn t h e film wiis rapidly stretched about threefold anti ciiol~titi) ~ I I O I I I teiiiperature i i i t h i i contlition. Dichroic f i l i i 1 5 ( i f rliloropli! 11 il c'in 'ilhn be obtuiiirtl b y this technique, 1))- ,ititling :I Ill~tll~IlllJl solution of c l i l ~ ~ r ~ ~ p lni yt lol a rnethan(il solution of p~~lyviiiylhut!-ral. T h e ilicliroism of these filins, h o w riiiuncetl than t h o s e for chlorophylliii c:--pcil~-vi d u e to poorer orierit:itioii possibilitii~srr.ith ~ ~ i ~ l ~ ~ v i r i ~ ~ l ~ ~ u t ~ ~ r ~ Fluoresccnce ,pectr,i ribtaineil by e\citutiori Lvitli lengths u e r e deteriniiied in the laboratory of I)r. S the IBM Watson Research Lab. I l l i s instrument is ;I niotlificiitiim of t h a t described" :uid consists of ;i yenoii w u r c e w h ~ i \ eo u t put passes through a iiiorioclir(jm~itorand a n xusiliary filter. 'l'lie fluorescent light which is itt right angles tu tlic cycititig hc~tiii is esnrnineti froin tlie front surface i i f tlie ianipie by lxi>\iiig it through a second tii~1110cI1ro111at1~r t o ;i c o ~ ~ l rrntl-on d inultipli~~r phototube (Dtirnoiit T y p e 6911). .I11 data obtained :irc corrected for the sniall amount of scattered exciting light :$\ tlcterrninetl in "blank" runs. Queriching of the tluorescerice w a s studied in :in hi tic^^ light scattering instrument" using t h e 4.76 in@rriercury line a n d inemuring with a yellow filter the relative light intrnsity fallirig on a n IZCh T y p e 1P2X rnultiplier phototube. \vas
r
n
p
'
1.
0..
*
i
1:
I.0
/
1
1 1 t a*1
,
u
¶ II
¶ 1a
L 1O
,a,
,
i
,
IT . I1
,
,a
-
.I
i
I1
I
,I
,I
L
-
9p
L
.
ID
FREPUENCY~CM','x IO..*
Fig 1
-Infrared absorption spectrum of chlorophyllin n in KBr pellet
work ' and its relevance to photosynthesis5 has been presented. X comparison of some spectral and photochemical properties of chlorophyllin (1 and b has been reported rece ti tl y . ti One objective of the present study was to compare tlie spectral properties of chlorophyll arid chlorophyllin since these two pigments differ in important structural respects. notably the absence of the cyclopentanone ring i i i this latter coinpouticl. &Is will he shown, a study of the spectral properties of chlorophyllin helps to elucidate the spectral characteristics of chlorophyll itself. X subsequent paper' will discuss the relevance of the spectral properties of chlorophyllin to its photocheniical properties. Experimental A . Preparation of Chlorophyllin a.-~-Chlorophyll is extracted from frozen spinach and the chioropli)-ll n component is obtairietl b y chromatographic purification on a powdered sucrose The pigment is diss~ilvedin benzene atid diluted 1 11 with pentane. Saponificatiori is curried out by the addition of 1 i n l . of a filtered T r i solution i i f KO11 in i i i e t h n o l t o 50 nil. of the chlorophyll i i solution. C h i vigorous sti king the solution turns lirown ni1~11ient~iri1~Sloliscli p h r c teyt , I c , ) , anti after ;I few iriinutcs bluisli crystals of c l i l ~ i r ~ q ~ l i ~ -izl lw i rtit l e w t , T h e crystals :ire ;epartttetI, \\-asliecl with pentane, ,iiiil clissolved in ;inti)-tlrous rneth:riicil. Carbon dioxide is then bubbled through the solution ( i n the rl:irk'i for 1 h r . in order t o remove tiny remaining KOH as the insiiluble nieth! I c:irbon:iti.. .llie solution is then filtercd through :I very tine sintered glass filter .tnd evaporated t o dryness. Partitioil chrorriatography o f the pigment (j11 paper using either pyridine -phosphate buffer or pyridiiic rth!