Photosynthesis Research 28: 89-93, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.
Technical communication
Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography Hans E.M. Christensen, Lars S. Conrad & Jens Ulstrup
Chemistry Department A, Building 207, The Technical University of Denmark, DK-2800 Lyngby, Denmark Received 9 October 1990; accepted in revised form 29 April 1991
Key words: capillary electrophoresis, Phenyl-Sepharose, plastocyanin, purification, spinach, Q Sepharose High Performance Abstract
A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio m278/m597of 1.10 +-0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.
Abbreviations: MES - 2(N-morpholino)ethanesulfonic acid; Tris - Tris(hydroxymethyl)aminomethane; FSCE - free solution capillary electrophoresis Introduction
Plastocyanin, a 'blue' single-copper metalloprotein with a molecular mass of 10500, is involved in photosynthetic electron transfer in higher plants and in some algae and bacteria. It transports electrons between the membrane-bound cytochrome f and photosystem I (Haehnel 1984). During the last decade, plastocyanin has been extensively studied, in particular with respect to its electron transfer properties having become a 'model' metalloprotein in biological electron transfer investigations (McArdle et al. 1977, Brunschwig et al. 1985, Skyes 1985, Adman 1985, Pladziewich and Brenner 1987, Jackman et al. 1988, Farver and Pecht 1989, Christensen et al. 1990). Since plastocyanin cannot be obtained commercially, it is important that fast and convenient procedures for obtaining this protein are available.
Isolation and purification procedures for spinach plastocyanin described in the literature are laborious and either based on organic solvent extraction (Borchert and Wessels 1970, Davis and San Pietro 1979, Ellefson et al. 1980) or the use of fresh spinach leaves (Matthijs et al. 1987, Yocum 1982). We present here a fast and convenient procedure for isolation and purification of plastocyanin from spinach leaves which have been stored in the frozen state and where use of organic solvents has been avoided. This preparative procedure is based on batch adsorption of plastocyanin on ion-exchange resin and (NH4)2SO4-precipitation , followed by purification using only a single hydrophobic interaction and a single ion-exchange column. Pure plastocyanin in high yields is obtained. The homogeneity of purified plastocyanin is for the first time analyzed by free solution capillary electrophoresis (FSCE).
90 Materials and methods
Sources
two batches of DE-23 were separated from cell particulate material by washing with 20mM phosphate buffer, pH 7.2, until the supernatant was only faintly coloured.
Spinach (Spinacea oleracea) was obtained from the local fruit and vegetable market. DEAEcellulose DE-23 was obtained from Whatman and pre-treated in accordance with the manufacturer's recommendations. Phenyl-Sepharose CI4B column material and the HiLoad 16/10 Q Sepharose High Performance column were obtained from Pharmacia. All chemicals were of analytical grade.
3. Elution of ptastocyanin. The DE-23 batches were packed in two 4.5 cm diameter columns. Plastocyanin was eluted with 20 mM phosphate buffer, pH 7.2, 0.2 M NaCI. Plastocyanin in the eluate was detected by a blue/green colour on addition of hexacyanoferrate(III).
Storage of spinach Spinach leaves were deveined, washed, drained and stored below - 2 5 ° C . Spinach has been stored in this way for eight months with no reduced yields of plastocyanin using the isolation and purification procedures described below.
Isolation of plastocyanin All procedures were carried out at 2-4 °C. The following isolation and purification procedure is scaled to 15 kg of fresh spinach giving approximately 12 kg of washed spinach leaves. The spinach leaves were thawed overnight at 2-4 °C.
