Two-way paper chromatography of the chloroplast pigments of leaves

16

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 4 5 2 4 4

TWO-WAY P A P E R CHROMATOGRAPHY OF T H E CHLOROPLAST PIGMENTS OF LEAVES H A R O L D H. S T R A I N , J O S E P H AND J O S E P H J. K A T Z

S H E R M A * , F R A N C I S L. B E N T O N * * ,

Argonne National Laboratory, Argonne, Ill. (U.S.A.) ( R e c e i v e d N o v e m b e r 23rd, 1964)

SUMMARY

I. Two-way paper chromatography of leaf extracts provides a very effective method for the separation of most of the chloroplast pigments. Separations are most effective at low loading of the paper. At high loading, multiple-zonation effects were frequently observed. These multiple zones cannot be attributed to the presence of a different pigment in each zone. 2. As in the one-way paper chromatography, the two-way method indicated that the principal pigments of leaves are chlorophylls a and b, neoxanthin, violaxanthin, lutein with or without zeaxanthin and carotene (fi-carotene with or without a-carotene). No carotenoid pigments corresponding to lutein epoxide, neozeaxanthin or eloxanthin were found.

INTRODUCTION

The two-way paper chromatography of leaf pigments reported by KKUDAIRI1 and by WOLF2 yielded several pigment zones in addition to those usually separated by other chromatographic methods, as summarized in the preceding article a. In principle, the two-way chromatograms should be the summation of the two oneway migrations, but the one-way chromatograms described in the preceding article did not indicate that there were additional pigments in the leaf extracts 3. The oneway migrations did show, however, that under some conditions, paper chromatography of the leaf extracts leads to double tailing and multiple zoning of the separated pigments. If these effects occur in the two-way migrations, then additional pigment zones might be attributed to this anomalous zone formation rather than to the occurrence of a very large number of pigments. To test this possibility, we have now studied the two-way paper chromatography of the individual leaf pigments and the two-way paper chromatography of the pigments in leaf extracts and in the saponified extracts. * P r e s e n t a d d r e s s : D e p a r t m e n t of C h e m i s t r y , L a f a y e t t e College, E a s t o n , Pa. (U.S.A.). ** P r e s e n t a d d r e s s : D e p a r t m e n t of C h e m i s t r y , S a i n t M a r y ' s College, N o t r e D a m e , Ind.

(u.s.a.). Biochim. Biophys. Aela, Io9 (1965) I6--22

17

CHLOROPLAST PIGMENTS

PROCEDURES

The plant materials, the extraction procedures, the pigment preparation, the saponification method, the paper, the chromatographic conditions, the identification m e t h o d s and the abbreviations were those employed in the preceding article 3. The chromatographic development in the first direction was carried out in covered jars, as described 3. W h e n the wash liquid h a d flowed into the paper to a migration distance of 15.2 cm, the paper was dried in air for 5 rain, then restapled in the form of a second cylinder with the axis at right angles to t h a t of the first one. This cylinder was placed in the second solvent (500 ml) until the liquid had risen 15.2 cm above the first line of pigment migration. OBSERVATIONS AND INTERPRETATIONS

Two-way chromatography of individual pigments I n the one-way c h r o m a t o g r a m s of individual pigments, multiple zoning was most often encountered a m o n g the xanthophylls 3. For this reason we have made chromatographic studies of the t w o - w a y migration of ]utein under conditions similar to those employed with the leaf extracts l& A saturated solution of lutein in e t h e r light petroleum ether (i : I, v/v) approx. 0. 7 m g lutein per ml, was added to the paper, and the washing was performed with light p e t r o l e u m - p r o p a n o l , then with light p e t r o l e u m - c h l o r o f o r m . W i t h only 3/,1 of the lutein solution, two pigment zones were formed as shown in Fig. I. One zone m a y be attributed to the "soluble" lutein, the other to the lutein retained at the starting point during the washing with the first solvent 4. W i t h IO t~l of the lutein solution, three or four lutein zones were formed. Two zones corresponded with those in Fig. I. Another zone remained at the starting point, and a large diffuse zone extended from the small zone adjacent to the starting point. Because colorless substances in the extract of white morning glory flowers have a great effect on the one-way c h r o m a t o g r a p h y of lutein 3, t w o - w a y migrations were I

a__~

Crp

Y

3/M

LUTEIN

PE + Ch (3:1)

Fig. i. Pure lutein, approx, o.oo2 mg in 3 l~1 ether-light petroleum (i:i, v/v), subjected to twoway migration. For abbreviations see Table I.

