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Characterization of ferredoxins on a nanomole scale
ANALYTICAL
BIOCHEMISTRY
90,
Characterization J. G. Department
501-509
(1978)
of Ferredoxins
HUISMAN, of Plant
S. STAPEL, Physiology. Amsterdam.
AND
on a Nanomole M. G.
TH.
University of Amsterdam. The Nrtherlands
Scale
GEBBINK IJdijk
26
Received May 2. 1978 Two methods are described for the characterization of ferredoxins. First, mapping of tryptic peptides from 2 to 3 nmol of carboxyniethylated ferredoxin by two-dimensional thin-layer electrophoresis and chromatography. Second, gel electrophoresis of tryptic digests of apoferredoxins. The latter method discriminates between ferredoxins of closely related species.
The iron-sulfur proteins known as ferredoxins act as electron carriers in metabolic processes. In higher plants, eukaryotic algae and prokaryotic blue-green algae, they have molecular weights of about 11,000, while in bacteria only 6000. It is believed that in the evolution of photosynthetic organisms blue-green algae occupy an intermediate position between photosynthetic bacteria and green plants (1). Hase et al. (2) designed a phylogenetic tree of ferredoxin, illustrating possible branching points and ancestral origins of ferredoxin types. In order to provide insight into this biochemical evolutionary process it is necessary to characterize and compare ferredoxins from a wide range of species. Amino acid sequence studies of plant-type ferredoxins revealed a high degree of conservation (1). with the final stage of development being reached with the appearance of the primitive blue-greens. Thus one needs a sensitive technique to be able to differentiate between higher-plant ferredoxins. In view of the fact that chloroplasts possess their own genetic material, many attempts have been made to demonstrate the site of synthesis of ferredoxin and the location of the genetic information (3-6). Again what is needed for such studies is the ability to distinguish between different ferredoxins from closely related interfertile species. For studying the mode of inheritance in Nicotiana species Kwanuyen and Wildman (5) described the application of conventional paper electrophoresis and chromatography to separating the peptides from partially hydrolyzed ferredoxins. They obtained peptide maps with 3 mg of protein (200-300 nmol); however, the resolution was insufficient to discriminate between ferredoxins of related species. 501
0003-2697/78/0902-0501$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
502
HUISMAN.
STAPEL.
AND
GEBBINK
Another interesting point arose recently from the discovery by Hase et al. (7,s) and by us (9). of two molecular species of ferredoxins in higher plants. A comparative study of these isozyme-like molecules may give important data about gene-duplication and evolution of ferredoxins. The finding has prompted us for that reason to develop techniques which allow us to compare the structure of these ferredoxins. The techniques should require only small amounts of protein since the purification of ferredoxin is rather difficult because of its low content, especially of the minor components (7,&g). In this report we describe two methods by which ferredoxins can be characterized and identified. First, we have developed a peptide mapping technique which requires only 2 to 3 nmol of protein and yet has satisfactory resolving power. Second, a conventional technique for gel electrophoresis of tryptic peptides (3-4 nmol) was used by which one also can distinguish ferredoxins from closely related species. We consider both techniques to be highly applicable to the study of the problems mentioned above. MATERIALS
AND
METHODS
Cellulose MN 300 thin-layer plates, Polygram, 0.1 mm, and cellulose, 20 x 20 cm, were obtained from Mackery and Nagel and Company, Diiren, Germany. Paper bridges, 185’50 mm, were from Desaga Heidelberg, Germany. Acrylamide and N,N’-methylene-bisacrylamide were obtained from Bio-Rad, Richmond, California. Chymotrypsin-free trypsin was obtained from E. Merck, Darmstadt, Germany. All other reagents were of analytical grade. Ferredoxins were isolated and purified from the higher plants Petunia axillaris Fries., P. inflata Lam., Nicotianu tabacum L., N. rustica L., N. alata L., N. clevelandii L., N. glutinosa L., Phaseolus vulgaris I.,., Spinaceci oleracea L., and the green alga Chlamydomonas reinhardii Dang., using the method described by Huisman et al. (6) The 8Fe-8S ferredoxin of Clostridium pasteurianum was purchased from Sigma Ltd., St. Louis, Missouri. Carboxymethylation of ferredoxin was carried out using the method of Kung et al. (10) with slight modifications. One milligram of ferredoxin was lyophilized in a Thunberg tube. The tube was evacuated and the air replaced by nitrogen. Then 1 ml of N,-saturated 0.5 M Tris-HCl buffer, pH 8.6, containing 8 M urea (Tris-urea medium), 1 mM EDTA, and 1 mg of dithiothreitol was added and the incubation was allowed to proceed for 2 h at 25°C. To this solution was added 0.2 ml of Tris-urea medium containing 1 mg of iodoacetamide. The mixture was incubated for 10 min at 25°C in the dark. It was then dialyzed exhaustively against cold distilled water for at least 48 h and the protein was collected and lyophilized.
