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Purification and properties of cytochrome f from parsley leaves
33
BIOCHIMICA ET BIOPI-IYSICA ACTA
BBA 45 215 PURIFICATION AND PROPERTIES OF CYTOCHROME f FROM PARSLEY LEAVES GIORGIO FORTI, MARIA LUISA BERTOL]~ AND GIULIANA ZANETTI
Laboratory of Plant Physiology, Department of Botanical Sciences of the University of Milan (Italy) (Received February I l t h , 1965)
SUMMARY
A method for the extraction and purification of cytochrome f from parsley leaves is described. Cytochrome f is obtained in a high state of purity. A molecular weight of 245 ooo is indicated by Sephadex G-2oo chromatography. The absorption spectrum of pure cytochromefin the reduced state shows peaks at 554.5 m~, 532.8 mr*, 524 m~, 422 mr* and 330 m/~ and a shoulder at 402 mt*. Cytochrome f possesses catalase (EC 1.11.1.6) activity, with a Kat. f value of 4.7" IO~.
INTRODUCTION
Cytochromef, a c-type cytochrome of photosynthetic tissues, was discovered by HILL AND SCARISBRICK1 in 1951. This haem protein, found only in the green tissues of plants 1, has been extensively purified by DAVENPORT AND HILL 2, and its presence demonstrated in chloropast preparations from a variety of plants 2. The participation of cytochrome f in photosynthetic electron transport has been demonstrated by differential spectrophotometry in intact cells~-e and by the light-dependent reduction of substrate amounts of the pigment by isolated chloroplast grana 7. The photooxidation of parsley cytochrome f (ref. 8) and algal f-type cytochrome° by detergenttreated chloroplasts has also been reported. It was shown in this laboratoryv that the photo-reduction of cytochromef by chloroplasts is coupled to phosphorylation. Furthermore, a flavoprotein enzyme of chloroplasts has been shown to be a highly specific NADPH-cytochrome f reductasel°, 11, with high affinity for NADPH and cytochrome f. Based on the above mentioned lines of evidence, a number of hypothetical schemes on the role of cytochrome f in photosynthetic electron transport have been proposed: although these may differ in some details, all of them indicate that cytochrome f mediates electron transport between the two photochemical reactions of photosynthesis4,x2,13, being photo-oxidized by the "pigment system I" and photoreduced, though not directly, by the "pigment system II". Although '~/-type cytochromes" have recently been obtained in pure form from algae14,15, the purification and characterization of cytochromef from higher plants has been limited to the excellent pioneering work of HILL and his collaboratorsl,L It is the purpose of this paper to describe a new method of obtaining reproducibly and with satisfactory yield, pure cytochrome f from parsley leaves. Some of the chemical and physical properties of this haem protein are also described. Biochim. Biophys. Acta, lo9 (1965) 33-4o
34
G. FORTI, M. L. BERTOL~2, G. ZANETTI
METHODS All spectrophotometric measurements were performed in i - c m light-path cuvettes, in a double-beam, recording spectrophotometer (Optica CF4). Protein determination was performed either according to KALCKAR16 or according to LOWRY et al. 17. The preparation of the columns of dextran gels, Sephadex G-ioo and G-2oo (Pharmacia, Uppsala), was performed as described b y ANDREWSls. After the column is filled with gel, the appropriate buffer is allowed to flow t h r o u g h overnight, in the cold-room, to obtain a properly packed gel bed. The concentration of cytochrome f is followed during purification b y measuring the difference in absorbance at 555 m ~ minus 54 ° m/,. The pigment is extracted from leaves in the completely reduced form 2. The ratio A422m/~/A228m# is checked in all fractions as a criterion of purity 2. The ratio increases progressively with the separation of cytochrome from contaminating proteins. RESULTS
Cytochrome f purification The m e t h o d described here has been applied reproducibly, over a period of more t h a n 2 years, to the purification of cytochrome f from parsley leaves. The application of the m e t h o d to other tissues has not been thoroughly investigated. The extraction and the initial steps of the purification are substantially identical to the procedure of DAVENPORT AND HILL2. Fresh parsley leaves {obtained commercially or cultivated in a garden) are depetiolated, and carefully washed with tap water. Excess water is blotted away. Portions of IOO g of leaves are ground in the cold-room in a Waring blendor with IOO ml of ethanol containing i % (v/v) Triton X - I o o (a non-ionic detergent} and I. 5 % (v/v) a m m o n i a {specific g r a v i t y 0.88). The blelldor is operated at full speed for approx. 2 min. The homogenate is then squeezed through cheesecloth, and centrifuged 2o min at approx. 