Biochim. Biophys. Acta, lO9 (1965) 16-22

18

H. H. STRAIN et al.

TABLE I ABBREVIATIONS AND SYMBOLS EMPLOYED IN THE FIGURES Br, Ch, Chl, E, Ext, F, f, F 1, F 2, Gr, G,

brown chloroform chlorophyll diethyl ether extract front, w a s h liquid faint, front, first wash front second wash green gray

O, PE, ppt., Pr, T, t, x, Y, \\\, ///,

orange petroleum ether precipitate ~,-propyl alcohol top of adsorbent trace starting point yellow blue b y HC1 vapor blue-green b y HC1 vapor

carried out w i t h pure lutein plus the flower extract. For these migrations, 2o ~1 of a I : I m i x t u r e of the lutein solution and the flower extract were added to the paper, and the migrations were carried out as indicated in Fig. 2. This resulted in quintuple zoning as shown. The formation of these five zones m a y be explained in terms of the "precipitation", double zoning and double tailing produced in the one-way migration 3. Multiple zoning of a single substance must, therefore, be considered in the t w o - w a y chromatography of the pigments in leaf extracts. 19cm

1

~Ft ¥0 Corofenes Chl o

@

Y Luteln

VioIoxclnt hin

OY

YG Chl b

I y

Y

Y Neoxonthin

3 pt

LEAF

EXT

LUTEIN-I- FLOWER EXT. IOpA eoch

PE + C h ( 3 : l )

F2

~

PE + Oh { 3: 1)

Fig. 2. Pure lutein, approx, o.oo 7 m g in IO/~1 e t h e r - l i g h t petroleum ( i : i , the extract of morning glory flowers subjected to t w o - w a y migration.

F2

v/v), plus Io Etl of

Fig. 3. P i g m e n t s of leaf extract separated b y t w o - w a y chromatography. Initial zone formed w i t h 3/*1 of light petroleum solution of the leaf e x t r a c t as described in the text.

Two-way chromatography of leaf extracts The chromatograms obtained by t w o - w a y migration of the leaf extracts 3 varied with the a m o u n t of extract added to the paper, with the solvent employed, and with the wash solvents. W i t h an initial zone formed from 3 t~1 of the light petroleum solution, t w o chlorophyll zones, three xanthophyll zones and one carotene zone were obtained as shown in Fig. 3- The reaction with HC1 vapors indicated that the xanthophylls were neoxanthin, violaxanthin and lutein (with or without zeaxanthin). Biochim. Biophys. Acta, lO9 (1965) 16-22

CHLOROPLAST

19

PIGMENTS

When smaller quantities of the leaf extract (2/~1) were adsorbed, as in Fig. 3, the separation of the chlorophylls was complete. The violaxanthin zone was then so pale that it could not be located. Otherwise the chromatogram was essentially the same as that indicated by Fig. 3. When larger quantities of the leaf extract were added to the paper, the two-way chromatogram yielded several additional zones. With IO F1 of the extract, which corresponds to the loading employed b y KHUDA~RI1, a two-way chromatogram like that in Fig. 4 was obtained. This chromatogram differs in a number of ways from 19 cm

I

| i

.

.

.

.

.

.

.

-Y2

Y2

.

2 Violoxont hin 3 Lutein Chlorophyll b Chlorophyll o Pheophytin Corotenes

4 5 6 7

-YI

I ~2,3

.

Y3 I 0 NL

LEAF

EXT

PE + Ch ( 5 : 1 )