-l acetate-phosphnte huffrr , i > t h e eluent reveals the prewiicr o f only one component. B. Other Materials.--Polyvirl!.iI,!.rrolidoiie (PI.1') used for tlie binding studies was obtained from t h e Xntara Chemical Iliviiioti i i f t h e General . h i l i n e and Filni C m p . 'The particular w i n i p l t . employed i S P - K 9 0 ) has i i weight average molecular weiglit o f 3.6 X 105. Polyvin!~l alcohol used as a medium for dichroic ,tutlies wa; obtained from E . 1 , du Pont d e Sernours and C o . , [ t i c . , < i s Elvanol 72--fU. All other cherniciils were reagent gr:itlc obtained from Fisher Scientific Co. C. Procedures.--Infrared absorptiiin spectra of t h e pigments dirwdin KBr pellets were deteririined on :t Perkin-Elmer XIotlel 2 1 ,pectrophotometer. .1-.i y i l i l c :ihiiiriition spectra \%-?reobtained on the Cary Model 11 rrcorcliiig l i a r r i w i i iiiscrted for concentrated solutions. IXchroic spectra i n tlic \-ibiljlc rcgirJn were carried out o n oriented films of polyviiivl i ~ l c o l i o l containing chlorophyllin cz, Polaroid dichroic filter, !~r!-rje H S R ! were inserted in both t h e analyzing and rrf( l i t e r a n d S R B r o y d e , S n l i i r r , 192, I 3 2 ,1961) . 1 a~ n d S D Rroyde, Conference on Photosynthesis C Y K S . Gif.it.ttr l . r i i n c ~ . ' J u l y 1962, to be p u b l i s h e d 'ti 1 (; i a \ L i n : i ant1 X- I? Evstixneev. Z h , , f i z i k n , 8 , 3 3 5 (1963) ,7' ~ ~ % t vT r 5 n c l l i n . a n d S €3 Rros.de, J A m C h e i n Soc , 8 6 , 1313 19' I 4 f < 1-nttrr a n d .A 5 H o l t , A r c h Riochrm Hznohys , (;
':
Ills
Infrared spectra.-^ The infrared spectrum of chlorophyllin n is illustrated in Fig. 1 . In corninon with chlorophyll a , there are hands at 3125, 2950, and 1055 c i n - ' , The bands at 1740, I T O O . and 1610 cm. ~i which are present i n the spectrum of chlorophyll a are absent in the case of chlorophyllin a. T w o peaks at 1 S T O and 1400 crn.-' which appear in the chlorophyllin a spectrum are absent i n that for chlorophyll (L. -1spectrum we obtained for chlorophyll a i n L B r pellets is identical with t h a t obtained for chlorophyll ( L in organic solvent^'^ except t h a t the 3423-cm. I band which we observed is associated with the moisture content of the sample. 'The absorption hand a t 2030 cni. --I is due to CH stretching found both with chlorophyll and with chlorophyllin. The absorption a t lM5 cm.-' in chlorophyllin n which in chlorophyll ( I itself appears a t IMiO cm. has, for the latter substance, been attributed to either C-C in phytoli4 or to a perturhed carbonyli3 l 5 of the cyclopen tanorie ring. Since chlorophyllin a possesses neither of these structures. they cannot be the origin of this vibration. L1-e therefore attribute the band at lii3.5 CITI. - for both chlorophyll (L and chlorophyllin a to CI-C bonds in the porphyrin structure. The absorption band a t 161I) cm. - I present i n chlorophyll (z hut ahsent i n chlorophyllin a has been attributed t o C-C bonds in the chlorophyll ringI3 h u t tnore likely it is associated with the enol form of the cyclopentanone ringi6 since
0
(;
,
Results and Discussion
A
\\'t-ll~r .I
fclier, a n t i E P l f J t z , .4x7> ( ~ ' h ~ r n495, . . 8 (1932) A m ( h i , w z .Sw , 7 6 , ,5819 f I R , j 4 ) .