1. Crude extract. One kilogram portions of leaves in two litres of cold 10 mM phosphate buffer, pH 7.2, were ground for 30 s in a Waring blender (CB-6, four-litre capacity) at medium speed. The homogenate was filtered by use of a press through a heavy cloth sack to remove debris. The resulting dark green aqueous extract was subsequently filtered through two layers of fine cotton mesh. 2. Batch adsorption of proteins. 400 ml of DE23 ion-exchange resin, equilibrated with 20 mM phosphate buffer, pH7.2, was added to the aqueous extract and stirred for an hour. The suspension was allowed to stand for half an hour after which the supernatant was easily decanted (or siphoned) off. Another batch of 400 ml DE23 ion-exchange resin was added to the supernarant and the procedure repeated as before. The
4. (NH4)2SO4-precipitation. All fractions containing plastocyanin were pooled and brought to 75% saturation with (NH4)2SO 4 (516 g per liter solution). After one hour of gentle stirring, the preparation can conveniently be left overnight. The mixture was then centrifuged for 10 min at 10 000 g and the precipitate discarded. Purification of plastocyanin Steps 5 and 6 were carried out at 2-4 °C and step 7 at room temperature.
5. Phenyl-Sepharose Cl-4B
chromatography.
Plastocyanin is easily recognized on the column if a small fraction is oxidized. A small amount of hexacyanoferrate(III) was therefore added to the supernatant from the (NH4)2SO4-precipitation. The supernatant containing the plastocyanin was loaded on a Phenyl-Sepharose C1-4B column (2.6 cm x 16 cm), equilibrated with 20 mM phosphate buffer, pH7.2, 2.0M (NH4)2SO 4. After loading, the column was washed with one column volume of 20 mM phosphate buffer, pH 7.2, 2.0M (NH4)zSO 4 and the plastocyanin eluted with 20mM phosphate buffer, pH7.2, 1.0M (NH4)2SO 4. The flow rate was 2.3 ml min -1.
6. Buffer change. The plastocyanin in the eluate from the hydrophobic interaction column was reduced by addition of a five-fold molar excess of sodium ascorbate, and the buffer was changed to 20mM Tris/HC1 buffer, p H 7 . 5 by diafiltration (Amicon model 8200, YM5 membrane). 7. Q Sepharose High Performance chromatography. A HiLoad 16/10 Q Sepharose High Performance column equilibrated with 20 mM Tris/
91 HCI buffer, pH 7.5, was loaded with up to 25 mg reduced plastocyanin. Pure plastocyanin was eluted with a flow rate of 2.0ml min -~ using a 600 ml gradient from 16 to 40% buffer B. (Buffer A: 20mM Tris/HCl, pH7.5. Buffer B: 20 mM Tris/HCl, pH 7.5, 0.5 M NaC1). The reduced form was then oxidized with hexacyanoferrate(III). The concentration of plastocyanin was calculated using 6597 4500 M-1 cm-i for the oxidized protein (Sykes 1985).
buffer. These compounds are removed in the buffer change step during the purification. The blue/green colour observed on addition of hexacyanoferrate(III) to the eluate from the DE23 columns is not stable unless extensive amounts of hexacyanoferrate(III) are added. The yield of plastocyanin after the ( N H 4 ) 2 S O 4p r e c i p i t a t i o n is overestimated due to impurities absorbing in the 600 nm region (Table 1).