Fig. 4. Pigments of leaf extract separated by two-way chromatography. Initial zone formed with io/~l of light petroleum solution of the leaf extract. This corresponds with the loading employed by KHUDAIRI. that reported by K~IUDAIRI1. The chlorophylls are incompletely separated and exhibit double tails, whereas he shows a complete separation with little tailing. Xanthophylls appear to have migrated from the starting point with the second solvent, but KHUDAIRI shows no similar effect. The neoxanthin and the violaxanthin are incompletely separated, whereas he finds them as contiguous but discrete zones. There is no indication of two zones corresponding to lutein and eloxanthin, yet he shows the zones to be distinct. Pheophytins were seldom detectable, but he reports two conspicuous, contiguous zones. Yet, when prepared from chlorophylls a and b and examined by the two-way procedure, pheophytins a and b yielded two well-separated zones. Our identification of the several yellow zones, as in Fig. 4, is very different from that reported by KHUDAIRI1 and by WOLF2. On the basis of color reactions of the pigments, from the chromatography of the individual pigments, and from the examination of the saponified extracts (described below), each zone cannot be attributed to a single pigment. On the contrary, lutein appears in three or four zones, violaxanthin in three or four and neoxanthin in two. The two-way chromatograms also varied with the wash liquids employed for formation of the initial zone and for formation of the chromatogram. Two examples of these effects are illustrated by Figs. 5 and 6. The former illustrates extensive double Biochim. Biophys. Acta, lO9 (196.5) 16-22

20

H . H . STRAIN et al.

tailing and multiple zoning attributable largely to the limited solubility of the pigments in light petroleum and to colorless contaminants. The latter illustrates reduced double tailing, attributable, in part, to better solubility of the pigments in the light p e t r o l e u m - e t h e r mixture. Variation of the solvent from which the mixture was applied to the paper influenced the separations. When 2/A of the extract dissolved in ether were applied to the paper, other conditions remaining the same as in Figs. 3 and 4, separate zones of carotene, lutein, chlorophyll a, chlorophyll b and neoxanthin were obtained, but

S Y I

LEAF

I

Neoxonthin

3 4 5 6 7

Luteln Chlorophyll b Chlorophyll o Pheophytin Corotenes

EXTRACT

3 4 5 6

(IOkd E)

LEAF

Lutein Corotenes CMorophyll b Chlorophyll o

EXTRACT

(IOFI

PE)

/

L_J PE + Ch

F2

~

PE ÷ C h ( 3 : l )

F2

Fig. 5- Pigments of leaf extract, ill IO /21 Of ether, separated by two-way migration. Fig. 6. Pigments of leaf extract, in IO #1 of light petroleum, separated by two-way migration.

the violaxanthin could not be detected. When the amount of the extract in ether was increased to IO/,1, a series of double-tailing, overlapping zones was observed as shown in Fig. 5. Two-way chromatograms were also formed after the leaf extract had been added from solution in light petroleum. To improve the solubility of the pigments, the first wash liquid was e t h e r - l i g h t petroleum ether ( I : i , v/v) plus propanol, the second was light petroleum-chloroform. With 3/A of the extract, the carotene, lutein, violaxanthin, chlorophyll a and chlorophyll b formed separate zones. The neoxanthin could not be detected. Presumably it was obscured b y the chlorophyll b. With IO ~1 of the leaf extract, the pigments yielded a series of zones as shown in Fig. 6. The most probable identification of the pigments in these zones is also indicated in the Fig. 6. Extracts of the leaves of barley, wheat, oats, spinach, violets and Elodea "~yield chromatograms similar to those obtained with extracts of cocklebur and reproduced as Figs. 3-6.

Two-waypaper chromatography of pheophytins In the two-way chromatogram reported by KHUDAIRI1, pheophytins a and b in the leaf extracts formed contiguous zones between lutein and carotene. In our experiments, pheophytins were not detectable unless the chlorophylls had undergone Biochim. Biophys. Acta, lO9 (I965) I6-22

CI-ILOR0PLAST PIGMENTS

2I

secondary reactions during preparation of the leaf extracts for adsorption. When the pheophytins were formed by the addition of acids to the extracts, the two pheophytins were separated completely by the two-way paper chromatography as used by KHUDAIRI 1.

Two-way chromatography of saponified leaf extracts Saponified leaf extracts, similar to those used for one-way migrations a, were also examined by two-way chromatography, both with small amounts and with ]

I

4-

FI

Car°'e°°° l I

Carotenes

) Lutein (3) ~Lutein

(3)

Y Cy

2:[I Violoxonthin (2) Neoxonthin

, ppt. 1±2'31.5Fd

Y~/

SAPONIFIED

Viol . . . .

,Y~

(I) LEAF

N .....

thin (2) thio el)

y, pp~ ~±2,3= I0/~I SAPONIFIED LEAF EXT. PE + Ch(3:l)

EXT.