CHARACTERIZATION
OF FERREDOXINS
503
To prepare apoferredoxin, samples of native protein were converted using the method previously described by Huisman et al. (6). Tryptic digestion of ferredoxin was accomplished by dissolving carboxymethylated ferredoxin or apoferredoxin in 0.05 M ammonium bicarbonate, pH 8.0, and adding trypsin [l trypsin:50 ferredoxin (w/w)] and incubating for 4 h at 37°C. The peptides were recovered by freeze-drying in Eppendorf microtubes and dissolving them in 0.5 M acetic acid and 40 mM pyridine (pH 3.5) to a final concentration of 25 PgIpl. The solution was allowed to stand for 15 min at 37°C. The preparation of fingerprints was as follows: Cellulose plates were pre-sprayed with electrophoresis buffer, using a vaporizator and dichlorodifluormethane as carrier gas. The electrophoresis was carried out in a Desaga thin-layer electrophoresis chamber. To ensure even application of the electrophoresis buffer to the plate, two filter paper bridges, wetted previously, were placed about 10 mm from both sides of the plate. The buffer was then allowed to infiltrate the plate by capillary action. Preelectrophoresis was carried out at 400 V for 60 min. To minimize solvent loss by evaporation the filter bridges were covered with glass strips, 20 x 0.5 cm, which also ensured contact with the cooling plate. One microliter (2-3 nmol) of tryptic digest was then applied using a micropipet. Electrophoresis was carried out at 5 mA, and for 120 min the temperature was maintained at 2 to 4°C. The cellulose plates were dried in a stream of warm air and equilibrated in a chromatography chamber at 20°C. For this purpose, a small beaker containing the water phase of the elution solvent [butan-I-ol:acetic acid:water, 60:15:75 (v/v/v)] and a roll of Whatman 3MM filter paper were placed in the chamber. Ascending chromatography was carried out for 6 h, using the butan-l-01 phase. The sheets were dried thoroughly and the tryptic peptides were stained with 0.5% ninhydrin in ethanol containing 1% collidine and 1% acetic acid. Polyacrylamide gel electrophoresis was carried out with 15% acrylamide and 0.1% N,N’-methylene-bisacrylamide made in 0.375 M Tris-HCl buffer, pH 8.6 [the Tris buffer also containing 5% N,N,N’,N’-tetramethylethylenediamine, (v/v)]. Polymerization was started by adding freshly prepared solution of ammonium persulphate to a final concentration of 0.01%. All solutions were made in deaerated distilled water. The gels were made in glass tubes, 10 x 0.6 cm. To remove unpolymerized products, the gels were pre-electrophoresed in 0.075 M glycine buffer (adjusted to pH 8.3 with Tris) at 4°C. for 16 h at 0.2 mAige1. After changing the Tris-glycine buffer, 40 Kg of tryptic digest was dissolved in sample buffer and subjected to electrophoresis as described by Huisman et al. (6). Gels were fixed in 10% trichloroacetic acid for 30 min and stained for 16 h. The staining solution was prepared as follows: Ten percent trichloroacetic acid was heated at 60°C and a 1% Coomassie brilliant blue solution was added to it slowly umil a final concentration of 0.05% Coomassie was reached. The mixture
504
HUISMAN,
STAPEL,
AND
GEBBINK
was allowed to cool to room temperature and then was filtered through four layers of filter paper. Destaining was accomplished first in hot tapwater for 2 h, and then by soaking in 10% trichloroacetic acid. The gels could be stored in the dark in staining solution diluted 1:l with water. RESULTS
AND DISCUSSION
In Fig. 1 we show representative maps of tryptic digests of S-carboxymethylated ferredoxins. In all cases 25 pg of digest was applied to the tic plate. From the maps we conclude that ferredoxins of higher plants have comparable structures. Ferredoxin of spinach consists of 97 residues, five of which are arginine and lysine (1 l), which should lead to formation of six peptides upon tryptic digestion. The fingerprint in Fig. 1A shows five separated spots and a double spot near the origin. We consider the occurrence of seven spots instead of six as probably due to variants in protein molecules, presumably resulting from duplication of genes followed