Water content of chlorophyll hydrate

ANALYTICAL

BIOCHEMISTRY

112,

282-286 (1981)

Water Content HERBERT

BLAHA,*

of Chlorophyll

Hydrate

PETER KIS,? AND HELMUTH SPRINGER-LEDERER~

*Institute of Inorganic Chemistry and ‘Iinstitute of Physical Chemistry, University of Vienna, Vienna, Austria. Received October 15, 1980 Thermogravimetry shows that polycrystalline chlorophyll a is a chlorophyll dihydrate. Neither thermogravimetry nor differential thermal analysis indicates the existence of a stable chl a monohydrate. Moreover it is found that larger amounts of water-free chlorophyll a (>50 mg) cannot be prepared by application of vacuum and heat in a reasonable time, if chemical decomposition is to be avoided. Thin layers of polycrystalhne chlorophyll a undergo spectroscopic changes depending on temperature and vacuum.

hydrate. Moreover, our data show the influence of some drying procedures on the preparation of water-free chl a.

Since the experiments of Jacobs et al. (1) we know of the intimate relationship between chlorophyll a (chl a)’ and water. These workers reported first that water must be present for preparation of “crystalline” chl a (chl 740). Because of its outstanding photoactive behavior chl 740 recently received renewed interest (2-5). Unlike solutions of chl u , which absorb red light near 660 nm, suspensions of chl 740 show a pronounced absorption band at about 740-750 nm, probably depending on crystal size (6). chl 740 has also been termed polycrystalline chl a, microcrystalline chl a, chl a hydrate, chl u-water adduct, or, loosely, chlu-water aggregate. Infrared studies on chl u-water interactions led to the proposal that absence of the 740-nm peak is a criterion for the absence of water and that chl 740 is a chlorophyll monohydrate (chl a.H,O), (7). In contrast, other workers inferred from water titration experiments (8) and X-ray photoelectron spectroscopic studies (9) that chl 740 is a chlorophyll dihydrate (chl a.2 H,O),. Here we attack the water problem by a thermoanalytical (thermogravimetric) method and demonstrate that chl740 is a di-

MATERIALS

chl740 was prepared from the blue-green alga Anucystis niduluns (strain L 1402-1, Culture Collection of Algae, Gbttingen, FRG). Two recently described preparation procedures (10,ll) were combined with a short chromatographic step. A. niduluns does not contain chlorophyll b (12). Algae were harvested, extracted shortly (2-3 min) with cold acetone, and chl a precipitated as a chl a -dioxane adduct (11). The centrifuged crude mass of the pigment was dissolved in a little acetone and purified by two precipitation steps with water (10). To remove remaining traces (< 1%) of pheophytin a the chl a was chromatographed on commercial powdered sugar, and eluted with acetone-petroleum ether 6 : 94. The solution of chl a was washed several times with water-methanol mixtures as described in (13), chl a precipitated as chl a hydrate and centrifuged. Final precipitation of chl 740 was always done from 2methylbutane by dropwise addition of water and gentle shaking. This low-boiling solvent (28°C) is easy to remove during dry-

1 Abbreviations used: chl a, chlorophyll a; chl 740, “crystalline” chl a; tic, thin-layer chromatography. OOO3-2697/81/060282-05$02.00/O Copyright All rights

Q 1981 by Academic Press, Inc. of reproduction in any form reserved.

AND METHODS

282

CHLOROPHYLL

L-~,,L,

0

HYDRATE

,/,._,,

15

WATER

‘,I,

30

L5 Tme [hours]

283

CONTENT

,,,,,,,

60

69 72

FIG. 1. Loss of weight of chl 740 on a thermobalance during drying at 2s”C in a current of argon. Weight remained constant at subsequent application of vacuum (7 X 10m4Pa) during 23 h.