i l l ) S S R r o d y and 41 B r o d y , A r i h B r o r h r m Riii,hhyq , 8 2 , I l i l (11159) ( 1 2 ) G . O s t e r , A n a l C hem , 2 5 , 11GS (19.5.3) (13) .A S H a l t :and E . E Jacobs, A J J~. A o l a x y , 4 1 , 710 (1!J.54) 114) J \I'\VeigI a n d I< 1.ii-ingiton. J A m C h e m .Snr , 75, 2173 11053) 11,;) 1% A Strain. >f K T h o m a s , H I, C r e \ p i , S I I F3Iakv, and J J K a t z . A , i i i .V I' ;iciz,i . s ' r t , 84, 617 (19601 ( 1 6 ) .I."L' Sidoros and .A S 'rerenin, O,$l i , 5 ' p e k l v o r k o / i i y n . 8 , 2.i4 :l!W, sec. IIOWPICT, A 5 I I i ~ l t .Pr!ic. 1 i : l r r n Copier B t o c h r m , 6, i U 1'JlX%)
April 5, 1964
SPECTRA O F CHLOROPHYLLIK
this band occurs only in those porphyrins containing a cyclopentanone ring. l i In agreement with U'illstatter's contention (ref, 3 , p, 803) t h a t chlorophyllin is the salt of a tricarboxylic acid. we find t h a t the band a t 1740 c m - ' present in chlorophyll and ascribed to ester groups is absent in chlorophyllin and is replaced by bands at llO(! and ljT0 cm. - I characteristic of carboxylate ion. I * The strong absorption band a t IT00 c m - ' for chlorophyll n is also seen i n the spectruiii of chlorophyllin b. I t s appearance as a shoulder i n Fig. 1 is due to trace amounts of chlorophq-lliii b as an impurity since only the b component possesses the strongly absorbing carbonyl (aldehyde) group. This provides the basis of a useful technique for detecting trace amounts of chlorophyll b in samples of chlorophyll a. The infrared spectrum of the saponified sample establishes the proportion of the two components. Visible Absorption Spectra.----The visible light absorption spectra of chlorophyllin a in water and in methanol are illustrated in Fig. 2 . For comparison the spectrum of chlorophyll (1 (in methanol) is also given. The principal bands of chlorophyll n a t 4% and 660 mp are shifted to 11s and (i40 rnp (or ( 6 5 mp) for chlorophyllin a. The shoulder on the blue band of chlorophyll n is absent in the chlorophyllin n spectra, presumably due to loss of the cyclopentanone ring since it is also absent i n the allonierized chlorophyll where the Chlorophyllin a in water ring has been transformed. also lacks fine structure. The relative height of the blue to red peaks is about three t-imes greater than for chlorophyll. At concentrations of chlorophyllin a .V the absorption a t 418 mp X (but not a t ii40 mp) does not follow Beer's law unless the path length of the absorption cell is reduced so as to maintaiti the optical density below 1.5. The same behavior was observed for chlorophyll n . This phenomenon is also characteristic of dichroic systems1$ ( ~ fbelow). . Since crystals of chlorophyllin a are hygroscopic and may also contain traces of potassium methyl carbonate, it was found more practicable to determine its molar extinction coefficient by saponification of a known concentration of chlorophyll a . Typically a pellet of KOH is added to a methanolic solution of chlorophyll a of about 5 X 10P6 31 (concentration obtained from its known niolar extinction coefficient). The solution is then treated with COz to remove excess alkali as the insoluble methyl carbonate. The spectrum in methanol is given in curve B in Fig. 2 where we have assumed complete conversion of chlorophyll a to chlorophyllin a. The tnethanolic solution is then evaporated to dryness and redissolved in buffer to give curve A of Fig. 2. Chlorophyllin a , as does chlorophyll, readily loses magnesium under acid conditions to produce chlorin e6 n acid, hereafter referred to as chlorin a. This is manifested by a shift in absorption spectrum to about 400 and (%!I inp for the two maxima.' Chlorin a is less soluble in aqueous media than is chlorophyllin ( 1 7 ) J E F a l k and J B \Villis. A ? i s l ? a i t a nJ S r i R P A .4, , 579 (1951) (181 1. J Bellamy, "The Infrared Spectra of Complex Molecules." John Wile)- and Sons. I n c . S e w York. S Y , 1958, p 174 (19) t: H [.and and C D West in "Colloid Chemistry," Vol V I , J Alexander, Ed Keinhold Publishing Corp , Kew I'ork, N Y , 1946, p 160 A mathematical formulation is given in Sect 14.6 of Ivf Born and E \Tolf. "Principles of Optic and K A K r o m h ~ r ~ t
GERALDOSTER,SUSE B. BROYDE, AND
1312
JUDITH
TABLE I DICHROISM O F CHLOROPHYLLIN 4;8
,
,
DO - D~
D8/Dpo
1 15 1.06 0.74 .53 .24 13
1 77 1.71 1.50 1.35 1.16 1.09 1.00
0 15 30 45 60 75 90
Vol. 86
U
-
A, ma-
7
8, degrees
S.BELLIN
..