by small differentiations which gave rise to a few amino acid changes (cf. 12). Figure 1B shows the peptide map from tryptic digests of Phaseolus vulgaris ferredoxin. There are eight spots visible as expected from seven lysine and arginine residues (unpublished results from Huisman). Seven spots were detected in tryptic digests of N. tabacum ferredoxin (Fig. lC), but from the amino acid analysis (5,6) only six were expected. Possibly the digestion was not complete or trypsin cleaved the protein at other than lysine and arginine residues as reported previously (13). From both Petunia species, a major and minor component of soluble ferredoxin were isolated and purified (9). In this report only the major components will be considered. The peptide maps of both Petunia major components show patterns consistent with the number of susceptible bonds as calculated from the amino acid compositions (9); see Fig. 1D for the peptide map of P. uxilluris ferredoxin. Figure 1E shows a peptide map from Chlamydomonus reinhardii ferredoxin. Although the amino acid composition is unknown, it is clear that this algal ferredoxin differs in qualitative degree from the higher planttype. Only two main cathodic peptides could be resolved, one having a characteristic form, and two anodic peptides. Some minor spots could also be resolved but stained faintly. Clostridium ferredoxin upon trypsin hydrolysis seems to yield two peptides (Fig. 1F). This agrees with the calculated presence of one lysine residue (14). We conclude that the technique has sufficient resolving power to be applicable to analysis of Clostridium 8Fe-8S ferredoxin, which is considerably smaller than higher plant ferredoxins. The high resolution potential of this technique is confirmed by the results obtained with tryptic digests of Chlamydomonas ferredoxin mixed with digests from S. oleracea and N. rustica ferredoxin, respectively (see Figs.
CHARACTERIZATION
OF FERREDOXINS
505
E
ELECTROPHORESlS
+
FIG. 1. Peptide maps produced from 25 kg of tryptic digests of S-carboxymethylated ferredoxins. (A)Spinaceu oleracea ferredoxin; (B)Phaseolus vulgaris ferredoxin; (C) Nicotiuna tuhacum ferredoxin; (D) Petunia axillaris ferredoxin; (E) Chlamydomonas reinhardii ferredoxin; (F) Clostridium pusteuriunum ferredoxin. Electrophoresis and chromatography as described under Methods. (Arrows indicate faintly stained spots).
2A and B). Peptide maps such as shown here proved to be highly reproducible and contained some characteristic spots, recognizable in either single digest. Peptide maps obtained from different Nicotiana ferredoxins were almost identical, even though some differences in amino acid compositions have been calculated (5,6). In Fig. 3, however, we show a map obtained from
506
HUISMAN,
STAPEL.
+
AND
GEBBINK
-
+
FIG. 2. Peptide maps obtained from mixtures of tryptic digests from several ferredoxins. About 15 pg of digest of each ferredoxin was analyzed. (A) Mixture of tryptic digests from spinach and Chlamydomonas ferredoxins; (B) mixture of tryptic digests from N. rustica and Chlamydomonas ferredoxin. Direction of electrophoresis and chromatography as Fig. 1.
combined digests of N. glutinosa and N. clevelandii which is recognizable as a mixture. At the anodic site there are four visible spots which were found for N. tabacum alone (cf. Fig. lC), but the double cathodic spot proved to be specific for this mixture and not present in either single digest. We consider this technique useful in distinguishing ferredoxins from different species because it is rapid and yields reproducible results. We have employed the procedure already to compare two plant-type ferredoxins isolated from each of two Petunia species (9). We could demonstrate that the number of spots were in agreement with the number of lysine and arginine residues. None the less the technique appears not to be suited to distinguish between ferredoxins which differ only in a few amino acids
mb FIG. 3. Peptide map of mixed ferredoxins of Nicotiana. N. clevelandii and N. glutinosa ferredoxin. respectively, Direction of electrophoresis and chromatography as Fig. minor spots.)