19000 x g in a refrigerated centrifuge at --12 °. Particular care has to be taken to avoid any undue standing of the ethallolic extract at temperatures above o °. The supernatant obtained is clear and yellow-colored. Cytochrome f is then precipitated b y the addition of I . I vol. of cold (--20 °) acetone. During the addition of acetone, the solution is continuously stirred. Kieselguhr is added to aid subsequent filtration. The extract is stored for approx. 1-2 h at --20 ° to allow the sedimentation of the precipitate, a reddish-yellow powder at the b o t t o m of the precipitation vessel. The precipitate is collected b y filtration (at - - 2 ° to - - 5 ° ) on W h a t m a n No. I paper, covered with a very thin layer of Kieselguhr. Suction is applied to accelerate filtration. The precipitate is washed on the filter with a cold ( - - i o ° to --15 °) mixture of a c e t o n e - e t h a n o l - w a t e r (2 : I : I, V/V), made alkaline with a m m o n i a (0. 5 % N H 3 of o.88 specific gravity}. During the filtration and the washing step the temperature of the cold-room is kept at - - 2 ° to --5 ° . The washing is discontinued when the filtrate is almost colorless. The precipitate is then washed on the filter with o.66 saturated (NHa) ~SO~ brought to p H 8 with ammonia. (NH~)~SO 4 removes variable amounts of reddish pigments, including ferredoxin. The filter cake is then removed, and resuspended in o.5 saturated (NH4)2SO 4 of p H 8.o. The suspension is again filtered, and the filtrate is discarded. Cytochrome f is then eluted from the filter cake with 0.06 M Biochim. Biophys. Acta, lO9 (1965) 33-4o
CYTOCHrOME / FRO~t P A R S L E Y
35
Na~HP04. Elution is continued until an appreciable amount of cytochrome f is present in the filtrate. Rather large volumes of eluent are required, probably owing to the adsorption of cytoch'rome by materials on the filter. Up to this step, the procedure is identical to that described by DAVENPORT A N D H I L L s , except for the addition of Triton X-ioo to the extraction mixture. This addition is essential in order to obtain a good yield of cytochrome f by means of Waring-blendor extraction. The cytochrome f extracted with Na2HPO 4 (approx. 15oo-2ooo ml, extraction of 8 kg leaves, Fraction I) is precipitated by slow addition of saturated (NH4)2SO 4 (pH 8) until the concentration of the salt corresponds to 0.45 saturation. The precipitated cytochrome is allowed to sediment overnight at 0% and the precipitate is collected by centrifugation in the cold. The precipitate is redissolved in 0.05 M Tris buffer (pH 8) and small amounts of insoluble materials are separated by centrifugation and discarded. The (NH4)~SO 4 fraction (Fraction II) is now subjected to a second (NH4)2SO 4 fractionation by addition of saturated (NH4)zSO 4 of pH 8. The fraction sedimented between 20 % and 45 % saturation (Fraction III) contains most of the cytochrome f, rendered free of most of the diaphorase present in Fraction I (see Table I). The diaphorase has properties very similar to those of spinach flavoprotein endowed with NADPH-cytochrome f reductase activityt°, 11. Considerable amounts of yellow pigments are also removed by the (NH4) 2SO 2 fractionation. Further purification is achieved by column chromatography on the dextran gel Sephadex G-Ioo. Fraction III, dissolved in 0.05 M Tris buffer (pH 8) is applied on a Sephadex G-Ioo column
I
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Fig. I. C h r o m a t o g r a p h y of c y t o c h r o m e ]', F r a c t i o n I I I , o n S e p h a d e x G-ioo. Conditions: o.o5 5I Tris buffer (pH 8.o). F r a c t i o n s of 5 m l collected a u t o m a t i c a l l y ,
Biochim. Biophys. Acta, l o 9 (1965) 33-40
36
G. FORTI, 2¢I. L. BERTOLE, G. ZANETTI
(2.5
X 8o cm) equilibrated with the same buffer. A disc of filter paper (Whatman No. i) is placed on top of the column, and the cytochrome solution (5-6 ml) is allowed to penetrate into the gel. Elution is then started with Tris buffer and 5-ml fractions are automatically collected. Cytochrome f moves ahead of yellow pigments, flavoprotein and other proteins, and emerges from the column with a volume of eluent equal to the void volume of the column. This indicates that the molecular weight is larger than io 5. Some retention is, however, observed as indicated by the dilution of c y t o c h r o m e f i n the eluate. Fig. I illustrates the chromatographic procedure. The best fractions are pooled (Fraction IV), while less purified fractions are concentrated by
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0.80
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Fracfi0n number Fig. 2. C h r o m a t o g r a p h y of c y t o c h r o m e f , F r a c t i o n IV, on S e p h a d e x G-2oo. Conditions as for Fig. i. TABLE I PURIFICATION
OF CYTOCHROME
f
FROM
PARSLEY
Preparation from approx. 7 kg of parsley leaves. Diaphorase activity is measured in Tris buffer (pH 8.9) in the presence of o.5 m M N A D P H and o.04 mlV[ 2,6-dichlorophenolindophenol.