---525--DO - DW
0 07 . 07
-----575---De - DSO
Do/Dw
0 07 07 05 04 01 01
3 34 3.34 2.66 2.27 1.27 1.16 1.00
,05 ,04 .Ol .Ol ..
causes reversion to the original chlorophyllin a. If the colloidal system is extracted with chloroform and the dried extract is suspended in KBr, the infrared spectrum is identical with that of ordinary chlorophyllin a described above except for the presence of bands at 1250 and 1739 cm.-' which are characteristic of undissociated carboxylic acids 2.0
I. 5
---646----
De/&
2 67 2 67 2 20 1 95 1 33 1 23 1 00
DO
-
Dm
0 52
48 35 24 10 05
Do/DR,
2 2 2 1 1 1 1
80 65 20 83 34 18
---688-DO - Dso
--DB/Dso
0 17 1; 14 11 05 02
00
2 00 2 00 1 82 1 65 1 30 1 12 1 00
the absorption is greatest when both axes coincide, A similar result, but less pronounced, was obtained with chlorophyll a in stretched films of polyvinylbutyral. In oriented micelles of ammonium oleate chlorophyll exhibits dichroism.25 Some representative data for chlorophyllin a are given in Table I where the angle 0 is that between the vibrational axis of the polarizer and the direction of stretch of the sample. It should be noted that the dichroism is greater for the blue peak than for the red region. The fluorescence spectrum of chlorophyllin a varies markedly with the wave length of excitation and to some extent with the nature of the solvent. The value for the maxima in the fluorescence spectra are given in Table 11. In pyridine or in methanol excitation with TABLE I1 FLUORESCENCE SPECTRA OF CHLOROPHYLLIN Exciting light,
0 W
Solvent
z a
Methanol
1.0
::
m
Phosphate buffer ( p H 7 )
4
Above with 10% pyridine
0.5
n
400
XK)
800
?oo
800
WAVELENGTH (mp).
Fig. 3.-Dichroic spectra of chlorophyllin a in stretched film of polyvinyl alcohol : ---, parallel (e = 0 ' ) ; - - - -, perpendicular ( 0 = 9 0 ' ) ; - . -, unstretched film.
Addition of water-miscible organic solvents such as methanol or pyridine dissolves the colloid t o give a red fluorescent solution absorbing maximally at 688 and at 640 mp. The same effect can be obtained by adding trace amounts of PVP. It would appear that the 688mp absorbing species is a dimer formed via hydrogen bonding of the carboxylic acid groups and that the 735mp absorbing species is a colloidal aggregate of these dimeric species arid is only poorly molecularly dispersed in purely aqueous media. The dichroic absorption spectrum of chlorophyllin a in a stretched film of polyvinyl alcohol is illustrated in Fig. 3 . The dichroism is always positive regardless of the relative orientations of the sample axis with respect to the direction of vibration of the polarizer, and
ma
436 640 436 640 436 640 678
f2
Fluorescence maxima, mp (rel. intensities in parentheses)
660 ( 1 . 6 ) 647 ( 1 4 . 8 )
656 (0.6)
705 ( 1 . 9 ) 705 ( 4 . 5 ) 705 ( 0 . 4 )
645 ( 2 7 . 8 ) 655(5.1) 645 (26 3 ) 680 (100)
705 (0) 709(5.0) 715 ( 8 . 1 ) 709 ( 2 1 . 8 )
blue light produces less of the deep red (705-713 mp) component than does excitation with red light. I t is possible that this deep red fluorescence arises from the 688 mp absorbing species which also absorbs light a t 640 and 436 mp. This species does not exist in aqueous buffer alone but does contribute to the fluorescence a t ca. 710 mp when pyridine is added. Here its contribution to the fluorescence is particularly evident when 678-mp light is used for excitation. The data further illustrate that the shorter wave length red fluorescence is produced with a greater fluorescence efficiency when excited with red light than with blue light. I n addition one also observes that excitation with blue light produces a fluorescence band a t slightly longer wave