t About 15 pg of tryptic digest of were applied to the tic plate. I. (Arrows indicate the position of
CHARACTERIZATION
507
OF FERREDOXINS
origin
C
D
E’
FIG. 4. Resolution of tryptic digests of apoferredoxins by gel electrophoresis. About 40 pg of digests were applied to 15% polykrylamide gels and electrophoresed as described under Methods. (A) S. olrrcccea ferredoxin; (B) P. vulgaris ferredoxin; (C) N. glutinosu ferredoxin; (D) P. inflata ferredoxin; (E) C. rcinhardii ferredoxin; (F) 40 pg of pure native ferredoxin of P. i&to. Only the negatively charged peptide bands show up on the gel patterns.
such as Nicotiuna (5,6). To achieve that goal we use the gel electrophoresis technique. In Fig. 4 we compare the electrophoretic mobilities on polyacrylamide gels of tryptic digests obtained from apoferredoxins of five different species. The gel patterns show only negatively charged peptide bands which have moved at different rates in the electric field. The different peptide mobilities are due to slight differences in primary structure. The number of peptides was either 2 or 3. The gel pattern of tryptic digests from spinach ferredoxin shows three bands of apparently negatively charged peptides. This is in agreement with the number of acidic tryptic peptides as could be deduced from the amino acid sequence (12). The two large peptides bearing a negative charge (position 5 to 40 and 53 to 91 of the primary structure) presumably show up in our gel system as the two heavily stained fast-moving bands, while the faintly stained band represents the small terminal peptide, position 92-97. It is clear that positively charged peptides refuse to be analyzed in this way. If we compare the resolution of anodic peptides in these gels with the resolution on tic fingerprint plates, it is clear that this method achieves a much higher resolution. In addition, the time-consuming preparation of S-carboxymethylated
HUISMAN,
508
STAPEL,
AND GEBBINK
+ FIG. 5. Resolution of tryptic digests from several Nicohana ferredoxins. About 40 pg of digests were analyzed. (A) N. tabacum var Samsun ferredoxin; (B) N. alata ferredoxin; (C) N. glutinosa ferredoxin; (D) N. rustica ferredoxin.
ferredoxin is replaced by the simple conversion of ferredoxin into apoferredoxin. The procedure has enabled us to characterize the small differences between ferredoxins of closely related species of Nicotiana, of which the amino acid composition shows differentiations in only a few amino acids (see Fig. 5, cf 6,9).
A
B
C
FIG. 6. Resolution of tryptic peptides of several ferredoxins mixed prior to electrophoresis. (A) spinach (1) and Chlamydomonas (2) ferredoxins; (B) N. tabacum (1) and Chlamydomonas (2) ferredoxins; (C) P. axillaris (1) and Chlamydomonas (2) ferredoxins; (D) N. tabacum (1) and N. glauca (2) ferredoxins.
CHARACTERIZATION
OF FERREDOXINS
509
The species-specific patterns persisted even when digests of several ferredoxins were mixed prior to analysis. This is illustrated in Fig. 6A-D, for Chlamydomonas digest mixed with spinach, N. tabacum and P. injiata digest, respectively, and for N. tabacum var Samsun digest mixed with N. glauca digest. We conclude that the described thin-layer procedure is a very sensitive method if compared with conventional paper technique and convenient for characterizing whole ferredoxin molecules, but that the one-dimensional resolution of anodic peptides on gels is more suitable for the characterization of ferredoxins from closely related species. ACKNOWLEDGMENTS The authors thank Prof. Dr. D. Stegwee for his encouragement and valuable review of this paper. We thank Dr. A. 0. Muijsers (Laboratory of Biochemistry, University of Amsterdam) for his advice and constructive discussions. This work was supported in part by a grant. No. 86-35, from The Netherlands Foundation for Biological Research (BION) with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO).