Free solution capillary electrophoresis
The large-scale purification of plastocyanin described has been developed and optimized using a Pharmacia FPLC-setup equipped with Mono Q HR 5/5 and Phenyl Superose 12 HR 5/5 columns. The hydrophobic interaction column has proved convenient for concentration of the plastocyanin after the (NH4)zSO4-precipitation. Upon reducing the (NH4)2SO 4 concentration, remaining ferredoxins are eluted ahead of the plastocyanin due to the gel-properties of the Phenyl-Separose C1-4B gel. The binding strength of proteins of hydrophobic interaction columns depends strongly on temperature. It is therefore necessary to decrease the (NH4)2SO 4 concentration if the column is run at higher temperatures. Plastocyanin was purified on the Q Sepharose High Performance column in the reduced state to obtain optimal separation from impurities still present (see Fig. 1). Figure 1 shows a HiLoad 16/10 Q Sepharose High Performance column chromatogram of reduced plastocyanin. Pure plastocyanin with an absorbance ratio Azv8/A597 of 1.10 + 0.02 in the oxidized state was obtained. With the loading
=
The homogeneity of the purified plastocyanin was evaluated by free solution capillary electrophoresis (Karger et al. 1989), using an Applied Biosystems Model 270A analytical capillary electrophoresis system. The column was a fused silica capillary (Polymicro Technologies, Phoenix, AZ, USA) with a total length of 75 cm and an internal diameter of 50/zm with 50 cm to the detector and the running buffer was 25 mM MES, pH 5.5 Samples of 0.3mg plastocyanin per ml in 10mM MES, pH5.5, were introduced by a 17 kPa vacuum for 0.5 s. Sample 'stacking' for enhanced resolution was performed at 2 kV for 5 min (A. Vinther, personal communication), and the electropherograms were recorded at 200 nm and 30 °C using a 20 000 kV potential.
Results and discussion
Isolation of plastocyanin The breakdown of cell walls was initiated by the freeze-thaw cycle (Schmitt et al. 1985) and aided by lysis of the cells in the low ionic strength phosphate buffer during the grinding in the blender. During the batch adsorption some cell fragments aggregate. Unfortunately this excludes filtration on sintered glass by suction, and the ion-exchange resin was therefore separated by siphoning. When the ion-exchange resin is separated from the debris some faintly yellowcoloured compounds are still released into the
Purification of plastocyanin
Table 1. Purification of spinach plastocyanin Step
Volume ml
Plastocyanin yields, mg
Abs. ratio A278/A597
1. Crude extract 3. DE-23 columns 4. (NH4)2SO 4precipitation 5. Phenyl-Sepharose chromatography 7. Q Sepharose H.P. chromatography
24 000 375
-
-
430
120
>10
140
100
3
98
1.10
92
75
0.15
5C
0.10
I~0 I'2 I'4 m
°
Technical communication
Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography Hans E.M. Christensen, Lars S. Conrad & Jens Ulstrup
Chemistry Department A, Building 207, The Technical University of Denmark, DK-2800 Lyngby, Denmark Received 9 October 1990; accepted in revised form 29 April 1991
Key words: capillary electrophoresis, Phenyl-Sepharose, plastocyanin, purification, spinach, Q Sepharose High Performance Abstract
A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio m278/m597of 1.10 +-0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.
Abbreviations: MES - 2(N-morpholino)ethanesulfonic acid; Tris - Tris(hydroxymethyl)aminomethane; FSCE - free solution capillary electrophoresis Introduction
Plastocyanin, a 'blue' single-copper metalloprotein with a molecular mass of 10500, is involved in photosynthetic electron transfer in higher plants and in some algae and bacteria. It transports electrons between the membrane-bound cytochrome f and photosystem I (Haehnel 1984). During the last decade, plastocyanin has been extensively studied, in particular with respect to its electron transfer properties having become a 'model' metalloprotein in biological electron transfer investigations (McArdle et al. 1977, Brunschwig et al. 1985, Skyes 1985, Adman 1985, Pladziewich and Brenner 1987, Jackman et al. 1988, Farver and Pecht 1989, Christensen et al. 1990). Since plastocyanin cannot be obtained commercially, it is important that fast and convenient procedures for obtaining this protein are available.
Isolation and purification procedures for spinach plastocyanin described in the literature are laborious and either based on organic solvent extraction (Borchert and Wessels 1970, Davis and San Pietro 1979, Ellefson et al. 1980) or the use of fresh spinach leaves (Matthijs et al. 1987, Yocum 1982). We present here a fast and convenient procedure for isolation and purification of plastocyanin from spinach leaves which have been stored in the frozen state and where use of organic solvents has been avoided. This preparative procedure is based on batch adsorption of plastocyanin on ion-exchange resin and (NH4)2SO4-precipitation , followed by purification using only a single hydrophobic interaction and a single ion-exchange column. Pure plastocyanin in high yields is obtained. The homogeneity of purified plastocyanin is for the first time analyzed by free solution capillary electrophoresis (FSCE).