Fraction
Crude o - 5 o ~o 20-45 % G-Ioo G-2oo
1 II III IV V
Protein (mg)
Cytochrome f (ffmoles)
A4~mt*/A~Tsml*
Diaphorase activity (#moles/min)
-640 54 ° 14o 54-3
3.800 3.000 2.800 1.89 ° I.O34
-0.74 0.85 1.98 2.9o
34.2 8.3 2.I o.16 o.oo
Biochim. Biophys. Acts, lO 9 (1965) 3 3 - 4 °
CYTOCI-IROME
f FROM PARSLEY
37
(NH4)~SO 4 precipitation (45 % saturation, p H 8) and subjected to a second Sephadex G-Ioo chromatography. The more purified fractions of the second chromatography are added to Fraction IV, and cytochrome f is again precipitated at 45 % saturation of (NH4) 2S04, at p H 8. (NH4)~SO 4 precipitation achieves now almost complete recovery of the cytochrome, provided the precipitation is allowed to continue for several hours in an ice-bath. The following step is column chromatography on Sephadex G-2oo (see Fig. 2) in 0.05 M Tris buffer (pH 8.0). C y t o c h r o m e f i s partially retained on this gel, as will be discussed later. The best fractions are pooled and concentrated either b y (NH4) ~S04 precipitation or by freeze-drying. The less pure fractions are subjected to a second chromatography on Sephadex G-2oo. Table I summarizes the procedure. As can be seen, 34 % of the c y t o c h r o m e f present in the crude extract is recovered in Fraction V. Higher recoveries are achieved if the less pure fractions from the Sephadex columns are repeatedly pooled and rechromatographed.
Purity tests Cytochrome f obtained in Fraction V migrates as a single peak in the ultracentrifuge. The ultracentrifuge runs were performed in o.I M phosphate buffer (pH 7.1). The sedimentation coefficient value (s20, w) is 6.5 • lO-13, as shown in Fig. 3. This value is very close to the one reported b y DAVENPORT AND HILL2. As a further criterium of purity the eytochrome f of Fraction V was applied to a column (I cm × 8o cm) of Sephadex G-2oo. The pigment appeared in the eluate as a symmetrical peak, and the ratio A422In~/A278 mt~ was constant in all fractions, and equal to 2.8. Molecular weight was determined b y the dextran gel filtration methodlS, 19. Cytochrome f (A422 mt~/A'~Tsmt,= 2.8) dissolved in o.I M phosphate buffer (pH 7.1) was applied to a Sephadex G-2oo column (o.5 cm x 8o cm) equilibrated with the same buffer, and its elution volume was compared with that of pure trypsin, phosphoglueomutase and triose phosphate dehydrogenase (Fig. 4). If the ratio of elution
2.5"t
o
Trypsin
~o 2.0o~luilmutose 7654210 0
o
0
0
O 0
1.5"
:~ 3 4 mg profein/ml
5
4'.1
413
415
4'.7 419 511 I_o(Jmol.wt
513
515
517
Fig. 3. Sedimentation coefficient of cytochrome f. Conditions: see text. Fig. 4. Determination of molecular weight of cytochromef by the gel filtration method. Conditions : o.i M phosphate buffer (pH 7.1). Temperature I5 °.
Biochim. Biophys. Acta, lO9 (1965) 33-4 °
38
G. FORTI, M. L. BERTOLE, G. ZANETTI
volume (V) to void volume of the column (V0) is plotted against the decimal logarithm of the molecular weight according to W H I T A K E R 19, & molecular weight of 245ooo is found for cytochrome f (Fig. 4). The three enzymes used to determine the slope of the line according to WHITAKERm are on a straight line intersecting the abscissa (V/Vo = I) at the point corresponding to a molecular weight of 437 ooo (Fig. 4). This value is in close agreement with the data reported by ANDREWSls.
optical properties The absorption spectrum of pure cytochromef, in the reduced form, is shown in Fig. 5. The spectrum differs from the one published by DAVENPORT AND HILL2 in a 10
20
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40
~o Lo
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. . . .
350
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450
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550
Wavelength (. m/u)
Fig. 5. A b s o r p t i o n s p e c t r u m of r e d u c e d c y t o c h r o m e f. C o n d i t i o n s : 0.05 M Tris buffer (pI-I 8.o); q u a r t z c u v e t t e s of i cm l i g h t - p a t h . TABLE II EXTINCTION
COEFFICIENTS
O l~ C Y T O C H R O M I g f
Redox state
Wavelength (ml,)
Extinction coel~cient*
Reduced Reduced Reduced minus oxidized Reduced minus oxidized
554.5 m i n u s 580 554-5 m i n u s 54 ° 554.5 554.5 m i n u s 54 °
26 20 19 22
125 25 ° 68o o5o
* Molar e x t i n c t i o n coefficient in i - c m l i g h t - p a t h c u v e t t e s of c y t o c h r o m e f s o l u t i o n s c o n t a i n i n g I M haematin.
Biochim. Biophys. Acta, lO9 (1965) 33-4 °
CYTOCHROME
f
FROM
PARSLEY
39
small peak at 532.8 m/z, not reported by these autlaors. Furthermore, in the previously described cytochrome f preparation s the shoulder at 402.4 m/~ was not shown. The ratio .4422m~,/A278m ~ reported previously z was 1.92, while pure cytochrome f shows a value of 2.8. Upon oxidation the spectral changes previously reported ~ were confirmed in this laboratory. Table I I summarizes the spectral properties of parsley c y t o c h r o m e f i n the reduced and oxidized form. The figures are referred to haematin concentration. If one assumes a molecular weight of 250 ooo, I molecule of cytochrome f contains 4 haematin groups, all of which are oxidized b y ferricyanide or H20 2 and reduced by a number of reductants, including ascorbate, sodium dithionite and sulphydryl compounds. Enzymatic reduction by N A D P H and the cytochrome f reductase of chloroplasts 1° produces spectral changes identical to those obtained with non-enzymatic reduction. TABLE I[I CYTOCHROM•
f ANALYSIS
Protein (mg) Kalchar's method
Lowry's method
9.69
i o. 14
Dry weight (mg )
Cytochrome f haematin (ttmoles)
Total Fe* (#atom)
9.36
o. 155
o. 16o
* Measured b y the orthophenallthroline method, after wet-ashing 22.