REFERENCES 1. Dayhoff, M. 0. (ed.), (1972) Atlas of protein sequence and structure, 5, D-40, National Biomedical Research Foundation, Washington, D. C. 2. Hase, T., Wada, K., and Matsubara, H. (1976) J. B&hem. 79, 329-343. 3. Matson, R. S., and Kimura, T. (1975) Biochim. Biophys. Acta 3%, 293-300. 4. Sluiters-Scholten, C. M. Th., Mall, W. A. W., and Stegwee, D. (1977) Planta 133, 289-294. 5. Kwanuyen, P., and Wildman, S. G. (1975) Biochim. Biophys. Acta 405, 167-174. 6. Huisman, .I. G., Gebbink, M. G. Th., Modderman, P., and Stegwee, D. (1977) Planta 137, 97- 105. 7. Hase, T., Wada, K., and Matsubara, H. (1977) J. Biochem. 82, 267-276. 8. Hase, T., Wada, K., and Matsubara, H. (1977) J. Biochem. 82, 277-286. 9. Huisman, J. G., Stapel, S., Muijsers, A. 0. (1978) FEB.5 Lett. 85, 198-202. 10. Kung. S. D., Sakano, K., and Wildman, S. G. (1974) Biochim. Biophys. Acta 365, 138- 147. 11. Matsubara, H., Sasaki, M. R., and Chain, K. R. (1967) Proc. Nat. Acad. Sci. USA 57, 439-445. 12. Matsubara, H., and Sasaki, R. M. (1968) J. Biol. Chem. 243, 1732-1757. 13. Keil-Dlouha, V., Zylber, N., Imhoff, J. M., Tong, N. T., and Keil, B. (1971) FEBS Lett. 16, 291-295. 14. Tanaka, M., Nakashima, T., Benson, A., Mower, F., and Yasunobo, K. T. (1964) Biochem. Biophys. Res. Commun. 16, 422-427.
BIOCHEMISTRY
90,
Characterization J. G. Department
501-509
(1978)
of Ferredoxins
HUISMAN, of Plant
S. STAPEL, Physiology. Amsterdam.
AND
on a Nanomole M. G.
TH.
University of Amsterdam. The Nrtherlands
Scale
GEBBINK IJdijk
26
Received May 2. 1978 Two methods are described for the characterization of ferredoxins. First, mapping of tryptic peptides from 2 to 3 nmol of carboxyniethylated ferredoxin by two-dimensional thin-layer electrophoresis and chromatography. Second, gel electrophoresis of tryptic digests of apoferredoxins. The latter method discriminates between ferredoxins of closely related species.
The iron-sulfur proteins known as ferredoxins act as electron carriers in metabolic processes. In higher plants, eukaryotic algae and prokaryotic blue-green algae, they have molecular weights of about 11,000, while in bacteria only 6000. It is believed that in the evolution of photosynthetic organisms blue-green algae occupy an intermediate position between photosynthetic bacteria and green plants (1). Hase et al. (2) designed a phylogenetic tree of ferredoxin, illustrating possible branching points and ancestral origins of ferredoxin types. In order to provide insight into this biochemical evolutionary process it is necessary to characterize and compare ferredoxins from a wide range of species. Amino acid sequence studies of plant-type ferredoxins revealed a high degree of conservation (1). with the final stage of development being reached with the appearance of the primitive blue-greens. Thus one needs a sensitive technique to be able to differentiate between higher-plant ferredoxins. In view of the fact that chloroplasts possess their own genetic material, many attempts have been made to demonstrate the site of synthesis of ferredoxin and the location of the genetic information (3-6). Again what is needed for such studies is the ability to distinguish between different ferredoxins from closely related interfertile species. For studying the mode of inheritance in Nicotiana species Kwanuyen and Wildman (5) described the application of conventional paper electrophoresis and chromatography to separating the peptides from partially hydrolyzed ferredoxins. They obtained peptide maps with 3 mg of protein (200-300 nmol); however, the resolution was insufficient to discriminate between ferredoxins of related species. 501
0003-2697/78/0902-0501$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
502
HUISMAN.
STAPEL.
