The preparation and characterization of chloroplast fragments having an absorbancy maximum at 7400 A

.4RCMI~‘ES

OF

BIOCHEYISTRY

The Preparation Having

.4ND

BIOPHYFICY

98,

1i-2i

(1962)

and Characterization of Chloroplast an Absorbancy Maximum at 7400

.JAMES A. LIPPIXOTT,” .4SD

,JACQIYES AGHIOS, EVELYNE WALTER F. HERTSCH’

Rcceircd

November

Fragments A.’ PORCILE:’

27. 1961

rl method is described for the preparation of aqueous suspensions of chloroplast fragments having a major absorhancy maximum at 7400 -1, These chloroplnst fragments arc obtained by brief treatment of leaves wit)h hot methanol, or se\-era1 other water-miscible chlorophyll solvents, follo\yed by homogenization at 2-4°C. and cenkifugal purification. Optimum conditions for the reaction are described. An inrersc anti the refractive relation was observed bct,xcen the optimal solvent concentration index of the solvent. The 7400-A. absorbancy maximum was obtninetl from members of all major plani groups ercrpt the gymnosl~erms. Various properties of the 7400-A. absorbing suspensions arc described :ml their possible significance discussed. The data indicate that the 7400-A. absorhancy maximum is due to chloroplast fragments with a highly aggregated form of chlorophyll. Drspitc similarities between the absorbanap of t,he materials in these prcparat~ions am1 the action spectra for the red, far-red photomorphogenic response, no rridcnce relating i he two phenomena Teas obtained in these investigations.

crystal having a two-dimensional conduction band, several authors (5-7) have adopted similar concepts to provide for the necessary efficiency required for this energy migration. Evidence that such a condit,ion may mist has been supplied by the cspcrinlents of Arnold and dhcrwood (5) and A4rnold and Clayton (7)) which show that t,he chloroplast may function as a seiniconductor. The dcnionstration of light-induccd free radicals in clilorol~last pieparations, by Coinuioncr et nl. (8, 9) and by Calvin and Sogo ( JO) is also consistent n-ith SllCll a conclusion. Since semiconductors are typically crystallirw, these results suggest that, the chloroplast may contain crystalline areas. At present, hoverer, there is little eritknce that such aggregations occur or that, t’lie arlnugeuicnt of the cliloropl~yll with relation to other chloroplast components n-ould allow such it plicnonicmon. The tlatn presenkxl beloJv describe the Jwcparatiori of cliloiol~last fragnicnts liaring ab-

INTRODUCTION

The concept of a photosynthetic unit, as a functioning portion of the cliloroplxst (-3) has led to the probleins associated with the transfer of light quant,a from one point within the unit’ to a receptor point. Since Katz’s (4) original suggestion that the chlorophyll-containing lamellac in tht chloroplast might form a t’~~o-tliiiicnsionul 1 This investigation Teas aided in part by g:rant~ from The Jane Coffin Childs Memoi~ial Fund foi hletli~nl Research and the IT, S. National SAcnc~v I~‘oimd:rtion. ’ Fellow of The Jane Coffin Childs Memorial l~untl for Medical Research. Presrnt address: Dcpartment of Biological Sciences, Northwestern University, Eranst,on, Ill. 3 Present address: Lnhoratoire de Microsropie I~~le~troniclue, FacultP de M6derinc. 45 Rue tlrs Saints-P&s, Paris, France. ‘Fellow of t.he IT. S. National Svienc~e Fountlation. EVesent, address: Biology Division. Oak Kiclpv Sa tional T,:rhoratory, Oak Ridge, Term. 17

18

LII’PINCOTT,

AGHIOK,

PORCILE

AND

BERTSCH

gencmlly required between 1 and 2 min., aftci which the mixture was placed in an ire-watrr bat11 until the tempernttuc fell to about, j”C. The mixttnc was then homogenized in :a Waring blendoi at 24’C. with three 15-sec. Iiomogenizstion pcriods, interspaced with I-min. cooling pc>riods; I his was followed by a I-min. homogenization. The homogenate was then ccntrifugr4 at. 2000 j< g for 10 min., and the sulwnntnnt was discarded. The precipitate was suspended in 200 ml. of cold distilled water, centrifuged for 10 min. at 2000 X g, and the supwnatant was again diwnrded. The latter step was repcatcd, the final precipitate being suspended in either 2 M NaCl or writer and centrifuged at 500 X g for 5 min. to remove most of the ccl1 debris. Similar results were obtained with both suspension media. The final supernatant contains the chloroplast fragments described below. It is possible to eliminate the washing and purification proccdurr, since similar result:: may he

sorbancy and fluorescence properties consistent with the supposition that they contain highly aggregated or crystalline cllloropllyll. MATERIALS