Table I I I shows the analytical data for purified cytochrome f. As can be seen, the determination of protein based on ultraviolet absorption 16 is in close agreement with that obtained by the combination of biuret and Folin reagents ~7and with the dryweight estimation. The absorption spectrum analysis indicates I mole of haematin per 63 ooo g protein, in close agreement with the total iron analysis (Table III). The value of I mole of haematin per 68ooo g of dry weight was previously reported b y DAVENPORT AND HILL2.
Catalase activity Purified cytochrome f possesses catalase (EC 1.11.1.6) activity. The activity, measured according to VON EULER AND JOSEPHSON 2°, corresponds to a Kat. f value of 4-7"1o4, which, expressed ill terms of the first-order reaction constant per mole of haematin per 1 (kl) 21, is equal to 1.14. lO 5. This figure is in close agreement with the value obtained in the rapid spectrophotometric method 21. The catalatic activity of cytochrome f is inhibited b y cyanide and b y 2,4-aminotriazole, as with other wellknown catalases. It is of interest to note t h a t chloroplasts and also washed grana contain a very active catalase, recently considerably purified in this laboratory (FORTI A:,'D ZANET~IS), which is readily separated from cytochrome f. ACKNO~,VLEDGEMENT
This work was supported by Grant GM-o986i-oi/o2 from the U.S. Public Health Service, National Institutes of Health. Biochim. Biophys. Acta, lO9 (1965) 33-4 °
40
G. FORTI, M. L. BERTOLI~, G. ZANETTI
REFERENCES I ~2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22
R. HILL AND R. SCARISBRICK, New Philologist, 5 ° (1951) 98. H. E. DAVENPORT AND R. HILL, Proc. Roy. Soc. London, Ser. B, 139 (1952) 327. J. AMESZ AND L. N. IV[. DUYSENS, Biochim. Biophys. Acta, 64 (1962) 261. B. CHANCE AND W. D. BONNER, ill Photosynthetic Mechanisms of Green Plants, P u b l i c a t i o n 1145, N a t l . Acad. S c i . - N a t l . Res. Council, W a s h i n g t o n , 1963, p. 66. W. D. BONNER AND R. HILL, in Photosynthetic Mechanisms of Green Plants, p. 82. J. ~JI. OLSON AND M. SMILLIE, in Photosynthetic Mechanisms of Green Plants, p. 56. G. FORTI, L. M. BERTOL~ AND B. PARISI, Biochem. Biophys. Res. Commun., i o (1963) 384. G. FORTI AND G. ZANETTI, u n p u b l i s h e d results. B. KOK, H. J. RURAINSKI AND E. A. HARMON, Plant Physiol., 39 (1964) 513 . G. FORTI, IV[. L. BERTOL~ AND B. PARISI, in Photosynthetic Mechanisms of Green Plants, P u b l i c a t i o n 1145, N a t l . Acad. S c i . - N a t I . Res. Council, W a s h i n g t o n , 1963, p. 284. G. ZANETTI AND G. FORTI, J. Biol. Chem., in t h e press. L. N. M. DUYSENS, in Photosynthetic Mechanisms of Green Plants, P u b l i c a t i o n 1145, N a t l . Acad. S c i . - N a t l . Res. Council, W a s h i n g t o n , 1963, p. i. R. HILL AND W. D. BONNER, in W. D. MCELROY AND B. GLASS, Light and Life, The J o h n s H o p k i n s Press, B a l t i m o r e , 1961, p. 424 . S. KATOH, J. Biochem. Tokyo, 46 (1959) 629. J. J. WOLKEN AND J. A. GROSS, J. Protozool., i o (1963) 189. H. IV[. I~ALCKAR, J. Biol. Chem., 167 (1947) 461. O. H. LOWRY, N. J. ROSEBROIJGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . P. ANDREWS, Biochem. J., 91 (1964) 233. J. R. WHITAKER, Anal. Chem., 35 (1963) 195°H. VON EULER AND K. JOSEPHSON, Ann. Chem., 452 (1927) 158. B. CHANCE, in D. GLICK, Methods of Biochemical Analysis, I n t e r s c i e n c e , N e w Y ork, 1954, p- 408. R. BALLENTINE AND D. D. BURFORD, in S. P. COLO~VlCK AND N. O. I~APLAN, 2VIethods in Enzymology, Vol. 3, A c a d e m i c Press, N e w Y o r k , 1957, p. lOO2.
Biochim. Biophys. Acta, lO9 (1965) 33-4 o
BIOCHIMICA ET BIOPI-IYSICA ACTA
BBA 45 215 PURIFICATION AND PROPERTIES OF CYTOCHROME f FROM PARSLEY LEAVES GIORGIO FORTI, MARIA LUISA BERTOL]~ AND GIULIANA ZANETTI
Laboratory of Plant Physiology, Department of Botanical Sciences of the University of Milan (Italy) (Received February I l t h , 1965)
SUMMARY
A method for the extraction and purification of cytochrome f from parsley leaves is described. Cytochrome f is obtained in a high state of purity. A molecular weight of 245 ooo is indicated by Sephadex G-2oo chromatography. The absorption spectrum of pure cytochromefin the reduced state shows peaks at 554.5 m~, 532.8 mr*, 524 m~, 422 mr* and 330 m/~ and a shoulder at 402 mt*. Cytochrome f possesses catalase (EC 1.11.1.6) activity, with a Kat. f value of 4.7" IO~.