AND
GEBBINK
Another interesting point arose recently from the discovery by Hase et al. (7,s) and by us (9). of two molecular species of ferredoxins in higher plants. A comparative study of these isozyme-like molecules may give important data about gene-duplication and evolution of ferredoxins. The finding has prompted us for that reason to develop techniques which allow us to compare the structure of these ferredoxins. The techniques should require only small amounts of protein since the purification of ferredoxin is rather difficult because of its low content, especially of the minor components (7,&g). In this report we describe two methods by which ferredoxins can be characterized and identified. First, we have developed a peptide mapping technique which requires only 2 to 3 nmol of protein and yet has satisfactory resolving power. Second, a conventional technique for gel electrophoresis of tryptic peptides (3-4 nmol) was used by which one also can distinguish ferredoxins from closely related species. We consider both techniques to be highly applicable to the study of the problems mentioned above. MATERIALS
AND
METHODS
Cellulose MN 300 thin-layer plates, Polygram, 0.1 mm, and cellulose, 20 x 20 cm, were obtained from Mackery and Nagel and Company, Diiren, Germany. Paper bridges, 185’50 mm, were from Desaga Heidelberg, Germany. Acrylamide and N,N’-methylene-bisacrylamide were obtained from Bio-Rad, Richmond, California. Chymotrypsin-free trypsin was obtained from E. Merck, Darmstadt, Germany. All other reagents were of analytical grade. Ferredoxins were isolated and purified from the higher plants Petunia axillaris Fries., P. inflata Lam., Nicotianu tabacum L., N. rustica L., N. alata L., N. clevelandii L., N. glutinosa L., Phaseolus vulgaris I.,., Spinaceci oleracea L., and the green alga Chlamydomonas reinhardii Dang., using the method described by Huisman et al. (6) The 8Fe-8S ferredoxin of Clostridium pasteurianum was purchased from Sigma Ltd., St. Louis, Missouri. Carboxymethylation of ferredoxin was carried out using the method of Kung et al. (10) with slight modifications. One milligram of ferredoxin was lyophilized in a Thunberg tube. The tube was evacuated and the air replaced by nitrogen. Then 1 ml of N,-saturated 0.5 M Tris-HCl buffer, pH 8.6, containing 8 M urea (Tris-urea medium), 1 mM EDTA, and 1 mg of dithiothreitol was added and the incubation was allowed to proceed for 2 h at 25°C. To this solution was added 0.2 ml of Tris-urea medium containing 1 mg of iodoacetamide. The mixture was incubated for 10 min at 25°C in the dark. It was then dialyzed exhaustively against cold distilled water for at least 48 h and the protein was collected and lyophilized.
CHARACTERIZATION
OF FERREDOXINS
503
To prepare apoferredoxin, samples of native protein were converted using the method previously described by Huisman et al. (6). Tryptic digestion of ferredoxin was accomplished by dissolving carboxymethylated ferredoxin or apoferredoxin in 0.05 M ammonium bicarbonate, pH 8.0, and adding trypsin [l trypsin:50 ferredoxin (w/w)] and incubating for 4 h at 37°C. The peptides were recovered by freeze-drying in Eppendorf microtubes and dissolving them in 0.5 M acetic acid and 40 mM pyridine (pH 3.5) to a final concentration of 25 PgIpl. The solution was allowed to stand for 15 min at 37°C. The preparation of fingerprints was as follows: Cellulose plates were pre-sprayed with electrophoresis buffer, using a vaporizator and dichlorodifluormethane as carrier gas. The electrophoresis was carried out in a Desaga thin-layer electrophoresis chamber. To ensure even application of the electrophoresis buffer to the plate, two filter paper bridges, wetted previously, were placed about 10 mm from both sides of the plate. The buffer was then allowed to infiltrate the plate by capillary action. Preelectrophoresis was carried out at 400 V for 60 min. To minimize solvent loss by evaporation the filter bridges were covered with glass strips, 20 x 0.5 cm, which also ensured contact with the cooling plate. One microliter (2-3 nmol) of tryptic digest was then applied using a micropipet. Electrophoresis was carried out at 5 mA, and for 120 min the temperature was maintained at 2 to 4°C. The cellulose plates were dried in a stream of warm air and equilibrated in a chromatography chamber at 20°C. For this purpose, a small beaker containing the water phase of the elution solvent [butan-I-ol:acetic acid:water, 60:15:75 (v/v/v)] and a roll of Whatman 3MM filter paper were placed in the chamber. Ascending chromatography was carried out for 6 h, using the butan-l-01 phase. The sheets were dried thoroughly and the tryptic peptides were stained with 0.5% ninhydrin in ethanol containing 1% collidine and 1% acetic acid. Polyacrylamide gel electrophoresis was carried