lengths, These observations indicate that internal conversion between the two electronic states associated with the two principal absorption bands does not readily take place (compare ref. 26). The photochemical manifestations of these phenomena are considered elsewhere.' The over-all red fluorescence of chlorophyllin a is quenched by a number of substances. In methanol (25) Cf. H. Zocher, Trans. Faraday Soc., 56, 34 (1939); J . C Goedheer, Biochim. Biophys. A d a , 16, 471 (1955); compare W. D. Bellamy, G I, Gaines, and A. G Tweet, 1.Chem. P h y s . , S9, 2528 11963) (26) G . Oster a n d G. K . Oster in "Luminescence of Organic a n d Inorganic Materials," H. Kallman and G. M . Spruch, E d . , John Wiley a n d Sons, Inc , New York, N. Y.,1962
PHOTOCHEMISTRY OF CHLOROPHYLLIN a
L\pril 5, 1964
the fluorescence of chlorophyllin a is quenched by nitrobenzene following the Stern-Volmer relation with a quenching constant of 29 1. 'mole. If every encounter (1.1 X 101c 1. sec.) leads to a quenching, then the lifetime of excited chlorophyllin a is 2.8 X lop9 sec. The intrinsic lifetime obtained by integrating over the red absorption band and using the Ladenburg formula gives a lifetime of 2.2 X 1OF8 sec.. indicating that the fluorescence efficiency is about 10%. X more powerful fluorescence quencher for chlorophyllin (as well as for chlorophyll"), p-quinone, gives a SternYolnier constant of 440 1. mole. Here, since the quenching constant is so high, the quenching can obviously not be attributed to diffusional encounters and must be due to complex formation. Direct t 2 i ) K I.iving\trm and C . K e , J . Ani Chrin. Soc., 73, 909 (1950)
[COSTRIBUTION FROM
THE
1313
evidence of this is seen by the fact that p-quinone, in amounts sufficient for appreciable quenching, causes a shift in the red absorption band of chlorophyllin a to shorter wave lengths by 15 mp, Upon continuous strong illumination (from a high pressure mercury lamp) and in the absence of oxygen, both chlorophyll a and chlorophyllin n in plastics a t room temperature exhibit a reversible brown coloration with a lifetime of about 1 sec.?O Apparently this metastable species is identical with that observed in flash ~ p e c t r o s c o p y ? ~and + ~ ~attributed to triplettriplet absorption. (28) G (29) R (30) H . (31) S . (195Y).
Oster, t o he published Livingston, J A m Chem Soc., 7 7 , 2171) (1955) Linschitz and K . Sarkanen, ibid , 80, 4826 (1958). Claesson, L Lindqvist, and B Holmstrom, S a l u y e , 183, 661
DEPARTMEST OF CHEMISTRY, POLYTECHNIC INSTITUTE OF BROOKLYN, BROOKLYN 1, N . Y . ]
Photochemical Properties of Chlorophyllin a1 BY GERALD OSTER, JUDITH S. BELLIN,*A K D SUSEB. BROYDE RECEIVED JULY 3, 1963 Chlorophyllin a can be photoreduced and can sensitize photoreductions and photooxidations in a manner similar to t h a t of chlorophyll a . Iieither t h e cyclopentanone ring nor magnesium is essential for photochemical activity. Chlorophyllin a (or chlorin a) undergoes a reversible photoreduction in t h e presence of ascorbic acid t o give a product which can in a subsequent reaction either complex with pyridine to give a stable pink substance ithe Krasnovsky intermediate) or reduce a n azo dye. T h e kinetics of these reactions are presented and require t h e participation of a long-lived metastable state of chlorophyllin. T h e quantum yield of t h e Krasnovsky reaction is greater for red light excitation t h a n for blue light. In sensitized photooxidation the metastable species react with oxygen to give a n unstable intermediate which oxidizes t h e substrate. Binding of chlorophyllin t o a high polymer enhances its photochemical activity.