INTRODUCTION
Cytochromef, a c-type cytochrome of photosynthetic tissues, was discovered by HILL AND SCARISBRICK1 in 1951. This haem protein, found only in the green tissues of plants 1, has been extensively purified by DAVENPORT AND HILL 2, and its presence demonstrated in chloropast preparations from a variety of plants 2. The participation of cytochrome f in photosynthetic electron transport has been demonstrated by differential spectrophotometry in intact cells~-e and by the light-dependent reduction of substrate amounts of the pigment by isolated chloroplast grana 7. The photooxidation of parsley cytochrome f (ref. 8) and algal f-type cytochrome° by detergenttreated chloroplasts has also been reported. It was shown in this laboratoryv that the photo-reduction of cytochromef by chloroplasts is coupled to phosphorylation. Furthermore, a flavoprotein enzyme of chloroplasts has been shown to be a highly specific NADPH-cytochrome f reductasel°, 11, with high affinity for NADPH and cytochrome f. Based on the above mentioned lines of evidence, a number of hypothetical schemes on the role of cytochrome f in photosynthetic electron transport have been proposed: although these may differ in some details, all of them indicate that cytochrome f mediates electron transport between the two photochemical reactions of photosynthesis4,x2,13, being photo-oxidized by the "pigment system I" and photoreduced, though not directly, by the "pigment system II". Although '~/-type cytochromes" have recently been obtained in pure form from algae14,15, the purification and characterization of cytochromef from higher plants has been limited to the excellent pioneering work of HILL and his collaboratorsl,L It is the purpose of this paper to describe a new method of obtaining reproducibly and with satisfactory yield, pure cytochrome f from parsley leaves. Some of the chemical and physical properties of this haem protein are also described. Biochim. Biophys. Acta, lo9 (1965) 33-4o
34
G. FORTI, M. L. BERTOL~2, G. ZANETTI
METHODS All spectrophotometric measurements were performed in i - c m light-path cuvettes, in a double-beam, recording spectrophotometer (Optica CF4). Protein determination was performed either according to KALCKAR16 or according to LOWRY et al. 17. The preparation of the columns of dextran gels, Sephadex G-ioo and G-2oo (Pharmacia, Uppsala), was performed as described b y ANDREWSls. After the column is filled with gel, the appropriate buffer is allowed to flow t h r o u g h overnight, in the cold-room, to obtain a properly packed gel bed. The concentration of cytochrome f is followed during purification b y measuring the difference in absorbance at 555 m ~ minus 54 ° m/,. The pigment is extracted from leaves in the completely reduced form 2. The ratio A422m/~/A228m# is checked in all fractions as a criterion of purity 2. The ratio increases progressively with the separation of cytochrome from contaminating proteins. RESULTS
Cytochrome f purification The m e t h o d described here has been applied reproducibly, over a period of more t h a n 2 years, to the purification of cytochrome f from parsley leaves. The application of the m e t h o d to other tissues has not been thoroughly investigated. The extraction and the initial steps of the purification are substantially identical to the procedure of DAVENPORT AND HILL2. Fresh parsley leaves {obtained commercially or cultivated in a garden) are depetiolated, and carefully washed with tap water. Excess water is blotted away. Portions of IOO g of leaves are ground in the cold-room in a Waring blendor with IOO ml of ethanol containing i % (v/v) Triton X - I o o (a non-ionic detergent} and I. 5 % (v/v) a m m o n i a {specific g r a v i t y 0.88). The blelldor is operated at full speed for approx. 2 min. The homogenate is then squeezed through cheesecloth, and centrifuged 2o min at approx. 19000 x g in a refrigerated centrifuge at --12 °. Particular care has to be taken to avoid any undue standing of the ethallolic extract at temperatures above o °. The supernatant obtained is clear and yellow-colored. Cytochrome f is then precipitated b y the addition of I . I vol. of cold (--20 °) acetone. During the addition of acetone, the solution is continuously stirred. Kieselguhr is added to aid subsequent filtration. The extract is stored for approx. 