ASD

METHODS

Unless otherwise cited, leaves from Rumcx acctosn and Nicotinnn tabacu7n (Hebana 38) mere utilized in the experiments to be dcscrihed; similar results mere obtained with both plants. The leaves were cut from the plant, washed in tap water, the midrib removed, and the tissue frozen at -25OC. for later use. The standard extraction procedure used is shown in Fig. 1. Redistilled methanol (500 ml. in a 3-1. beaker) was brought to a boil in a water bath; 100 g. of frozen tissue was pulverized at -25°C. and added to the boiling methanol, and the leaf-methanol mixture vvas heated in the water bath until it boiled. Boiling

500 ml. of boiling

methanol

I 100 g. of chopped

I

tissue added

Heated

to boiling

Cooled

I in an ice-water

Homogenized

(l-2 min.)

in a Waring

bath blendor

10 min. at 2000 X g I Supernat.ant discarded

Pkilet I Suspended in 200 ml of cold dist . water 10 min. at 2000 X g (This I

step repeated

2-3 times)

Pellet from 3rd centrifugatiom cycle

Supernatant, discarded

I Suspended in 2 111SaCI or water I 5 min. at 500 X Q

Pelliet discarded

Material

FIG. 1. Standard leaves.

procedure

for the preparation

I

described

of 7400-A. absorbing

material

from green

obtained directly from crude cell homogenates of fresh (or frozen) leaf tissue samples as small :IS 0.1 g. Because of t,he obvious advantages of using small samples and simple homogenates, the follow ing “small sample procedure” was utilized in those experiments II-here effects of varying temperature and solvents were compared. LI 1r:rf sample of 0.25 g. was placed in 1.25 ml. methanol in a glass homogenizer tube.” The tissue and solvent were brought to the desired temperatures by placing the homogenizer in a water bath for cithcr a I-min. or 2-min time period as specified. The homogenizer was then removed to an ice-nntrr bnth and lhe sample homogenized. After a 2-min. homogtkation period, the crude cell extract was nllow-c~t to st,and for 5-10 min., then diluted lOO-foltl \vi-i(h tlk tilled water and the nbsorbancy spectrum d&ermined. -1bsorbancy spectra were obtained with :I &cl
1. ABSORBANCY

SPECTRA OF CHLOROPLA~T FRAGMESTS EXTRACTED FROM LEAVES WITH HOT METHANOL

Microscopic examination of the supernatant obt’ained by t,he abore procedure showed it to consist mainly of chloroplast fragments. The absorbnncy spectrum of such a supernatnnt fluid from R. ncetoscz diluted sixfold with 2 X NaCl is shown in Fig. 2 (curve -4). The absorbancy spectrum of the same final supernatant suspension, diluted sixfold with methanol (final conccntration about 837; mcthwnol), is also shown in Fig. 2 (curve B) . The two absorbancy spectra show marked differences in the blue, red) and ultraviolet regions of the

Wavelength

,

A

FIG. 2. Absorbnnry spectra of final sul)ernniant material isolated from R’unwl: ncetosn as dcsrribed in text,. Curve A : sixfold dilution with 2 JI iYaC1; Curve B: sixfold dilution lyith methanol.

far-red nbsorbancy maximum at about 7350-7400 -4. (hereafter referred to as 7400 A.), and only a small absorbancy maximum at about 6780 A. mherc chlorophyll normally absorbs. In some instances, an nbsorbancy maximum occurs at wavelengths as high as 7600 A. When diluted with methanol, however, this material has an absorbancy spectrum typical of alcoholic extracts of chlorophyll-containing leaves. Smaller differences in absorbancy between the aqueous and nictlinnolic dilutions which occur at 2600, 3400, 3800, and 4600 A. are I)robably due in part to changes in chlorophyll absorbancy, although it is not possible at present to assign specific chloroplast pigment:: to all of ilicse changes. It is important to note t’hat the addition of large amounts of any chlorophyll solvent to aqueous suspensions shon-ing a 7400-h. absorbancy results in the immcdiat~e loss of this absorbancv band. as well as associated absorbancp diffcrcnces, and the appcarancc of the typical chlorophyll spectrum illustrated in Fig. 2 (curve l3).

spectru111.