Introduction 111 the previous paper3 we demonstrated that many of the spectral properties of chlorophyll a are shown by its water-soluble derivative chlorophyllin a despite the absence of the cyclopentanone ring in this latter compound. In fact, a comparison of the spectral properties of the two pigments helped to explain those previously reported for chlorophyll a . The present paper will demonstrate t h a t the photochemical properties of the two substances likewise resemble each other. I t is particularly convenient to study the photochemistry of chlorophyll-like pigments in aqueous solution, since many physiological substances involved in photosynthesis are water soluble. The photochemical properties of chlorophyll, notably its photoreduction and its sensitization of photoreduction and photooxidation, in organic 'solvents have been extensively investigated. 4 - 7 The addition of water to organic (1) ( a ) Supported b y t h e United S t a t e s Air Force through t h e Air Force Cambridge Research Laboratories under Contract No. A F 19(628)-475 a n d by t h e S a t i o n a l Institutes of Health under Research G r a n t No. (26351. ( b ) Taken in p a r t from t h e dissertation of Suse B. Broyde, submitted June, 1963, t o t h e Faculty of t h e Polytechnic I n s t i t u t e of Brooklyn in partial fulfillment of t h e requirements for t h e degree of Doctor of Philosophy. (2) Public Health Service Research Career Awardee ~~, (3) G . Oster, S . B. Broyde, and J. S . Bellin, J . A m . Chem S O C . , 1309 (1964) (4) E . I . Rabinowitch, "Photosynthesis a n d Related Processes," Vol. I , Interscience Publishers, I n c . , S e w York, 9. Y , 1945, C h a p t e r 18. (5) J . L Rosenberg. A n n . Rev. Plant Physzol., 8 , 115 (1957). (6) A . A Krasnovsky, ibid., 11, 363 (1960). ( 7 ) ( a ) R . Livingston, Rad Res. Suppl., 3 , 196 (1960); (b) R . Livingston in "Handbuch der Pflanzenphysiologie," Vol. 5, P a r t I, W. R u h l a n d , E d . , Springer-Verlag, Berlin, 1960.
solutions of chlorophyll yields colloidal systems whose photometry is difficult to define. Chlorophyll dissolved in pyridine containing a trace of water and in the presence of a reductant such as ascorbic acid undergoes a reversible photoreduction yielding a pink intermediate.8 This reaction (the Krasnovsky reaction) is also exhibited by chlorophyll analogs6 A photoreduced form of chlorophyll may be produced in photosynthesis since differential spectrophotometric studies of bacteria and algae show that under certain limiting conditions an intermediate is found which resembles Krasnovsky's pink substance.5 Since the precise conditions for the photoreduction of chlorophyll are difficult to reproduce and since the system is not completely reversible, l 1 a kinetic study of the Krasnovsky reaction has not been conducted. In the case of chlorophyllin, however, and especially for its magnesium-free analog chlorin, the reaction is easily reproduced and completely reversible. Sensitized photoreductions and photooxidations, as well as reduction of the pigment itself, are also exhibited b y synthetic dyes (see, for example, ref. 12-14). It has been shown t h a t these reactions (8) A . A. Krasnovsky, Dok! A k a d . Nalck S S S R , 60, 421 ( l Y 4 8 ) (9) A . N. Terenin, E x p r v i e i i i r a , S i i p p l , 7 , 343 (14.57). (10) J . W. Coleman and E I. R a h i n o n i t c h , J P h y i . Chun7 , 63, 30 (1959) (11) T . T. Bannister, Plarit Phyriol., 3 4 , 216 (l95Y). (12) G . K . Oster and G . Oster, J . A m . C h o n . S o c , 8 1 , ,5543 ( I % j % (13) G Oster, J . S . Bellin, R . W. Kimball, and hl. Schrader. tbtd , 81, 5095 (1959) (14) G. Oster and A . H . Adelman, i b i d . , 78, 913 11956).