90 Materials and methods
Sources
two batches of DE-23 were separated from cell particulate material by washing with 20mM phosphate buffer, pH 7.2, until the supernatant was only faintly coloured.
Spinach (Spinacea oleracea) was obtained from the local fruit and vegetable market. DEAEcellulose DE-23 was obtained from Whatman and pre-treated in accordance with the manufacturer's recommendations. Phenyl-Sepharose CI4B column material and the HiLoad 16/10 Q Sepharose High Performance column were obtained from Pharmacia. All chemicals were of analytical grade.
3. Elution of ptastocyanin. The DE-23 batches were packed in two 4.5 cm diameter columns. Plastocyanin was eluted with 20 mM phosphate buffer, pH 7.2, 0.2 M NaCI. Plastocyanin in the eluate was detected by a blue/green colour on addition of hexacyanoferrate(III).
Storage of spinach Spinach leaves were deveined, washed, drained and stored below - 2 5 ° C . Spinach has been stored in this way for eight months with no reduced yields of plastocyanin using the isolation and purification procedures described below.
Isolation of plastocyanin All procedures were carried out at 2-4 °C. The following isolation and purification procedure is scaled to 15 kg of fresh spinach giving approximately 12 kg of washed spinach leaves. The spinach leaves were thawed overnight at 2-4 °C.
1. Crude extract. One kilogram portions of leaves in two litres of cold 10 mM phosphate buffer, pH 7.2, were ground for 30 s in a Waring blender (CB-6, four-litre capacity) at medium speed. The homogenate was filtered by use of a press through a heavy cloth sack to remove debris. The resulting dark green aqueous extract was subsequently filtered through two layers of fine cotton mesh. 2. Batch adsorption of proteins. 400 ml of DE23 ion-exchange resin, equilibrated with 20 mM phosphate buffer, pH7.2, was added to the aqueous extract and stirred for an hour. The suspension was allowed to stand for half an hour after which the supernatant was easily decanted (or siphoned) off. Another batch of 400 ml DE23 ion-exchange resin was added to the supernarant and the procedure repeated as before. The
4. (NH4)2SO4-precipitation. All fractions containing plastocyanin were pooled and brought to 75% saturation with (NH4)2SO 4 (516 g per liter solution). After one hour of gentle stirring, the preparation can conveniently be left overnight. The mixture was then centrifuged for 10 min at 10 000 g and the precipitate discarded. Purification of plastocyanin Steps 5 and 6 were carried out at 2-4 °C and step 7 at room temperature.
5. Phenyl-Sepharose Cl-4B
chromatography.
Plastocyanin is easily recognized on the column if a small fraction is oxidized. A small amount of hexacyanoferrate(III) was therefore added to the supernatant from the (NH4)2SO4-precipitation. The supernatant containing the plastocyanin was loaded on a Phenyl-Sepharose C1-4B column (2.6 cm x 16 cm), equilibrated with 20 mM phosphate buffer, pH7.2, 2.0M (NH4)2SO 4. After loading, the column was washed with one column volume of 20 mM phosphate buffer, pH 7.2, 2.0M (NH4)zSO 4 and the plastocyanin eluted with 20mM phosphate buffer, pH7.2, 1.0M (NH4)2SO 4. The flow rate was 2.3 ml min -1.