In aqueous KaCl

(or water)

there id a

2. PREPAR.~TIOK MATERM.

“Homogenizers of the type used may be l’repared by plunging the molten end of a Pyrex rod into the bott,om of a Pyrex test tube with dimensions of 1.1 X 14 cm. The piston and tube arc then ground togcthrr with the addition of fine sand :mtt carborundum to form a rough-surfaced, loos+ fitting homogenizer.

OF 7400 A. AB~ORBISG FROM T’.~RIOCX PUNTS

A number of chlorophyll-containing plants have been extracted with hot’ mcthanol in the manner described, to determine whether the formation of this 7400-A. absorbancy maximum is species depcndcnt~. Table I lists the plants which wwc utilized.

20

LIPPINCOTT,

AGHION,

PORCILE

T.4BLE

Dicot,yledons Rudbeckia ?‘uraxacrLm xanthirm

+ +

Pteridophytac

Digitalis pcrrpIcrerc Nicotiana tabacnm Perilla crispa Ilex aqlcifolium Oenothera parvijlora Chenopodium polyspermum Ifumes acetosn

+ + + + + + +

Bryophytae

l’teritliunl

.p

.s Q 25 e

z 2, Q) a;

5“6

1.38

+ + + +

+

maximum

I .34

methono>o 25

minus

(-)

indicates

that

no

3. EFFECTIVENESSOF VARIOUS ORGANIC SOLVEKT~

1.36

1.33

was obtained;

may reflect major structural or chemical differences in these chloroplasts, or merely the presence of interfering substances.

oYb~~~opo”ol \

0

+

+

annun

n - proponol

02

aqdintcm

Marchantia polymorpha Mnilrrn hornuw Pol?ytrichunl fomo.stLtn Alg:te Chlorella pyrenoidosa

1.40 9 ,”

-

Ginkgo bilobn Picea excelsa l’h,,rya sp.

+ + +

a Plus (+) indicates a i&O-A. absorbancy 7400-A. absorhancy maximum was obtained

c

XBSORBASCY ?VIAXIMUW

(~,vnnosperms speciosa sp. pennsylv.

Monocotjyledons ~-lccca sp.

0-E No

7300-9.

Ch~ysnnthe?nltnl sp. SnYnbrtcus nigra

l’oa

BERTSCH

I

OF PLANTSTREATEDTO OBTAIS A

I~ESP~NSE

AND

50

75

Composition of extraction solvent (Mole per cent in water) to obtain 7400 A obsorboncy

FIG.3. The relation between the refractive index of solvents causing the formation of material having a 7400-A. absorbancy maximum, and the concentration of the solvents necessary to obtain this change. Leaf samples of 0.25 g. from R. acetosa were treated for 1 min. at 60°C. in 1.25 ml. of the appropriate concentrations of the various solvents. With the exception of gymnosperms, all green plants treated (14 higher plank 1 fern, 2 mosses, 1 liverwort, 1 alga) showed a 7400-A. absorbancy maximum. iZlthough repeated efforts were made, no 7400-A. absorbancy maximum was noted in extracts of gymnosperms. The failure to obtain such changw in chloroplasts of the gymnospcrms

To assess the possible role of methanol in the experiments described above, green leaves were treated in a similar manner with many organic solvents. Using the ‘Lsmall sample procedure” and appropriate dilutions of the organic solvents with water, it is possible to obt,ain a 7400-A. absorbancy maximum with methanol, ethanol, isopropyl alcohol, n-propanol, tert-butanol, and acetone (see Fig. 3). All are miscible with water at, the concentrations at which they are effective in producing this absorbancy change. These solvents are all Br@nstcd bases, or nucleophilic reagents, and all but one are alcohols. Representative concentrations of the following solvents were tested, but no 7400-A. maximum was obscrwd: carbon t,etrachloridc, chloroform, dioxanc, ethanolamine, ethyl ether, glycerol, methyl cellosolve, petroleum ether, and twt-amyl alcohol. In dioxane the absorbancy maximum of t,he chlorophyll was shifted to 6900 A. and in ethanolamine to 6380 A. The optimum solvent concentration fol production of a 7400-A. absorbancp mnximu1~1is different for each of the six organic