PE + Gh (3:1)

Fig. 7. Pigments of saponified leaf extract, in 1. 5/,1 of e t h e r - l i g h t petroleum ether (I : I, v/v), separated by two-way migration. Fig. 8. Pigments of saponified leaf extract, in io y1 of e t h e r - l i g h t petroleum ether ( i : i , v/v), separated by two-way migration. i I

I

Carotenes

(~Lutein Y Violoxonthin

O Y Neoxanthin

X 1.5 FI SAPONIFIED LEAF EXT. PE + Ch(3:l)

~

I

Carotenes I

~ ( 3 ) (2)

laxonthin

Neoxanthin (I)

( ~ Y, ppt.

1+-2'310hd SAPONIFIED LEAF EXT PE + Ch(3:l)

Fig. 9. Pigments of saponifted leaf extract, in 1.5/*1 of e t h e r - l i g h t petroleuln ether (I : I, v/v), separated by two-way migration. Fig. IO. Pigments of saponified leaf extract, in IO #1 of e t h e r - l i g h t petroleum (i : i, v/v), separated by two-way migration.

Biochim. Biophys. Acla, lO9 (1965) 16-22

22

H . I-I. S T R A I N

et al.

r e l a t i v e l y large a m o u n t s a d d e d to the paper. W i t h only 1.5/~1 of t h e solution of saponified leaf e x t r a c t , a series of zones like t h a t shown in Fig. 7 was o b t a i n e d . Two of these zones r e a c t e d w i t h the v a p o r s of c o n c e n t r a t e d HC1 yielding blue a n d bluegreen colors as indicated. Here there is no i n d i c a t i o n of m u l t i p l e zoning, a n d the four zones c o r r e s p o n d to t h e four principal c a r o t e n o i d p i g m e n t s of leaves. W i t h IO t,1 of the solution of the saponified leaf e x t r a c t , a b o u t six yellow zones were o b t a i n e d , as shown in Fig. 8. As i n d i c a t e d b y the color reaction with t h e v a p o r s of HC1, one of these zones was n o t homogeneous. Multiple zoning h a d o b v i o u s l y led to crossc o n t a m i n a t i o n of t h e zones. T w o - w a y c h r o m a t o g r a m s of the saponified leaf e x t r a c t were r u n w i t h e t h e r - l i g h t p e t r o l e u m e t h e r (I : I , v / v ) - 1 % p r o p a n o l as the first wash liquid. W i t h 1. 5 ~l of the p i g m e n t solution, four discrete zones were o b t a i n e d as shown in Fig. 9. These zones c o r r e s p o n d to the four p r i n c i p a l leaf carotenoids. W i t h larger q u a n t i t i e s of the saponified pigments, IO t,1, an a d d i t i o n a l zone a p p e a r e d at the s t a r t i n g point, a n d one of the zones was n o t homogeneous. This result is shown in Fig. IO. Saponified e x t r a c t s of the leaves of barley, oats, spinach, violets a n d E l o d e a y i e l d e d c h r o m a t o g r a m s similar to those o b t a i n e d w i t h the saponified e x t r a c t s of cocklebur a n d shown as Figs. 7-1o. The b e t t e r s e p a r a t i o n s in Figs. 9 a n d IO as c o m p a r e d to Figs. 7 a n d 8 m a y be a t t r i b u t e d to the b e t t e r solvent p r o p e r t i e s of the e t h e r - l i g h t p e t r o l e u m e t h e r m i x t u r e . This effect reduces t h e " p r e c i p i t a t e " f o r m a t i o n a n d the t r a i l i n g of t h e zones. ACKNOWLEDGEMENTS

Mr. W . CHORNEY of A r g o n n e ' s Division of Biology a n d Medicine generously grew the cocklebur a n d o a t p l a n t s used in this i n v e s t i g a t i o n . This w o r k was p e r f o r m e d u n d e r the auspices of the U.S. A t o m i c E n e r g y Commission. REFERENCES I 2 3 4

A. K. KHUDAIRI,Biochim. Biophys. Acta, 46 (1961) 344F. T. WOLF, Plant Physiol., 38 (1963) 649. H. H. STRAIN,J. SHERMA,F. L. BENTON AND J. J. KATZ,Biochim. Biophys. Acla, lO9 (1965) I. H. H. STRAI,'¢,Ind. Eng. Chem., 42 (195o) 13o 7.

13iochim. Biophys. Acta, lO9 (1965) 16-22