1-2 h at --20 ° to allow the sedimentation of the precipitate, a reddish-yellow powder at the b o t t o m of the precipitation vessel. The precipitate is collected b y filtration (at - - 2 ° to - - 5 ° ) on W h a t m a n No. I paper, covered with a very thin layer of Kieselguhr. Suction is applied to accelerate filtration. The precipitate is washed on the filter with a cold ( - - i o ° to --15 °) mixture of a c e t o n e - e t h a n o l - w a t e r (2 : I : I, V/V), made alkaline with a m m o n i a (0. 5 % N H 3 of o.88 specific gravity}. During the filtration and the washing step the temperature of the cold-room is kept at - - 2 ° to --5 ° . The washing is discontinued when the filtrate is almost colorless. The precipitate is then washed on the filter with o.66 saturated (NHa) ~SO~ brought to p H 8 with ammonia. (NH~)~SO 4 removes variable amounts of reddish pigments, including ferredoxin. The filter cake is then removed, and resuspended in o.5 saturated (NH4)2SO 4 of p H 8.o. The suspension is again filtered, and the filtrate is discarded. Cytochrome f is then eluted from the filter cake with 0.06 M Biochim. Biophys. Acta, lO9 (1965) 33-4o
CYTOCHrOME / FRO~t P A R S L E Y
35
Na~HP04. Elution is continued until an appreciable amount of cytochrome f is present in the filtrate. Rather large volumes of eluent are required, probably owing to the adsorption of cytoch'rome by materials on the filter. Up to this step, the procedure is identical to that described by DAVENPORT A N D H I L L s , except for the addition of Triton X-ioo to the extraction mixture. This addition is essential in order to obtain a good yield of cytochrome f by means of Waring-blendor extraction. The cytochrome f extracted with Na2HPO 4 (approx. 15oo-2ooo ml, extraction of 8 kg leaves, Fraction I) is precipitated by slow addition of saturated (NH4)2SO 4 (pH 8) until the concentration of the salt corresponds to 0.45 saturation. The precipitated cytochrome is allowed to sediment overnight at 0% and the precipitate is collected by centrifugation in the cold. The precipitate is redissolved in 0.05 M Tris buffer (pH 8) and small amounts of insoluble materials are separated by centrifugation and discarded. The (NH4)~SO 4 fraction (Fraction II) is now subjected to a second (NH4)2SO 4 fractionation by addition of saturated (NH4)zSO 4 of pH 8. The fraction sedimented between 20 % and 45 % saturation (Fraction III) contains most of the cytochrome f, rendered free of most of the diaphorase present in Fraction I (see Table I). The diaphorase has properties very similar to those of spinach flavoprotein endowed with NADPH-cytochrome f reductase activityt°, 11. Considerable amounts of yellow pigments are also removed by the (NH4) 2SO 2 fractionation. Further purification is achieved by column chromatography on the dextran gel Sephadex G-Ioo. Fraction III, dissolved in 0.05 M Tris buffer (pH 8) is applied on a Sephadex G-Ioo column
I
: i
8.o~
!
~90
7.0~i
6°I
\
I
3
[
I 7°
o 5.0~ o
i 100
i
oj
/i
o
~-
%
,
4.0'
-2
II..'
60-
50 .o, 40
t
o
2.0 /
3O
1
%
\
20 10
1"01 3~0
3~5 4'0 4'5 Fraction number
EP
50
•
P
Fig. I. C h r o m a t o g r a p h y of c y t o c h r o m e ]', F r a c t i o n I I I , o n S e p h a d e x G-ioo. Conditions: o.o5 5I Tris buffer (pH 8.o). F r a c t i o n s of 5 m l collected a u t o m a t i c a l l y ,
Biochim. Biophys. Acta, l o 9 (1965) 33-40
36
G. FORTI, 2¢I. L. BERTOLE, G. ZANETTI
(2.5
X 8o cm) equilibrated with the same buffer. A disc of filter paper (Whatman No. i) is placed on top of the column, and the cytochrome solution (5-6 ml) is allowed to penetrate into the gel. Elution is then started with Tris buffer and 5-ml fractions are automatically collected. Cytochrome f moves ahead of yellow pigments, flavoprotein and other proteins, and emerges from the column with a volume of eluent equal to the void volume of the column. This indicates that the molecular weight is larger than io 5. Some retention is, however, observed as indicated by the dilution of c y t o c h r o m e f i n the eluate. Fig. I illustrates the chromatographic procedure. The best fractions are pooled (Fraction IV), while less purified fractions are concentrated by
o/o- °Xo\
1.00[
!
0,90 0.80 /o
070
/
0.80
o 0.40 0.30 -I
/
\
\ 20
o..,°
Z
:
4
/
'0 0.50 -
~
/
/o
/
\
/
\
',
\
b
?i 2t / /
10,~,
"i
:/
/
/ t
o 0.20-!
A, .\
,.
f"" fl;//
I
/- -
0,10 1 I
o
i / L_z 25
15
5
~" Y"
,' ..," ,
.
4-" .... 30
-1 r
40
35
45
5'0
1
~P
55
Fracfi0n number Fig. 2. C h r o m a t o g r a p h y of c y t o c h r o m e f , F r a c t i o n IV, on S e p h a d e x G-2oo. Conditions as for Fig. i. TABLE I PURIFICATION
OF CYTOCHROME
f
FROM
PARSLEY
Preparation from approx. 7 kg of parsley leaves. Diaphorase activity is measured in Tris buffer (pH 8.9) in the presence of o.5 m M N A D P H and o.04 mlV[ 2,6-dichlorophenolindophenol.