CHLOROPLAST

21

FR.AGMENTS

solvents found to produce this change. These optima are inversely proportional to the refractive indices of the organic solvents. Figure 3 shows the approximat,e optimal concentration for each organic solvent, plotted against the refractive index of the pure solvent. Because of the technical difficulties involved, the optima were not determined exactly. However, concentrations of solvent 5% higher or 1oIver than those represented resulted in very little or no formaCon of a 7400-A. absorbancy maximum. These results suggest, that the effectiveness of the extraction solvent, in producing the 7400-A. absorbancy maximum depends on a physical property of the solvent! rather than any chemical property. Furt’her work mill be required before the significance of the correlation between the refractive index and t,he opt’imal concentrations of organic solvents effective in producing this change can be fully assesssed.

these absorbancy changes, therefore, is the concentrat,ion of organic solvent. Figure 5 shows the dependence of formation of t,lie 7400-,4. maximum on the volume

4. CHARACTERIZATIOA- OF THE REACTIOS Iiv METHAKOL

of varying methanol conccntration on the subsequent appearance of an absorbancy maximum at 7400 A. Leaf samples of

(a) Dependence on Methanol Concentration The formation of a 7400-A. absorbancy maximum in these preparations is dependent on the concentration of the organic solvent utilized during the original homogenization. The optimal concentration of methanol for extraction must be determined for a particular ratio of leaf weight to solvent volume, since the leaves themselves dilute the solvent in the homogenate. The standard “small sample procedure” was utilized to obtain the data presented in Fig. 4 which shows the relative height of the 7400-A. maximum obtained when various concentrations of methanol were used for extraction of a standard leaf sample. For this part’icular leaf-solvent ratio (1 g. leaf/5 ml. solvent), t,he optimal methanol concentration is about 85%. Below 75% methanol, very little 7400-A. absorbancy is obtained. Similar results were obtained with various leaf-solvent ratios, as long as the finnl methanol concentration (with aIIow:ince folk the mater content of the leaf) was about 855,. The important factor in obtaining

0.160{-

0.000

Jo 70

: 80

90

I( 0

Per cent methanol in water FIG.

4. The

effect

0.25 g. from R. acctosa were heated for 1 min. at 60°C. in 1.25 ml. of the wrious methanol conrenmasitratione, and the height, of the ;thuorhzrnc\mum at 7400 A. was determined. 04 8

.120..

0

F z E

.lOO.\

i ‘X

.080--

E 2 6 b :: 0 a z

,060..

i .rr

2

\

I’

.020-. .040..

’

2

0I 2

Milliliters

0

o-0

\

. 4

of methanol

0

8

6 per

gram

of

leaf

FIG. 5. The effect of varying thr> wtio of met,hanol to leaf material on the subwcl\:ont nppenrance of an nbsorbancy maximum at 7400 A. Leaf samples of 0.25 g. from I?. ncetosir mwe 811hjetted to a 1-min. heat treatment at BO’C. in varying volumes of 95% methanol. and thta height of the ;Ibeorb:mq maximum at 7400 .\. w;t,k detcrmined.

22

LIPPIFCOTT,

r

0.08 0.06

AGHlO?;,

1 ; 40

0

50

AND

BERTSCH

and absorbancy measurements. The experiment illustrated is typical of t,he results obtained and shows t)hat a temperature treatment of 50-60°C. is optimum. With different leaves from the same species similar curves have been obtained, however, which are shifted as much as 10°C. toward lower t’emperatures. It should also be noted that some 7400-A. absorbing material may IX formed when the ent,ire procedure is carried out at 24°C.

0.02 0.00 *

PORCIIJ:

60

Temperature (“Cl before homogenization FIG. 6. The effect of temperature on the abilit,) to obtain 7400-A. absorbing material from methanol-treated leaves. For these experiments leaf samples of 0.25 g. from R. ncetoscl were treated for 2 min. at various temperatures in 1.25 ml. methanol, and the height of the 7400-d. absorbancy maximum formed after homogenization was determined.

of 95% methanol present per unit weight of leaf. A 7400-A. maximum was obtained in all the proport,ions test’ed. The optimum is exhibited at a ratio of about 4 ml. methanol/g. tissue. These results are in fairly close agreement with those in Fig. 4 when the water content of the tissue and the respective volume of :~lcol~ol usccl aw taken into account. ib) Dependence on Methanol Tempera twe The amount of material having an absorbancy maximum at 7400-A. in methanoltreated leaf homogenates also depends on the temperature of the methanol treatment’. Figure 6 relates the amount, of 7400-A. absorbancy obt,ained using the “small sample procedure” to the temperature of the methanol before homogenization. The leaf samples were immersed in methanol for 2 min. at the tcmpcratures indicated, then homogenizcd for 2 min. at 2-4°C. Since absorption changes often occurred during the first fen minutes after homogenization, the samples were allowed to stand in an ice-water bath for 10 min. before making aqueous dilutions

5.