out with 15% acrylamide and 0.1% N,N’-methylene-bisacrylamide made in 0.375 M Tris-HCl buffer, pH 8.6 [the Tris buffer also containing 5% N,N,N’,N’-tetramethylethylenediamine, (v/v)]. Polymerization was started by adding freshly prepared solution of ammonium persulphate to a final concentration of 0.01%. All solutions were made in deaerated distilled water. The gels were made in glass tubes, 10 x 0.6 cm. To remove unpolymerized products, the gels were pre-electrophoresed in 0.075 M glycine buffer (adjusted to pH 8.3 with Tris) at 4°C. for 16 h at 0.2 mAige1. After changing the Tris-glycine buffer, 40 Kg of tryptic digest was dissolved in sample buffer and subjected to electrophoresis as described by Huisman et al. (6). Gels were fixed in 10% trichloroacetic acid for 30 min and stained for 16 h. The staining solution was prepared as follows: Ten percent trichloroacetic acid was heated at 60°C and a 1% Coomassie brilliant blue solution was added to it slowly umil a final concentration of 0.05% Coomassie was reached. The mixture
504
HUISMAN,
STAPEL,
AND
GEBBINK
was allowed to cool to room temperature and then was filtered through four layers of filter paper. Destaining was accomplished first in hot tapwater for 2 h, and then by soaking in 10% trichloroacetic acid. The gels could be stored in the dark in staining solution diluted 1:l with water. RESULTS
AND DISCUSSION
In Fig. 1 we show representative maps of tryptic digests of S-carboxymethylated ferredoxins. In all cases 25 pg of digest was applied to the tic plate. From the maps we conclude that ferredoxins of higher plants have comparable structures. Ferredoxin of spinach consists of 97 residues, five of which are arginine and lysine (1 l), which should lead to formation of six peptides upon tryptic digestion. The fingerprint in Fig. 1A shows five separated spots and a double spot near the origin. We consider the occurrence of seven spots instead of six as probably due to variants in protein molecules, presumably resulting from duplication of genes followed by small differentiations which gave rise to a few amino acid changes (cf. 12). Figure 1B shows the peptide map from tryptic digests of Phaseolus vulgaris ferredoxin. There are eight spots visible as expected from seven lysine and arginine residues (unpublished results from Huisman). Seven spots were detected in tryptic digests of N. tabacum ferredoxin (Fig. lC), but from the amino acid analysis (5,6) only six were expected. Possibly the digestion was not complete or trypsin cleaved the protein at other than lysine and arginine residues as reported previously (13). From both Petunia species, a major and minor component of soluble ferredoxin were isolated and purified (9). In this report only the major components will be considered. The peptide maps of both Petunia major components show patterns consistent with the number of susceptible bonds as calculated from the amino acid compositions (9); see Fig. 1D for the peptide map of P. uxilluris ferredoxin. Figure 1E shows a peptide map from Chlamydomonus reinhardii ferredoxin. Although the amino acid composition is unknown, it is clear that this algal ferredoxin differs in qualitative degree from the higher planttype. Only two main cathodic peptides could be resolved, one having a characteristic form, and two anodic peptides. Some minor spots could also be resolved but stained faintly. Clostridium ferredoxin upon trypsin hydrolysis seems to yield two peptides (Fig. 1F). This agrees with the calculated presence of one lysine residue (14). We conclude that the technique has sufficient resolving power to be applicable to analysis of Clostridium 8Fe-8S ferredoxin, which is considerably smaller than higher plant ferredoxins. The high resolution potential of this technique is confirmed by the results obtained with tryptic digests of Chlamydomonas ferredoxin mixed with digests from S. oleracea and N. rustica ferredoxin, respectively (see Figs.
CHARACTERIZATION
OF FERREDOXINS
505
E
ELECTROPHORESlS
+
FIG. 1. Peptide maps produced from 25 kg of tryptic digests of S-carboxymethylated ferredoxins. (A)Spinaceu oleracea ferredoxin; (B)Phaseolus vulgaris ferredoxin; (C) Nicotiuna tuhacum ferredoxin; (D) Petunia axillaris ferredoxin; (E) Chlamydomonas reinhardii ferredoxin; (F) Clostridium pusteuriunum ferredoxin. Electrophoresis and chromatography as described under Methods. (Arrows indicate faintly stained spots).
2A and B). Peptide maps such as shown here proved to be highly reproducible and contained some characteristic spots, recognizable in either single digest. Peptide maps obtained from different Nicotiana ferredoxins were almost identical, even though some differences in amino acid compositions have been calculated (5,6). In Fig. 3, however, we show a map obtained from
506
HUISMAN,
STAPEL.
