Esther Dujardin, Pierre Laszlo, and David Sacks'
The Chlorophylls
Universite de Liege au s a r t - T ~ I ~ ~ ~ 4000 Liege 1, Belgium
An experiment in bio-inorganic chemistry
There are a number of hiomolecules in which a metal ion is enclosed in a cavity inside the molecular structure. Some examples of these are enzymes such as carbonic anhydrase (Zn2+),antibiotics such as valinomycin or monensin
A
CH,COOH
which effect the active transport of the alkali metal cations (K+, Na+) through the cell membranes, or a respiratory pigment such as hemoglobin (Fez+).This paper describes a series of experiments appropriate for an undergraduate lahoratory in which the chlorophyll pigments extracted from green bean leaves have their Mg2+ metal ions removed and reolaced bv various other metal ions such as CU" and Zn". The experiments are designed more tu exnose the student to hioloeiral and metallic chemistr\r rather than to provide a thorough program of research, although during the laboratory questions are posed that could lead to further topics of research. The Chlorophylls
The chlorophylls are a very important group of metallic hiomolecules since, as the pigments responsible for the
green color of plants, they "trap" solar energy for us. Located in intracellular organelles called chloroplasts, the chlorophylls are attached to lipoproteins which form what are known as the chloroplast lamellae. These are membranes which enclose flattened "sacs" called thylakoids; some of them extend along the full length of the chloroplast, while other smaller ones cohere together to form the grana, which can be seen under an ordinary microscope as small dark-green grains. I t is in the lamellae of the chloroplast that the primary processes of photosynthesis take place, converting light energy into chemical energy. The chlorophylls, bound to proteins, play an essential part in this process. The basic structure of the chlorophylls is as follows
A tetrapyrrolic ring system surrounds a magnesium atom. The pyrroles are linked together by methene =CH-hridges. The tetrapyrrolic ring is characterized by a system of conjugated double bonds. Various suhstituents can attach themselves to the carbon atoms 1-8, as well as to the methene hridging carbons a, & r ,or 6. The more ahundant chlorophyll, chlorophyll a, was first synthesized by R. B. Woodward in 1960 (1). FH,
yH-nq
CWH,
H CH, IphytylllOC-H,C.H
The chlorophylls have a saturated bond between atoms 7 and 8, a fifth ring (ring V), and a long carbon chain joined by an ester link to the propionic acid group on carbon 7. CH,
I
phytol =CH,ICH-CH&H2CH,h-C
H,C
I- a W C H O H
Hard and Soft Acids and Bases
,
. .;, .
..
i . i i
. .
Figure 1. ElecVon micrograph of an unrathin longitudinal section in a spinach chloroplast (enlargement = 60.000:,courlesy of Professor R. Bronchart. University of Li6ge).
742 / Journal of Chemical Education
The exchange of the central Mg2+ ion in chlorophyll for other metal ions is interesting because of its relevance to Pearson's theory of Hard and Soft Acids and Bases (HSAB) (2). Hard acids, according to this theory, are those that are small in size with a high positive charge and do not contain unshared pairs of electrons in their valence shells, while hard hases similarly have high electronegativity, low polarizahility, and are hard to oxidize (3). Soft acids and bases exhibit the opposite behavior: lower electronegativity, greater electron density, greater polarizahility. Al'Visiting student from Princeton University, Summer 1974.
though theoretical explanations have been offered (4), the importance of the HSAB theory lies in its premise that hard acids prefer to hind with hard bases, while soft acids prefer soft bases. While innate acid or base strength is still important, the HSAB theory adds the parameters of hardness and softness in determining acid-base complex stability. This theory applies very nicely to the chlorophyll reactions described in this paper. While nitrogen is a hard base in a saturated molecule, the presence of extensive electron delocalization, as found in the unsaturated system of the chlorophylls, permits sufficient polarizahility that the nitrogen becomes softer, moving into the "borderline" category. Magnesium(II), however, being a small, doubly charged ion, is classified as a hard acid. Obviously this hard acid-soft base complex is sufficiently stable to exist, but chlorophylls with a softer central ion, such as Cu(I1) or Zn(II), form stabler complexes than the magnesium(I1) chlorophyll. I t is for this reason, that the central magnesium ion is easy to remove from the tetrapyrrolic ring hut very difficult to replace, while the copper and zinc analogs have central ions that are difficult to remove but extremely easy to insert. I t is found that a 10%acetic acid solution or a N HCI solution are sufficient to remove the Mg(I1) and form a pheophytin
Table 1.