ing. All solvents were pro analysi products of Merck. Purity checks confirmed high quality of the chl a. Thin-layer chromatography (tic) on cellulose (eluent: 90% petroleum ether, 9.6% acetone, 0.4% isopropanol) showed only one spot. Both the uv/visible spectra recorded on a Perkin-Elmer Hitachi 200 spectrophotometer and the absorption coefficient agreed with recent data (10). Elemental analysis for C, H, and N was carried out with a Perkin-Elmer Analyzer 240; the results will be given below. Thermogravimetric traces were recorded by a thermobalance (Mettler Thermoanalyzer TA 1) is connection with a low-temperature furnace. Good sensitivity of the balance required samples of at least 60- 100 mg chl740. The specimens were placed on a platinum dish (diameter, 13 mm), dried at room temperature, and subsequently heated at the slowest possible rate of 0.2”C min-’ up to 200-250°C. Differential thermoanalysis was done simultaneously with the same apparatus, with A1203 as a reference substance. RESULTS

In order to dry chl 740 the specimens were purged from adhering isopentane and water by flushing with argon (4 liters h-l) and/or by applying high vacuum (about 7 x lop4 Pa) isothermally at room temperature. Depending on sample preparation (grain size, packing density, amount, etc.)

and drying method (argon or vacuum) 30130 h were needed to reach, practically, constancy of weight (Fig. 1). During this procedure all samples lost about 10% of their original weights. Afterward temperature was raised. Loss of weight, obviously due to loss of water, set in shortly after heating began (Fig. 2). At 85-95°C loss of weight ceased (indicated by a negligible slope of the trace), signaling end of dehydration. Further rise of temperature led to a new, and rapid, loss of weight, clearly due to thermal decomposition of chl a. The six dehydration experiments with freshly prepared chl 740 samples yielded a mean loss of 2.04 + 0.51 mol water/m01 chl a. When the two extreme values (1.42 and 3.05 H,O) were left out, calculation gave 1.94 +- 0.36 HzO. In the sample dried 87 h at high vacuum (~5 X 1Oe4 Pa) 1.42 H,O was found, and in the sample dried by flushing with argon only, 3.05 HZ0 (see Figs. 1,2). No sample lost exactly 2.0 mol water/ mol of chl a. Apparently either small residues of solvent still had been present, or some water had been liberated in consequence of the applied high vacuum. Independent evidence for a content of 2 H,O in chl 740 were derived from the results of the elemental analysis done with one sample both at room temperature and at 100°C (Table 1). Further information about the dehydration of chl 740 was obtained from differential thermal analysis. This technique de-

284

BLAHA,

KIS, AND SPRINGER-LEDERER TABLE PERCENTAGES

Starting with chl 740 found

\

i

I,/,

50

sI/I

I 100 Temperature

I,/,,

I,, 150 ['Cl

FIG. 2. Loss of weight of chl740 during heating on a thermobalance at a rate of 02°C min-‘. Drying procedures: Sample 1 (1.8 H,O): 94 h in argon, 20 h at 3 x 10S4Pa; Sample 2 (2.1 HpO): 72 h in argon, 23 h at 7 X lo+ Pa.

tects temperature depending on heat effects associated with physical and chemical changes. Such enthalpic effects are caused by phase transitions, dehydration, decomposition, etc. With chl 740 small heat effects occurred near 90, 130, and 150°C. The first value corresponds to the end of dehydration and/or the beginning of decomposition of chl a. Checks by tic proved that samples heated in the thermobalance above 100°C were chemically altered. In contrast to pure chl a these samples showed immobile spots. Moreover, the absorption spectra in diethyl ether were slightly changed. Compared to pure chl a, the peak ratios 428 r&409 nm were decreased, and the “blue to red” ratios 428 nm/660 nm increased. Since removal of H,O is effected by heat we studied the influence of temperature on the absorption spectrum of chl 740. Microscopic slides were covered with thin layers (about 0.05-0.1 g me2) of chl 740 and heated up to a definite temperature (60, 80, or 90°C) on a Kofler hot stage. The absorption spectra of the thin layers are shown in Fig. 3. At 80°C a band at 680 nm appeared at the expense of the original band, at 745 nm. This change reflects destruction of the polycrystalline order of chl 740 (14), with concomitant loss of water. The conversion