+
AND
GEBBINK
-
+
FIG. 2. Peptide maps obtained from mixtures of tryptic digests from several ferredoxins. About 15 pg of digest of each ferredoxin was analyzed. (A) Mixture of tryptic digests from spinach and Chlamydomonas ferredoxins; (B) mixture of tryptic digests from N. rustica and Chlamydomonas ferredoxin. Direction of electrophoresis and chromatography as Fig. 1.
combined digests of N. glutinosa and N. clevelandii which is recognizable as a mixture. At the anodic site there are four visible spots which were found for N. tabacum alone (cf. Fig. lC), but the double cathodic spot proved to be specific for this mixture and not present in either single digest. We consider this technique useful in distinguishing ferredoxins from different species because it is rapid and yields reproducible results. We have employed the procedure already to compare two plant-type ferredoxins isolated from each of two Petunia species (9). We could demonstrate that the number of spots were in agreement with the number of lysine and arginine residues. None the less the technique appears not to be suited to distinguish between ferredoxins which differ only in a few amino acids
mb FIG. 3. Peptide map of mixed ferredoxins of Nicotiana. N. clevelandii and N. glutinosa ferredoxin. respectively, Direction of electrophoresis and chromatography as Fig. minor spots.)
t About 15 pg of tryptic digest of were applied to the tic plate. I. (Arrows indicate the position of
CHARACTERIZATION
507
OF FERREDOXINS
origin
C
D
E’
FIG. 4. Resolution of tryptic digests of apoferredoxins by gel electrophoresis. About 40 pg of digests were applied to 15% polykrylamide gels and electrophoresed as described under Methods. (A) S. olrrcccea ferredoxin; (B) P. vulgaris ferredoxin; (C) N. glutinosu ferredoxin; (D) P. inflata ferredoxin; (E) C. rcinhardii ferredoxin; (F) 40 pg of pure native ferredoxin of P. i&to. Only the negatively charged peptide bands show up on the gel patterns.
such as Nicotiuna (5,6). To achieve that goal we use the gel electrophoresis technique. In Fig. 4 we compare the electrophoretic mobilities on polyacrylamide gels of tryptic digests obtained from apoferredoxins of five different species. The gel patterns show only negatively charged peptide bands which have moved at different rates in the electric field. The different peptide mobilities are due to slight differences in primary structure. The number of peptides was either 2 or 3. The gel pattern of tryptic digests from spinach ferredoxin shows three bands of apparently negatively charged peptides. This is in agreement with the number of acidic tryptic peptides as could be deduced from the amino acid sequence (12). The two large peptides bearing a negative charge (position 5 to 40 and 53 to 91 of the primary structure) presumably show up in our gel system as the two heavily stained fast-moving bands, while the faintly stained band represents the small terminal peptide, position 92-97. It is clear that positively charged peptides refuse to be analyzed in this way. If we compare the resolution of anodic peptides in these gels with the resolution on tic fingerprint plates, it is clear that this method achieves a much higher resolution. In addition, the time-consuming preparation of S-carboxymethylated
HUISMAN,
508
STAPEL,
AND GEBBINK
+ FIG. 5. Resolution of tryptic digests from several Nicohana ferredoxins. About 40 pg of digests were analyzed. (A) N. tabacum var Samsun ferredoxin; (B) N. alata ferredoxin; (C) N. glutinosa ferredoxin; (D) N. rustica ferredoxin.
ferredoxin is replaced by the simple conversion of ferredoxin into apoferredoxin. The procedure has enabled us to characterize the small differences between ferredoxins of closely related species of Nicotiana, of which the amino acid composition shows differentiations in only a few amino acids (see Fig. 5, cf 6,9).
A
B
C
FIG. 6. Resolution of tryptic peptides of several ferredoxins mixed prior to electrophoresis. (A) spinach (1) and Chlamydomonas (2) ferredoxins; (B) N. tabacum (1) and Chlamydomonas (2) ferredoxins; (C) P. axillaris (1) and Chlamydomonas (2) ferredoxins; (D) N. tabacum (1) and N. glauca (2) ferredoxins.
CHARACTERIZATION
OF FERREDOXINS
509
The species-specific patterns persisted even when digests of several ferredoxins were mixed prior to analysis. This is illustrated in Fig. 6A-D, for Chlamydomonas digest mixed with spinach, N. tabacum and P. injiata digest, respectively, and for N. tabacum var Samsun digest mixed with N. glauca digest. We conclude that the described thin-layer procedure is a very sensitive method if compared with conventional paper technique and convenient for characterizing whole ferredoxin molecules, but that the one-dimensional resolution of anodic peptides on gels is more suitable for the characterization of ferredoxins from closely related species. ACKNOWLEDGMENTS The authors thank Prof. Dr. D. Stegwee for his encouragement and valuable review of this paper. We thank Dr. A. 0. Muijsers (Laboratory of Biochemistry, University of Amsterdam) for his advice and constructive discussions. This work was supported in part by a grant. No. 86-35, from The Netherlands Foundation for Biological Research (BION) with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO).
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