6. Buffer change. The plastocyanin in the eluate from the hydrophobic interaction column was reduced by addition of a five-fold molar excess of sodium ascorbate, and the buffer was changed to 20mM Tris/HC1 buffer, p H 7 . 5 by diafiltration (Amicon model 8200, YM5 membrane). 7. Q Sepharose High Performance chromatography. A HiLoad 16/10 Q Sepharose High Performance column equilibrated with 20 mM Tris/
91 HCI buffer, pH 7.5, was loaded with up to 25 mg reduced plastocyanin. Pure plastocyanin was eluted with a flow rate of 2.0ml min -~ using a 600 ml gradient from 16 to 40% buffer B. (Buffer A: 20mM Tris/HCl, pH7.5. Buffer B: 20 mM Tris/HCl, pH 7.5, 0.5 M NaC1). The reduced form was then oxidized with hexacyanoferrate(III). The concentration of plastocyanin was calculated using 6597 4500 M-1 cm-i for the oxidized protein (Sykes 1985).
buffer. These compounds are removed in the buffer change step during the purification. The blue/green colour observed on addition of hexacyanoferrate(III) to the eluate from the DE23 columns is not stable unless extensive amounts of hexacyanoferrate(III) are added. The yield of plastocyanin after the ( N H 4 ) 2 S O 4p r e c i p i t a t i o n is overestimated due to impurities absorbing in the 600 nm region (Table 1).
Free solution capillary electrophoresis
The large-scale purification of plastocyanin described has been developed and optimized using a Pharmacia FPLC-setup equipped with Mono Q HR 5/5 and Phenyl Superose 12 HR 5/5 columns. The hydrophobic interaction column has proved convenient for concentration of the plastocyanin after the (NH4)zSO4-precipitation. Upon reducing the (NH4)2SO 4 concentration, remaining ferredoxins are eluted ahead of the plastocyanin due to the gel-properties of the Phenyl-Separose C1-4B gel. The binding strength of proteins of hydrophobic interaction columns depends strongly on temperature. It is therefore necessary to decrease the (NH4)2SO 4 concentration if the column is run at higher temperatures. Plastocyanin was purified on the Q Sepharose High Performance column in the reduced state to obtain optimal separation from impurities still present (see Fig. 1). Figure 1 shows a HiLoad 16/10 Q Sepharose High Performance column chromatogram of reduced plastocyanin. Pure plastocyanin with an absorbance ratio Azv8/A597 of 1.10 + 0.02 in the oxidized state was obtained. With the loading
=
The homogeneity of the purified plastocyanin was evaluated by free solution capillary electrophoresis (Karger et al. 1989), using an Applied Biosystems Model 270A analytical capillary electrophoresis system. The column was a fused silica capillary (Polymicro Technologies, Phoenix, AZ, USA) with a total length of 75 cm and an internal diameter of 50/zm with 50 cm to the detector and the running buffer was 25 mM MES, pH 5.5 Samples of 0.3mg plastocyanin per ml in 10mM MES, pH5.5, were introduced by a 17 kPa vacuum for 0.5 s. Sample 'stacking' for enhanced resolution was performed at 2 kV for 5 min (A. Vinther, personal communication), and the electropherograms were recorded at 200 nm and 30 °C using a 20 000 kV potential.
Results and discussion
Isolation of plastocyanin The breakdown of cell walls was initiated by the freeze-thaw cycle (Schmitt et al. 1985) and aided by lysis of the cells in the low ionic strength phosphate buffer during the grinding in the blender. During the batch adsorption some cell fragments aggregate. Unfortunately this excludes filtration on sintered glass by suction, and the ion-exchange resin was therefore separated by siphoning. When the ion-exchange resin is separated from the debris some faintly yellowcoloured compounds are still released into the
Purification of plastocyanin
Table 1. Purification of spinach plastocyanin Step
Volume ml
Plastocyanin yields, mg
Abs. ratio A278/A597
1. Crude extract 3. DE-23 columns 4. (NH4)2SO 4precipitation 5. Phenyl-Sepharose chromatography 7. Q Sepharose H.P. chromatography
24 000 375
-
-
430
120
>10
140
100
3
98
1.10
92
75
0.15
5C
0.10
I~0 I'2 I'4 m
°