C H N

OF

1 C. H,

AND

Theoretical

N values of

At 20°C

At 100°C

chl (r.2HI0

chi a.HpO

chl a

70.90 8.32 6.01

73.66 8.06 6.33

71.07 8.24 6.03

72.47 8.18 6.15

73.93 8.12 6.27

was quantitative at 90°C and could be reversed within 13 h in an atmosphere containing saturated water vapor at 70°C. When chl740 in thin layers was exposed to vacuum, heating to 70°C (l-2 Pa, 3 h) led to disappearance of the 745nm band, and 50°C (same conditions) was sufficient to convert half of the chl740 into amorphous chl a, absorbing at 675-680 nm. Under high vacuum (1O-2 Pa) at room temperature the 745~nm peak vanished almost completely after 10 h. Interestingly, complete conversion into chl a could also be achieved by suspending chl740 in isooctane and heating to 45°C for 1 h. We also observed the melting behavior of chl 740 on a Kofler hot stage with a micro-

c

715 BOO 500 675745BCm 500 Wave lcnglh [nml

675

800

FIG. 3. Optical density of chl 740 in thin layers on glass slides, heated 4 h at 60°C (a); 4 h at 80°C (b); 12 h at 90°C (c).

CHLOROPHYLL

HYDRATE

scope. Heating at the low rate of 1°C min-‘, the first signs of melting, indicated by the loss of sharp edges of small particles, were seen at 93°C. DISCUSSION

Measurements with the thermobalance and elemental analysis demonstrate that chl 740 dried at room temperature in high vacuum is a chl a dihydrate. Surprisingly neither thermogravimetric analysis nor differential thermal analysis point to the existence of a stable chl n monohydrate. We therefore assume that removal of the first water molecule facilitates the immediate removal of the second water molecule to an extent that thermal analysis does not resolve a step involving the loss of one water molecule. This view can be rationalized through the proposed model for chlorophyllide a dihydrate, based on X-ray data (15,16). In this substance, which differs from chl a only by the absence of the phytyl chain, the two water molecules are interconnected by hydrogen bonds. Why does the drying of larger amounts of chl740 proceed so slowly? We suggest that release of water in chl 740 may propagate from molecule to molecule of chl a with simultanous dearrangement of the lattice (14) and formation of water-permeable pores. Complete removal of HZ0 leaves behind exposed unsaturated functional groups (keto and ester groups and coordinatively unsaturated Mg) and makes ch! a accessible to thermal decomposition. This view is consistent with the result that dehydration is not fully separated from thermal decomposition (Fig. 2). The chemical decomposition is inferred from tic. Further support comes from elemental analysis data. While measured C, N, and H values (Table 1) for the unheated sample are in excellent agreement with the calculated values of the proposed dihydrate (experimental C/N ratio 11.80, theoretical 11.79), the same sample heated to 100°C deviates noticeable from the value for water-

WATER

285

CONTENT

free chl a (experimental C/N ratio 11.64, theoretical 11.79). This suggests beginning liberation of a low-molecular N-free compound. We conclude that application of high vacuum and/or heat is not suitable for preparation of water-free chl a in bulk samples (e.g., 50- 100 mg) within a reasonable time. Pure water-free chl a has to be prepared by other methods, like repeated codestillation (17).

The absorbance changes of thin layers of chl 740 on glass slides express a close relationship between water content and absorption spectrum of chl a. An interesting feature is the acceleration of disappearance of the 745nm band under vacuum. Assuming that absence of 740 to 750-nm band guarantees absence of water in chl a samples (7), this finding seems to contradict our thermogravimetric traces. The contradiction may be explained through the difference in sample size between thermogravimetry (60100 mg) and thin-layer spectrometry (some pg). Probably in thermogravimetry even the smallest possible heating rate was still too fast for thermodynamical equilibrium to be attained at each moment of heating. Since it is impossible to measure loss of weight due to removal of water in microgram specimens, optical density at 745 nm and water content cannot be correlated directly at this time. ACKNOWLEDGMENTS This work has been supported by the “Fonds zur Forderung der wissenschaftlichen Forschung” of the Republic of Austria. We thank Professor E. Broda for his interest and Dr. H. Zak and Mr. S. Frenczko for excellent measurements of C, N, and H contents. The able technical assistance of Mr. 0. Kuntner is gratefully acknowledged.

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