PARTIAL

CHARACTERIZATION

MATERIAL

ABSORBING

AT

0F

Tm

7400 A.

We assume that the 7400-A. absorbancy maximum is due to an aggregated form of chlorophyll. The 7400-A. peak has invariably appeared with a concomitant loss of chlorophyll absorbancy at 6620-6800 A., and it is well known that some aggregated forms of chlorophyll show an absorbancy maximum at about 7400 A. (11). Further! in our experiments, the typical red nbsorbancy maximum of chlorophyll (6620 A.) has always been obtained with any prowtlure which resulted in loss of the 7400-A. niaxinlu111.

(a) Recipmxd

ilbsorbancy

Correlation, between Changes at 7400 A. rind 6620 .,I.

The addition of sufficient quantities of any chloropliyll solvent to aqueous suspensions of the 7400-A. absorbing material causes a reduct,ion of the 7400-A. maximum and an increase in t,he 6620-A. absorbancy maximum. All solvents which wre cffectivc in the original production of the 7400-A. absorbancy were also active, but at, higher concentrations, in reversing this absorbancy change. Dioxanc and cthanolnminc, watcrmiscible solvents which were not effective in the production of a 7400-A. absorbancy maximum were capable of reversing this change. boater-immiscible solvents such as ether and phytol were also effective in shifting the absorbancy of these preparations to 6620-A. With these solvents, the 6620-A. allsorbing material was extracted into the organic solvent phase. Thus, the loss of 7400-9. absorbancy on treatment with

CHLOROPLAST

FRAGMENTS

chlorophyll solvents appears to result from the solvation of the chlorophyll present. This conclusion is strengthened by the observation t’hat the newly formed 6620-A. maximum cannot be precipitated b$ centrifugation, although the 7400-A. maxmum always precipitates with the chloroplast fragments. With some aqueous suspensions showing a 7400-A. maximum, it has been possible to reversibly shift the absorbancy of the material from 7400 to 6620 A. by heating to 75°C. in the presence of an appropriate volume of met,hanol. These temperatureinduced absorbancy changes are repeatedly reversible by alternate temperature changes as shown in Fig. 7. The reciprocal nature of these absorbancy changes can be readily noted in this figure. This temperature ef’fect is obtained in 67% methanol, a concentration somewhat lower than that required to extract chlorophyll from these preparations at room temperature. Loss of the 7400-A. band and the appearance of a strong band at 6620 A. also results from lyophilization of preparations having an absorbancy maximum at 7400 A. This indicat’es t,hat either water, small amounts of t,he organic solvent, or perhaps both, are required to maintain the 7400-A. absorbancy.

0.80

(b) Relation of ?‘400-A. dbsodmncy to Totul Chlorophyll The relation between total chlorophyll absorbancy of a leaf sample and the maximum amount of 7400-A. absorbing material which could be formed was determined in the following manner. Similar leaf samples were obtained from opposite halves of a single leaf. One sample was trcatetl with met’hanol using the lismall sample procedure,” and the absorbancy was measured at 6620 and 7400 A. The second sample was extracted with cold acetone, and t,he total quantity of chlorophyll was dctermined from its red absorbancy maximum. In several such experiments, the total 7400A. absorbancy was 50-65s as great as the total chlorophyll absorbancy obtained in the acetonic extract.. In addition, 20-30% of the chlorophyll nbsorhancy remained un-

0.60 u” 6 a

8 2

0.40

0.20

0.00 Wavelength

, A

FIO. 7. Temperat~~re-induced :~bsorbnnq changes. Curve A wx obtained fl,orn an aqueous suspension of 7400-A. absorbing mntrrial diluted threefold with methanol at room temprr:iture. On hcnting to 75”C., the nbsorbancy spertruni shifts to that of curve H and is similar to that of :I iypical methanolic leaf extract with x red nbsorb:mcp maximum at 6620 A. Aftw cooling at room tcmperaturc for 15 min., the absorbancy sprcWun of curve C is obtninetl. Cur\ p C’ is typic’nl of prcl,;rrations having an :ilworbanc,y maximum :rt 7400 A. This spectral c,llnngcs (wr~c’s L1 and C) ow~~rrr~l on each sulxccltlr~nt heating :m(l c~ooling.