Fraction
Crude o - 5 o ~o 20-45 % G-Ioo G-2oo
1 II III IV V
Protein (mg)
Cytochrome f (ffmoles)
A4~mt*/A~Tsml*
Diaphorase activity (#moles/min)
-640 54 ° 14o 54-3
3.800 3.000 2.800 1.89 ° I.O34
-0.74 0.85 1.98 2.9o
34.2 8.3 2.I o.16 o.oo
Biochim. Biophys. Acts, lO 9 (1965) 3 3 - 4 °
CYTOCI-IROME
f FROM PARSLEY
37
(NH4)~SO 4 precipitation (45 % saturation, p H 8) and subjected to a second Sephadex G-Ioo chromatography. The more purified fractions of the second chromatography are added to Fraction IV, and cytochrome f is again precipitated at 45 % saturation of (NH4) 2S04, at p H 8. (NH4)~SO 4 precipitation achieves now almost complete recovery of the cytochrome, provided the precipitation is allowed to continue for several hours in an ice-bath. The following step is column chromatography on Sephadex G-2oo (see Fig. 2) in 0.05 M Tris buffer (pH 8.0). C y t o c h r o m e f i s partially retained on this gel, as will be discussed later. The best fractions are pooled and concentrated either b y (NH4) ~S04 precipitation or by freeze-drying. The less pure fractions are subjected to a second chromatography on Sephadex G-2oo. Table I summarizes the procedure. As can be seen, 34 % of the c y t o c h r o m e f present in the crude extract is recovered in Fraction V. Higher recoveries are achieved if the less pure fractions from the Sephadex columns are repeatedly pooled and rechromatographed.
Purity tests Cytochrome f obtained in Fraction V migrates as a single peak in the ultracentrifuge. The ultracentrifuge runs were performed in o.I M phosphate buffer (pH 7.1). The sedimentation coefficient value (s20, w) is 6.5 • lO-13, as shown in Fig. 3. This value is very close to the one reported b y DAVENPORT AND HILL2. As a further criterium of purity the eytochrome f of Fraction V was applied to a column (I cm × 8o cm) of Sephadex G-2oo. The pigment appeared in the eluate as a symmetrical peak, and the ratio A422In~/A278 mt~ was constant in all fractions, and equal to 2.8. Molecular weight was determined b y the dextran gel filtration methodlS, 19. Cytochrome f (A422 mt~/A'~Tsmt,= 2.8) dissolved in o.I M phosphate buffer (pH 7.1) was applied to a Sephadex G-2oo column (o.5 cm x 8o cm) equilibrated with the same buffer, and its elution volume was compared with that of pure trypsin, phosphoglueomutase and triose phosphate dehydrogenase (Fig. 4). If the ratio of elution
2.5"t
o
Trypsin
~o 2.0o~luilmutose 7654210 0
o
0
0
O 0
1.5"
:~ 3 4 mg profein/ml
5
4'.1
413
415
4'.7 419 511 I_o(Jmol.wt
513
515
517
Fig. 3. Sedimentation coefficient of cytochrome f. Conditions: see text. Fig. 4. Determination of molecular weight of cytochromef by the gel filtration method. Conditions : o.i M phosphate buffer (pH 7.1). Temperature I5 °.
Biochim. Biophys. Acta, lO9 (1965) 33-4 °
38
G. FORTI, M. L. BERTOLE, G. ZANETTI
volume (V) to void volume of the column (V0) is plotted against the decimal logarithm of the molecular weight according to W H I T A K E R 19, & molecular weight of 245ooo is found for cytochrome f (Fig. 4). The three enzymes used to determine the slope of the line according to WHITAKERm are on a straight line intersecting the abscissa (V/Vo = I) at the point corresponding to a molecular weight of 437 ooo (Fig. 4). This value is in close agreement with the data reported by ANDREWSls.
optical properties The absorption spectrum of pure cytochromef, in the reduced form, is shown in Fig. 5. The spectrum differs from the one published by DAVENPORT AND HILL2 in a 10
20
30
g
40
~o Lo
g
50
o~ 70
80-
90-
1 0 0
,
,
260
,~
. . . .
300
i
. . . .
350
~
J
400
450
. . . .
J
. . . .
500
~
,
,
'
550
Wavelength (. m/u)
Fig. 5. A b s o r p t i o n s p e c t r u m of r e d u c e d c y t o c h r o m e f. C o n d i t i o n s : 0.05 M Tris buffer (pI-I 8.o); q u a r t z c u v e t t e s of i cm l i g h t - p a t h . TABLE II EXTINCTION
COEFFICIENTS
O l~ C Y T O C H R O M I g f
Redox state
Wavelength (ml,)
Extinction coel~cient*
Reduced Reduced Reduced minus oxidized Reduced minus oxidized
554.5 m i n u s 580 554-5 m i n u s 54 ° 554.5 554.5 m i n u s 54 °
26 20 19 22
125 25 ° 68o o5o
* Molar e x t i n c t i o n coefficient in i - c m l i g h t - p a t h c u v e t t e s of c y t o c h r o m e f s o l u t i o n s c o n t a i n i n g I M haematin.
