586 SHORT COMMUNICATIONS

sc 43040

Preparation of a colloidal form of chlorophyll a from concentrated solution

Recent investigations z have demonstrated that a form of chlorophyll a ab- sorbing maximally in the red near 700 m/~ is reactive in one of the primary photo- reactions of photosynthesis. This species is thought to be a chlorophyll-protein complex 2 or an aggregated form of chlorophyll 3. Chlorophyll systems exhibiting similar long-wavelength red absorption bands in vitro are of interest in that they may shed some light on the nature of this pigment.

BANNISTER 4 has studied the properties of chlorophyll colloids prepared by adding water to dilute solutions of chlorophyll in solvents such as ethanol or acetone. He finds that the red absorption maximum of these colloids is in the vicinity of 672 m/z. When the water content is raised to 45-60 %, the 672 rn/~ band is broadened, and occasionally a shoulder is observed near 700 m/z. We find that when the chloro- phyll colloid is prepared from concentrated solutions (greater than 5" lO-4 M) a form with a red absorption maximum at 698 m/~ predominates 5 (see Fig. i, solid line). A search was made for fluorescence from this colloid using the apparatus described previously 3. No evidence for fluorescence was found upon irradiation with mono- chromatic blue light (436 m/,), either at room temperature or at 77 ° K .

I t was noted that the colloid exhibiting the 698 m/~ absorption band, which is prepared from concentrated chlorophyll solutions showed considerably more turbidity than the colloid formed from dilute solutions, which absorbs at 672 m/~. Therefore, it was necessary to record absorption spectra with a spectrophotometer (Cary model I4), equipped with an at tachment to minimize distortion due to scattering. To prepare the colloid, 2.75 mg of chlorophyll a (obtained according to the method of JACOBS,

VATTER AND HOLT 6) were dissolved in 0. 4 ml of ethanol to which o.i ml of water was added, giving a total chlorophyll concentration of 6.2" IO -3 moles/1. The absorption spectrum of the colloidal sample was examined in a quartz absorption cell having an optical path of 5" IO-Z cm.

I t is seen from Fig. I (solid line) that a large part of the chlorophyll in this system exists as a long wavelength absorbing form. The absorption spectrum of the

0.4

0.2 ,' \ S " , ;

0.0 I I I l 400 500 600 700 800

Wovelength (rol3)

Fig. I. Solid line: absorpt ion spectrum of chlorophyll a colloid prepared from concentrated ethanol solution: o. 4 ml of 4.9" lO-3 molar chlorophyll a in ethanol diluted wi th o.i ml of water. Broken line: absorpt ion spect rum of 698 m/~ colloid alone obtained by difference technique. Sample is same as for solid line. Reference is a solution of chlorophyll a in ethanol, abs0rbancy 0.2 a t 665 m/*.

Biochim. Biophys. Acta, 94 (1965) 586-588

SHORT COMMUNICATIONS 587

chlorophyE~ colloid alone (Fig. I , broken line) was obtained by the method of difference spectroscopy. In this method, the colloid is placed in the sample beam and a true ethanolic solution of chlorophyll a is placed in the reference beam. The absorbancy of the reference is adjusted to satisfy two criteria: first, that the difference spectrum show no negative absorption, and second, that no absorption by non-colloidal chloro- phyll be evident.

The major absorption maxima of the colloid in Fig. I (broken line) are at 698 and 45 ° m/~. I t is to be noted that the colloidal chlorophyll has a higher extinction for the red than the blue maximum. A similar relationship has been reported for chlorophyll dispersed in aqueous dioxane 4 where the absorption maxima are at 685 and 449 rn/~. The absence of a shoulder on the Soret band of the 698 m/~ colloid might result from our difference technique. If in the reference solution there was a slightly higher concentration of dissolved chlorophyll than in the sample, then a small shoulder near 428 m y would have been subtracted out in the difference spectrum.

The absorption maximum of the colloid prepared from concentrated solution is sensitive to small variations in the alcohol-water ratio. Maxima occurring anywhere from 690 to 71o m/~ were seen occasionally. On the other hand, the position of the red absorption band of the colloid formed from dilute solution is relatively insensitive to the proportion of water to alcohol 4.

In Fig. I , it can be seen that the absorbancy at 698 m/z is lower for the solid line curve (colloidal and dissolved chlorophyll) than for the broken line curve (colloid alone) the latter obtained by difference spectroscopy. This difference in absorbance occurred because formation of the colloid is time dependent. After the addition of water, the absorption near 700 m y increases while that of non-colloidal chlorophyll at 665 m/~ decreases. About an hour is required for the system to stabilize. The ab- sorption properties of the chlorophyll colloid then remain unchanged overnight.