A
ion
Table 2. Type
12.8
Borderline
2.8
I C I ~ P Ia1
-
A
ion
The Y Values of Metal Ions'
Range of Y
H a r d acid
Ionic Radii
3.2
Metal ions Li+ 10.36). N a + (0.931. Kt (0.92). ~ ~ ' + ( 0 . 8 7ca2+ ). (1.62). ~ n ' + 13.03). A I ~ +(0.70). I n w (2.241, Fe"(2.37). Co" (2.561. 5r2+ (2.08). cr3+ (2.70) Fez+ (3.091. cox+12.961. ~ i ' +
M i 6 0 n 0 . M.. et al.. 3. hers Nuel. Chem.. 29. 2685 119671.
(suhstituenta omitted for clarity) but strong hydrochloric acid is needed to remove the Zn(I1) ion. For the removal of Cu(I1) ion the even stronger acidic solution required oftep destroys the chlorophyll molecule. In addition to the hardness or softness of the cation, it is important also to consider the ionic radius (Table 1). Mg(I1) has an ionic radius of 0.65 A. It is expected that other ions must be of comparable size to fit into the cavity and form a stable complex. The above statement may be an over-simplification: the metal ion need not be in the cavity, as was pointed out for magnesium ion by J. L. Hoard. From Table 1, i t appears that Co2+, Co3+, Cu2+, Fez+, Fe3+, Ni2+, Pt2+, or Zn2+ could possibly be inserted. The Y values of hardness and softness listed in Table 2 indicate those ions which would be expected to form stable complexes. The stability sequence of the structurally related metalloporphyrins is experimentally found to be: Pt(II)>Pd(II)>Ni(II)>Co(II)>Cu(II)>Fe(II)>Zn(II)>Mg(II) (5). This agrees with our results for the modified chlorophylls. The stability of the Pt(I1) and Pd(I1) complexes is surprising in view of the relatively large size of the central ions compared to Mg(I1). Apparently, the softness of these ions permits a greater stability in the complex that far outweighs any destabilizing size effect^.^ The Laboratory Program
Depending upon time available for this laboratory session, the replacement of Mg(I1) by Zn(I1) and Cu(I1) can he attempted both indirectly and directly. The indirect method involves: the extraction of the chlorophyll pigments in an organic solvent, the removal of the Mg(I1) ion and consequent formation of the pheophytin, and then the inser21f rigorous square planarity of the molecule is not required, it is possible that large, soft metal cations such as Pt2+and Pd2+can maintain strong soft acid-soft base bonds while raising themselves out of the tetrapyrrolic plane, thus diminishing the importance of ionic size as a factor in complex stability.
tion of the new metal ion into the pheophytin to form the modified chlorophyll. While the initial extraction is simply a crushing of the green bean leaves in 80%acetone followed by decantation and filtration of the green solution, it is also possible in a cleaner experiment to perform a thin-layer chromatography to separate the various chlorophylls and then perform reactions with each extract, noting any differences in reaction behavior. Both the initial mixture of chlorophylls and the pheophytins formed by addition of acid (-pH 3) emit a red fluorescence, clearly seen under blue light (about 410-440 nm). I t should he noted that this low energy frequency of light absorbed is due to the extensive conjugation in the tetrapyrrolic ring, providing low energy transitions between energy levels. I t is interesting that while the Fez+ chlorophyll is green, the Fez+ hemoglobin molecule, which is also a tetrapyrrolic structure, is red. This is presumably due to extra conjugation in the chlorophyll due to the V ring. The pheophytins thus formed are then treated with concentrated salt solutions of Zn(I1) and Cu(I1) or other metal ions to form the modified chlorophylls, many of which are also fluorescent. The direct replacement in the leaves of the Mg(1I) by Cu(1II - . , or ZuiII) . . is a demonstration of the reaction that occurs when vegetables are cooked in a copper saucepan in the nresence of viueear. The leaves are heated in test tubes containing aqueous"acetic acid (10%) in the presence of copper or zinc acetate. The reaction can be verified by comparing the color changes with those observed previously. If there is time and if separation by thin-layer chromatography has been ~erformed,the spectra of the modified and original chlorophylls before and after chromatography can be compared in a spectrophotometer (Fig. 2). Experimental Extraction of the Chlorophylls
Cut green hean leaves into fine strips and crush them in a mortar containing washed sand and 80% acetone. Collect by decanting Volume 52, Number 11, November 1975 / 743
b, and the fifth hand, chlorophyll o, are separately scraped from the glass plate and placed in centrifuge tubes. The addition of 5 ml of ethvl ether forms a susoension which is eentrifueed. After eentrifu~ation,the clear green supernatan1 liquids are collected and their ahsorpt~onsmeasured hetwccn 400 and 3UO nrn and 600 and 700 nm in a spectrophorcrnetrr.