changed in tile Ilictlianol-tr~atcd samples. Thus the heights of the two absorbancy maxima in t,hc nictliunol-treated samples are equivalent in amount, to 70-95% of the height’ of the chlorophyll absorbancy in acet’onic extracts from control leaf samples. Many ‘(purified” preparations having an absorbancy maximum at 7400 A. and only a small maximum at, 6680 A. were treated with et,hanol, and the resulting absorbancy maximum at, 6620 A. was dctcrmincd. The heights of the 7400-A. maxima were found to vary between 35 and 955’, that of the 6620-A. maxima obtnincd in ethanol. Hecause of t,he different media in which tliesc measurements were matlc ant1 probable changes in aggregnt’ion of the cliloropliyll molecules in the two different solvents, these results probably have little meaning in terms of ii true extinct,ion value for the 7400-A. absorbing material. They do, nevertheless. provicle a basis for estimating the magnitude of these spectral changes and thtl extinction of thr 7400-A. absorbing material.

LIPPIKCOTT,

24

AGHION,

with aqueous suspensions haTing a 7400-A. a bsorbancy maximum and a minor 6680-A. maximurt~. These chloroplast, fragment suspensions gave no fluoreswnce in the region where rl~loropliyll n normally fluoresces n-hen excited with light. of 4500 or 6400 A. (,12). Aqueous suspensions of untreated isolatett chloroplast fragment,:: in the same medium and having about t,he same amount of cxtractztble 6620-A. absorbancy gave :I clear fluorescence. When ethanol was added to nlaterial having :L 7400-A. absorbancy maximum until a shift, in nbsorbancy to 6620 A. was obscrvecl, :I fluorescence spectrum identical to that of chlorot~li~-11 (I was obtained. The shift, in absorbancy maximum from 7400 to 6620 9. is thus accompaniecl by the appearance of a typical chlorophyll II fluoreswnce, indicating that the 7400-A. material is a nonfluorcsccnt form of chlorophyll. (d) Association Xnterinl with

of 7400-A. .-lbsovbiny Chlo?,oplnst

Fragments

The material responsible for the 7400-A. absorbancy maximum is pnrticulatc, and in all cases sediments after a centrifugation at 6000 x 9 for 30 min. The broken chloroplasts in tliesc 7400-A. absorbing suspensions may be clearly clistinguishrtl with the aitl of an oil-immersion microscope. If methanol is adtlcd to the edge of the cove] slip, the fragments may be observ(vl to t,urn first bright, green and then to bleach. This color transformation presumably reflects :I cliangc in the state of the chlorophyll (cl~loropl~yll having an absorbancy maximum at 6620 A. appears bright, green t followcd by elution of t)he chlorophyll from the particles. These result:: provide direct eyidcncc that the material responsible for the 7400-A. absorbuncy iti:~xiintuti may Iw located on portions of Clll0~OplilSt~.

PORCILE

AND

BERTSCH

has been obtained through cllroi~latogr:lt)lly. Chloroplast pignients from aqueous swpensions having ii 7400-8. absorhancy niaximum were oxtr:tctjcd (thus shifting the 7400-h. maximunl to 6620 A.) anti colupared cl~ron~atogr:~pl~ic:tlly with similar cxtractions from homogenates of control leaf tissue, using the methods of Strain (13). The pigments were ctiroiiirztograpllccl on powdered sugar columns, using petroleum c+hcr containing 0.05$? n-propanol for development. Positions of the colored bands were noted, and the various zones were cut from the column, elutetl, and the absorbancy tlctermined. The same colorecl zones obtained from ext,racts of 7400-A. absorbing suspcnsions were also present in the control leaf extractions. The mobility of chlorophylls n and b ctxtractcd from suspensions of 7400A. absorbing iiiatvrial was nlso siiiiilw to that of the same pigments ext,racted from the control leaf s:miples. IV0 differcnccs in absorbancy spectr:L between sample and control fractions were observed. The at)sence of typicnl ph:ieopliytin absorbancy (12) in the chlorophyll fractions indicated that’ t,lie magne.siuni was still present. Li tlvtermination of tllc, cl~loropl~yll HCl number (,12 1 showed that thcx phytol group was also piwent. These results imply that the 7400-A. absorbancy maximum 1s tluc: to chlorophyll. and that no lasting chemical change in the molcculc is relntett to the absorbancy maximum at, 7400 Ai. The variet’y of extraction solvents which may be usetl suggests that the solvent is not entering into direct chemical combination with the chlorophyll. If a chemical change in the chlorophyll is rcsponsiblc for the 7400-A. nbsorbancy maximum, then it, must be eit,lier ia) readily reversible on extraction with a chlorophyll solvent, or I bj affect only a small number of the rhlorophyll molecules anti hence cscapv detection by the methods uwtt in thaw cxperinients.