Biochim. Biophys. Acta, lO9 (1965) 33-4 °
CYTOCHROME
f
FROM
PARSLEY
39
small peak at 532.8 m/z, not reported by these autlaors. Furthermore, in the previously described cytochrome f preparation s the shoulder at 402.4 m/~ was not shown. The ratio .4422m~,/A278m ~ reported previously z was 1.92, while pure cytochrome f shows a value of 2.8. Upon oxidation the spectral changes previously reported ~ were confirmed in this laboratory. Table I I summarizes the spectral properties of parsley c y t o c h r o m e f i n the reduced and oxidized form. The figures are referred to haematin concentration. If one assumes a molecular weight of 250 ooo, I molecule of cytochrome f contains 4 haematin groups, all of which are oxidized b y ferricyanide or H20 2 and reduced by a number of reductants, including ascorbate, sodium dithionite and sulphydryl compounds. Enzymatic reduction by N A D P H and the cytochrome f reductase of chloroplasts 1° produces spectral changes identical to those obtained with non-enzymatic reduction. TABLE I[I CYTOCHROM•
f ANALYSIS
Protein (mg) Kalchar's method
Lowry's method
9.69
i o. 14
Dry weight (mg )
Cytochrome f haematin (ttmoles)
Total Fe* (#atom)
9.36
o. 155
o. 16o
* Measured b y the orthophenallthroline method, after wet-ashing 22.
Table I I I shows the analytical data for purified cytochrome f. As can be seen, the determination of protein based on ultraviolet absorption 16 is in close agreement with that obtained by the combination of biuret and Folin reagents ~7and with the dryweight estimation. The absorption spectrum analysis indicates I mole of haematin per 63 ooo g protein, in close agreement with the total iron analysis (Table III). The value of I mole of haematin per 68ooo g of dry weight was previously reported b y DAVENPORT AND HILL2.
Catalase activity Purified cytochrome f possesses catalase (EC 1.11.1.6) activity. The activity, measured according to VON EULER AND JOSEPHSON 2°, corresponds to a Kat. f value of 4-7"1o4, which, expressed ill terms of the first-order reaction constant per mole of haematin per 1 (kl) 21, is equal to 1.14. lO 5. This figure is in close agreement with the value obtained in the rapid spectrophotometric method 21. The catalatic activity of cytochrome f is inhibited b y cyanide and b y 2,4-aminotriazole, as with other wellknown catalases. It is of interest to note t h a t chloroplasts and also washed grana contain a very active catalase, recently considerably purified in this laboratory (FORTI A:,'D ZANET~IS), which is readily separated from cytochrome f. ACKNO~,VLEDGEMENT
This work was supported by Grant GM-o986i-oi/o2 from the U.S. Public Health Service, National Institutes of Health. Biochim. Biophys. Acta, lO9 (1965) 33-4 °
40
G. FORTI, M. L. BERTOLI~, G. ZANETTI
REFERENCES I ~2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22
R. HILL AND R. SCARISBRICK, New Philologist, 5 ° (1951) 98. H. E. DAVENPORT AND R. HILL, Proc. Roy. Soc. London, Ser. B, 139 (1952) 327. J. AMESZ AND L. N. IV[. DUYSENS, Biochim. Biophys. Acta, 64 (1962) 261. B. CHANCE AND W. D. BONNER, ill Photosynthetic Mechanisms of Green Plants, P u b l i c a t i o n 1145, N a t l . Acad. S c i . - N a t l . Res. Council, W a s h i n g t o n , 1963, p. 66. W. D. BONNER AND R. HILL, in Photosynthetic Mechanisms of Green Plants, p. 82. J. ~JI. OLSON AND M. SMILLIE, in Photosynthetic Mechanisms of Green Plants, p. 56. G. FORTI, L. M. BERTOL~ AND B. PARISI, Biochem. Biophys. Res. Commun., i o (1963) 384. G. FORTI AND G. ZANETTI, u n p u b l i s h e d results. B. KOK, H. J. RURAINSKI AND E. A. HARMON, Plant Physiol., 39 (1964) 513 . G. FORTI, IV[. L. BERTOL~ AND B. PARISI, in Photosynthetic Mechanisms of Green Plants, P u b l i c a t i o n 1145, N a t l . Acad. S c i . - N a t I . Res. Council, W a s h i n g t o n , 1963, p. 284. G. ZANETTI AND G. FORTI, J. Biol. Chem., in t h e press. L. N. M. DUYSENS, in Photosynthetic Mechanisms of Green Plants, P u b l i c a t i o n 1145, N a t l . Acad. S c i . - N a t l . Res. Council, W a s h i n g t o n , 1963, p. i. R. HILL AND W. D. BONNER, in W. D. MCELROY AND B. GLASS, Light and Life, The J o h n s H o p k i n s Press, B a l t i m o r e , 1961, p. 424 . S. KATOH, J. Biochem. Tokyo, 46 (1959) 629. J. J. WOLKEN AND J. A. GROSS, J. Protozool., i o (1963) 189. H. IV[. I~ALCKAR, J. Biol. Chem., 167 (1947) 461. O. H. LOWRY, N. J. ROSEBROIJGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . P. ANDREWS, Biochem. J., 91 (1964) 233. J. R. WHITAKER, Anal. Chem., 35 (1963) 195°H. VON EULER AND K. JOSEPHSON, Ann. Chem., 452 (1927) 158. B. CHANCE, in D. GLICK, Methods of Biochemical Analysis, I n t e r s c i e n c e , N e w Y ork, 1954, p- 408. R. BALLENTINE AND D. D. BURFORD, in S. P. COLO~VlCK AND N. O. I~APLAN, 2VIethods in Enzymology, Vol. 3, A c a d e m i c Press, N e w Y o r k , 1957, p. lOO2.
Biochim. Biophys. Acta, lO9 (1965) 33-4 o