Removal of the Magnesium Ion To 2 ml of the chlorophyll extract add 0.2 ml of 0.1 N HCI. Heat in the water hath to accelerate the reaction. Note the change in color and the fluorescence. This corresponds to absorption in whieh part of the visihle spectrum? Introduction of a Zinc or Copper Atom into Pheophytin To the pheophytin solution, add a salt of zinc or copper, using 1 ml of concentrated solution of the salt. Heat for at least 1 min in a water hath, until the solution turns green. Note the change of color and fluorescence and again determine the frequencies of absorbed light. Write the overall reaction that is taking place and determine whieh metal salts should " eive the best vields of the modified chlnrophylls. How could the importance of this variable be measured? For which metal ions would the effect be expected to he most or least important? Direct Replacement of Mg by Cu or Zn Heat leaf strips in diluted acetic aeid (10%).Separately, heat other leaf strips in diluted acetic aeid but in the presence of a copper salt. Repeat this operation with a zincsalt. Ohserve the color changes in the leaf strips in order to verify the reaction.
320350
4W
450
500
550
600
650
700
lnm
Fiowe 2. Absorotkm Soechum of un~urif'edextract from bean leaves. Ill a* etone exnact from leaves (2)me Fame extract aner a ~ ~ d d ~ a n(31 o na COP per sullate solullon has been aaaea Tne Cu-chloraphylls have men exmaed *nth
peholeum elner (40'-6O0C1
the green solution into a test tube. In this way a mixture (impure) of chlorophyll o and chlorophyll b is obtained. Observe the color and fluorescence, In whieh part or parts of the visible spectrum do the ahsorhed wavelengths lie? Chlorophyll can he regarded as an acid-base complex. The Mg(I1) ion is s Lewis acid, coordinated with the four nitrogen atoms of the porphyrin nucleus. Is it a square planar complex? Is the 18 electron rule fulfilled? Does it have to he fulfilled? What is the normal coordination of magnesium?
Separation of the Pigments Extract the pigments from the green bean leaves using ethyl ether rather than aqueous acetone, as the solvent, and filter. The concentrated ether extract is placed in long narrow streaks of 3 4 cm on cellulose tlc plates up to 1em from the edge. The plates are then placed in developing tanks containing the following mixture of solvents: petroleum ether (bp 60-80°C), acetone, n-propanol in the ratio of 90:10:0.45 by volume. The plates are developed in the dark at room temperature for ahout 30 min. The chromatography is stopped when the solvent front is about 2-3 em from the top edge of the plate. Immediately after removal from the tank, the plates are observed under natural and ultraviolet light. Of the five hands observed, only the green hands, the fourth and fifth from the top of the plate, are ehloraphylls. The fourth band, chlorophyll
Removing Cu(l1)From Cu Chlorophylland Zn(l1) From Zn Chlorophyll Take the test tubes that contain copper chlorophyll and zinc chlorophyll, respectively. With different, more or less concentrated solutions of hydrochloric aeid try to remove the copper and zinc. Cheek that pheophytins are obtained. Addnlonal Questions That May Be Asked from the Class Natural chlorophyll is apparently one of t h e most unstable forms of chlorophyll. B u t what is t h e distribution of metals in the earth's crust? Does t h e chlorophyll molecule need t o be very stable in order t o function properly? Can one, by using a solution of pheophytins, in t h e presence of the removed Mg(I1) ions, perceptibly lower t h e concentration of a n aqueous solution of a copper salt? Is t h e reaction immediate? Can a auick titration he verformed lls, a n d metal with a calorimeter? ( ~ h l o r o ~ h ~ pheophytins, c h l o r o ~ h v l l sare soluble in oetroleum ether which is n o t miscibie k i t h water). T h e replacement of Mg(I1) by other ions is a n equilibriu m reaction. How could t h e given list of modified chlorophyll stabilities he verified? T h e maximum light intensity from t h e s u n lies in t h e visible range. Would t h e modified chlorophylls he as effective in light absorption a s natural chlorophyll? Literature Cited (11 Woodward, R. B., Ayor. W. A., Beaton. J. M. Bickelhaupt, F., Bonnett. R., Buch8chacher.P.. Class, G.L.,Dutler.H.. Hannah, J., Hauek,F. P.. It&S.,Langemsnn. A,, Le coif,E., Leimgruber, W.. L w o ~ k i W., . sauer. J., Vale"*, 2.. and Vok. H., rl Amen Chem Soc, 82,38W 119601. (21 Pearson, 8. G.. "Hard and Soft Acids and Bawr:
1973.
(3) Pearson R. G., J. CHEM. EOUC.. 45,584 (19681. (11 Pcsrnon R. G., J. CHEM. EDUC.,45.643 (19681. (5) Phillips J. N..Comp Blochem., 9-70 11963).
John Wiley & Sans, Stroudsbuq,