(f,) Protein I e) Chrovzntography of C’hloroplnst Pigments Extrrrcted from 7&WLl. Absorbing Suspensions

Additional evidence that the 74OO-LL absorb:tnry maximum is due to chlorophyll

Absorbing

Protein chloroplast absorbancy of 7400-A.

(,‘ontent 0,i 7400-;l. Suspensions

determinations were made on fragments exhibiting a 7400-A. maximum. Aqueous suspensions absorbing ni:it~cri:~l w~rc furtlrvt

CHT,OROPIAST

purified t,hrough three cycles of centrifugation: the supernatant from 5 min. centrifugation at 500 X 9 was sedimented at 2000 x g for 15 min., t.he pellet resuspended, and the cycle repeated. The amount of chlorophyll present was determined from an acct,one extraction of an aliquot of the material and compared to the amount of protein present. Protein measurements were performed using t.he method of Lowry et crl. (14) and compared t.o a bovine serum albumin standard. The values are in general agreement with the protein estimations obtained by several authors for chloroplast fragment preparations, indicating the presence of from 5 to 10 ,ug. protein/pg. chlorophyll (or a protein molecular vcight of 5000-10,000 per molecule of cl~loropliyll~ (15). It remains to be determined whether this protein is necessary to the formation and maintenance of the 7400-.d?\.absorbancy maximum. 6.

PRESENCE

OF

r~RANSIEST

iIBWRB.4XCY

Figure 8, curve A, shows the absorbancy spectrum of a cell suspension prepared by the “small sample procedure” after 1 min. homogenization at 2-4”C., following which the sample was diluted with water and immediately placed in the recording spectrophotometer. This rather flat absorbancy spectrum is not st’ablc and changes with time until a stable spectrum is obtained having maxima at 6680 and 7400 A. (curve R). This change occurs in I-5 min. at room temperature, but is much sloww at 2-4°C. The original spectrum icurw J) show at least three small maxima bet,wecn 6500 and 7500 A. occurring at, 6680,7050, and 7400 A. The intermediate peak near 7000 A. has been found to vary in different preparations from 6900 to 7100 A., and in some casw may be large enough to obscure the maxima at 6680 and 7400 A. The absorbancy maximum at about 7000 A. is similar to that reported for colloidal aggregates of chlorophyll (16). Because of the nature of the material involved and the way in which thcsc absorbancy change:: may be obtained, it swms reasonable to assume that this absorbancy maximum is due to such colloidal aggregates.

6000

6500

7000

Wavelength

7500

6000

A

Frc. 8. ~~lbsorhanc~ spectra of a metlnmolic homogenate of Che,lopodiuwL polyspe~~~z~~m : cnr\-r A, shortly after preparation; curve H, the wne material after 1 hr. at room temprraturf~. DISCUSSIOK

The procedures utilized in this study are sufficient to produce a 7400-A. absorbing form of chlorophyll in tissue homogenates from most green plants, gymnosperms being the only exception observed. The formation of a 7400-A. absorbing chlorophyll may therefore be :I common property of the chloroplast~ st,ructurc. Two forms of chlorophyll have been r(‘portetl to have an absorbancy maximum in the region of 7400 A. These are crystalline chlorophyll rc monolayers (and ot,hcr highly contlcnsed forms of chlorophyll J (11. 17, 18)) and the unextractecl chlorophyll ( bactcrioviridin) of the green photosynthetic bacteria CJhZo~~obiunz((19). Both of these pigment:: exhibit, a shift of their red absorption maximum t>o about, 6620 A. when extractcd into a cliloropliyll solvent. It deems unlikely that the present 7400-A. absorbancy is due to 3 transformation of clilorophyll into “l)actc~~ioriritlin.” since Krasnovsky (16) has reported fluorescence from these bacteria which is attributed to ‘*b:tvterioriritlin,” and since the minor absorbanc;v maxima of “bacterioYiritlin” are s&iciently different. from those of chlorophyll 0 to 1w rcatlily tlistinguislwd (19-21 1. FLUther, d&a from cl~roiii:itogr:~l~liy, clet:aila of absorbancy spectra, and fluorwcenw :kll imlicate that the 7400--4. absorbancy masiIIILIIII in the 1)rewnt chloroplast fragment