The extinction coefficient, e698, of the chlorophyll colloid absorbing at 698 m~, is estimated in the following way: the overall chlorophyll concentration [ChlJ in any sample is known, as the concentrated solution is made by using a weighed amount of chlorophyll. The concentration of 698 m/~ colloid and of dissolved chlorophyll are not known. The concentration of colloid [Cs98~ is equal to Asgs/de698, and the concen- tration of dissolved chlorophyll [C~65] equals A665/de~6a, where A is the absorbancy at the indicated wavelength, e the extinction coefficient and d the cell path length. The absorbancy, A665, of non-colloidal chlorophyll in the sample is taken as equal to the absorbancy required in the reference beam to cancel out this absorption. The total chlorophyll concentration is equal to the sum of the concentrations of colloidal and of dissolved chlorophyll, i.e.,

[Chl] = [C698 ] 21- [C665] (I)

so that A ~gs/d

e698 A (665) " (2) [Chll

de (665)

In our sample, A698 = o.4I, [Chl; = 6.2. IO -z moles/l, A6~5 = o.20, d = 5" lO-3 cm, e665 = 6.6. IO 4, and e~98 is calculated to equal r.5- Io4. This relatively low extinction coefficient may arise from sieve effect ~ and mutual shading of chlorophyll molecules in the colloidal state.

Biochim. Biophys. Acta, 94 (1965) 586 - 588

588 SHORT COMMUNICATIONS

The 698 m/, colloid can readily be prepared only from concentrated chlorophyll solutions. Concentrated systems facilitate the formation of colloidal particles con- taining a large number of pigment molecules. Interactions between these molecules probably cause the large shift to longer wavelengths of the red band of chlorophyll in this colloidal state. It is well known that such spectral shifts accompany increasing pigment interactions 8-~. Since systems containing only chlorophyll, such as the colloid described in this paper, can absorb near 700 m~, the long wavelength absorption of chlorophyll a in vivo may likewise be due to interaction between neighboring chloro- phyll molecules. In view of the highly aggregated condition of chlorophyll in this colloid, its photochemistry should be of interest, since it is likely that aggregated chlorophyll participates in the photochemistry of photosynthesis l&

I B M Watson Laboratory, Columbia University, New York, N .Y . (U.S.A.)

SUSE B. BROYDE SEYMOUR STEVEN BRODY

i G. W. ROBINSON, Ann. Rev. Phys. Chem., 15 (1964) 323 . 2 B. KOK, Biochim. Biophys. Acta, 48 (1961) 527 . 3 S. S. BRODY AND M. BRODY, Natl. Acad. Sci.-Natl. Res. Council, Publ., 1145 (1963) 455. 4 T. T. BANNISTER, Physiol. Veg., I (1963) 115. 5 S. ICHIMURA AND E. RABINOWlTCH, Science, 131 (196o) 1314. 6 E. JACOBS, A. VATTER AND A. S. HOLT, Arch. Biochem. Biophys., 53 (1954) 228. 7 E. RABINOWlTCH, Photosynthesis and Related Processes. Interscience, New York, 1951 , p. 673. 8 E. RABINOWITCH, Photosynthesis ant Related Processes, Interscience, New York, 1951, p. 651. 9 D. G. HARRIS AND F. I. ZSCHEILE, Botan. Gaz., lO 4 (1943) 515 .

IO H. J. TRURNIT AND G. COLMANO, Biochim. Biophys. Acta, 31 (1959) 434. I I S. S. BRODY AND M. BRODY, Biochim. Biophys. Acta, 54 (1961) 495. 12 E. E. JACGBS AND 2~. S. HOLT, J. Chem. Phys., 22 (1954) 142.

Received November 2oth, 1964

Biochim. Biophys. Acta, 94 (I965) 586-588

sc 43o39

Plastocyanin photo-oxidation by detergent-treated chloroplasts

In previous publications1, 2, we reported observations concerning the photo- oxidation of cytochrome c, cytochrome f , and plastocyanin by detergent-treated chloroplasts. The two chloroplast enzymes proved to be effective catalysts of eyto- chrome c photo-oxidation, and were themselves photo-oxidized with high rate and efficiency. The effectiveness of long-wavelength light and the catalysis by viologen dyes suggested that only Photosystem I was involved. Presumably, ferrocytochrome and cuproplastocyanin are effective electron donors to primary photo-oxidant P7oo + and electron acceptors for cytochrome c.

The kinetics of the photo-oxidation of cytochrome f as a substrate proved to be peculiar. Rates in strong light as well as the quantum yield of the process were proportional to the fraction of the substrat~ pr~e~ent in the reduced state: [cyto- chrome flreaueed] ~cytochrome fltotal. This finding was interpreted to mean that P7oo and cytochrome f associate in a complex, and that for a successful photo-act, an

Biochim. Biophys. Acta, 94 (1965) 588-590