26

LIPPI~COTT,

AGHION,

suspensions is due to an aggregated form of chlorophyll a, or chlorophylls a and b. There is evidence that a form of chlorophyll a absorbing at 7400 A. occurs naturally in plants. Gorindjee et al. (22, 23) ha\-e reported an absorbancy maximum in ilnacystis at about 7400 A. which is apparently due to chlorol~l~yll u, since other chlorophylls have not been reported to exist in this organism, and also in Chlorelln which contains chloropliylls (1 and b. The size of this absorbancy at 7400 A. in dnacystis may be increased by the addition of proper amounts of methanol !C;ovintljce, personal coniniunication). The results of Coleman et al. (24) and Kok (25)) with difference spectrophotomerty, show minor light-induced changes in the 7300-7400-A. region which occur during photosynthesis. From these observations it appears fairly definite that small amounts of a pigment absorbing at 7300-7400 A. exist naturally, and that the pigment is related to photosynthesis. Although there is little direct, evidence that such in ~:ivo spectral differences arc due to an aggregated or crystalline-like form of cl~lorol~hyll, the present results indicate that at least i?, vitro a 7400-h. absorbing form of cl~loropl~yll may be attached to chloroplast fragments. Because of the similarity between these absorbsncy changes and t’hosc of the action spectra of the red, far-red reversible system (,26), the estract’ion of the 7400-h. absorbing material was carried out in a variety of ways designed to demonstrate possible correlations bet,ween these phenomena. Long- and short-day plants under inductire and noninductive light regimes when treated as described, all gave similar results. Further, no significant absorbancy changes resulted when the treatment was carried out in the absence of light or under light of various wavelengths. Since in these cxperiof the ments, the natural environment chlorophyll has received severe treatment, these observations do not rule out the possibility that similar chlorophyll absorbancy changes in villa are related to the red, farred reaction. The ability to reversibly change the absorbancy maximum of $hc cl~loropl~yll in thcsc prcI)nrations from 6620 to 7400 :\.

PORCILE

ASD

BERTSCH

through relatively minor changes in solvent or by Farming and cooling at appropriate solvent concentrations is very suggest,ive of an equilibrium situation in which the variable simply alters the relative proportion of molecules existing in two phases. A possible mechanism through which naturally occurring changes in chlorophyll absorbancy :tntl changes in the red, fur-red photomorphogenie pigment might occur is suggested by these results. From t,he extrapolation of the data in Fig. 3 t,o zero concentration it might bc cxpectcd that :I substance having a refractive index in the Ticinity of 1.40 nrjZO could support the formation of :t 7400-A. band when present at a very low concentration. Such concentrations might in fact approach the concentration of naturally occurring plast’id components. This posts the question of whether such substances exist and whether they support such changes in ZGVO.

The full significance of the data presented in this paper to the problems of energy transfer and to the st,rurturc of the chloroplast depends ul)on tlic rrlation of ttic cl~loropliyll to its natural matrix in the part,iclcs having an absorbancy maximunl at 7400 A. If the cliloropllyll molecules are naturally highly oriented. then the change in nbsorbancy to the 7400-A. absorbing form may occur without extraction of the chlorophyll from the chloroplast fragments. A slight rotation of highly oriented chlorophyll molecules, such as those described by Goedheer and Smith (27) in isolated holochromes, might cause the formation of crystalline arcas which would result, in profound changes in energy or charge transfer in the plastid. It secins possible that the hot methanolic t’reatment of chloroplasts provides solvation conditions such that the unextracted chlorophyll molecules can untlergo such a rotation, result,ing in a crystallization of the ctiloroptipll while it is htill attached to t,lie chlorol)last fragiuents.

The authors are indehtcrl to Professor P. Chouard and Dr. J. I’. Nitwh for nxking avnilnhlc Llre facilities of the Lnlmratoirr tlu l’hytotron, :ml for cont,inuecl irltcwd :lnd cncWllr:rgclll~nt.

WC wish to thank Dr. J. Lavorel for making the fluorescence measurements,. and for helpful discussions of the work. REFERENCES

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