- Email: [email protected]
Purification of cationic peanut peroxidase by immunoaffinity chromatography
Plant Science Letters, 26 (1982) 39--46
39
Elsevier Scientific Publishers Ireland Ltd.
P U R I F I C A T I O N OF CATIONIC PEANUT PEROXIDASE BY IMMUNOAFFINITY CHROMATOGRAPHY
J. 45OBARZEWSKI and R.B. van HUYSTEE a'*
Dept. of Biochemistry, University Maria Curie-Sktodowska, 20-031 Lublin (Poland) and aDept, of Plant Sciences, University of Western Ontario, London, Ontario N6A 5B7 (Canada) {Received July 29th, 1981) (Revision received October 12th, 1981) (Accepted October 12th, 1981)
SUMMARY
Antibodies, raised against the major cationic peroxidase from cultured peanut cells, were coupled to CNBr-activated Sepharose to serve as the affinity source. The conditions for purification o f peanut peroxidase on this antibody-coupled column were studied. The a m o u n t of peroxidase that could be purified on such affinity column depends on the purity and concentration of the antibodies coupled to Sepharose. Peroxidase from horseradish or the fungus (Trametes versicolor) did n o t bind to this column. This means that the peroxidase from these sources were n o t immunologicaUy identical to that of cultured peanut cells.
INTRODUCTION
Peroxidase is an enzyme of interest to a large variety of researchers. Higher plant peroxidase plays several roles [ 1] b u t its role in lignin synthesis has been emphasized primarily [2,3 ]. Fungal peroxidase is found to polymerize and depolymerize lignin [4]. Unfortunately detailed studies on peroxidase induction and structure have often been hampered due to impurities in the enzyme preparations [ 1]. The specific activity of peroxidase in the medium, supporting the growth of peanut cell, is a b o u t 20-fold greater than that in the crude cell extract [ 5]. Such high specific activity of peroxidase occurs also in the extracellular spaces in leaves [6]. The proteins were extracted from the suspension medium and fractionated by CM cellulose chromatography, which results in
*To whom correspondence should be addressed. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
40 the isolation of a single protein fraction with nearly 75% of all peroxidase activity [7]. Antibodies raised against this major cationic peroxidase subsequently were used for immunoprecipitation experiments to demonstrate in vitro peroxidase synthesis [8,9]. Antibodies in immunoaffinity chromatography have been used successfully in the purification of proteins that occur in major amounts in plants e.g. legumin [10]. The following study examines the feasibility of using antibodies for immunoaffinity chromatography of proteins, such as peroxidase, whose concentration is low. MATERIALS AND METHODS
Derivation of antibodies Peanut cultures in suspension medium were grown routinely and the medium was harvested as described [ 5]. The proteins were extracted from the medium by sequential precipitation with acetone and ammonium sulfate [11] and the major cationic peroxidase fraction was isolated by CM cellulose chromatography [ 7]. Antibodies were raised in rabbits against this peroxidase by procedures outlined [12]. The antiserum so obtained was either immediately used for immunoaffinity or was first freed from albumin and protease by passing 5 ml antiserum through 40 ml CM Affigel Blue (Biorad). In the latter case the antibodies were washed through the column with 0.01 M potassium phosphate (pH 7.25) containing 0.15 M NaC1 while the albumin and protease remained b o u n d to the column. The collected antibodies were precipitated in 45% ammonium sulfate and dialyzed against water overnight. The sample containing antibodies was then coupled to CNBr-activated Sepharose 4B (Pharmacia) by established procedures.
Immunoaffinity chromatography The CNBr-activated Sepharose was washed with 0.001 M HC1. The antibodies were coupled to the Sepharose in the presence of 0.1 M NaHCO3 containing 0.5 M NaC1 at 4°C for 16 h. The final p r o d u c t was washed with the saline coupling medium and the remaining active sites were neutralized with 1 M monoethanolamine (pH 8) for 2 h. Protein n o t covalently bound to the matrix were removed by washing three times successively in the two buffers; 0.1 M Na acetate (pH 4); and 0.1 M Na borate (pH 9). Prior to the application, peroxidase was dialyzed against water for at least 16 h. After the application of the peroxidase, the column was washed with 5 bed volumes of water. When the recorded absorption at 280 nm of the column effluent had returned to base line, the peroxidase was eluted from the column with 0.01 M Na acetate (pH 5).
Electrophoresis Disc electrophoresis was carried out for cationic proteins under nondenaturaing conditions according to procedures by Reisfeld et al. [ 13 ] and
41
the gels stained for peroxidase as reported [ 14]. Two-dimensional immunoelectrophoresis was carried out as described [ 12].
Determination of protein and peroxidase activity The protein concentration was measured either for absorption at 280 nm or by the technique of Lowry et al. [15]. Peroxidase presence was measured at 407 n m absorption and its purity determined by the 407/280 nm absorption values called RZ. Peroxidase activity was measured by incubating 0.1 ml of sample in a medium of 3 ml 0.05 M phosphate (pH 7.0), 1 ml 0.3% H202 and 1 ml 1% of guaiacol and measured as change of absorbance at 470 nm after 1 min [ 14]. One unit o f peroxidase activity was considered to be equal to AA470nm of 0.001 per rain [16]. RESULTS
The purity of antibodies for peroxidase in the serum used to couple to Sepharose was examined by two-dimensional immunoelectrophoresis (Fig. 1). The single reaction arc shows satisfactory purity in terms of peroxidase. It was noted that when antiserum containing 50 mg protein was coupled to 1 g CNBr-activated Sepharose at least 90% protein remained with the column during subsequent washing. The action of the column to purify peroxidase is expressed in terms of increase in RZ-values, as used before [7]. The values in Table I indicate a stepwise purification process. The upper limit for purification is set by the
Fig. 1. Two-dimensional immunoelectrophoresis o f all peanut peroxidase forms against s e r u m containing antibodies raised against pure major cationic fraction. The gel was w a s h e d extensively in saline, dried a n d t h e n s t a i n e d as b e f o r e [ 1 2 ] .
42 TABLE I PROGRESS OF PURIFICATION OF PEANUT PEROXIDASE ON IMMUNOAFFINITY COLUMN A n t i b o d i e s c o u p l e d to Sepharose were raised against a single c a t i o n i c protein with peroxidase activity [ 7,8 ]. The antiserum was purified b y passage through CM Affigel Blue. The 50 mg o f so o b t a i n e d protein was c o u p l e d to 1 g CNBr-activated S e p h a r o s e 4B. Sample
RZ (A407/~80 nm ) Before a
Aftera
A
1
B
1.5
1.5 2.5
aBefore or after chromatography. purity of the antigen used to elicit the antibodies, which serve for immunoaffinity. A more detailed view of the purification process is shown in Fig. 2. In this representative experiment most o f the non-heme proteins are washed through the column in the first peak, since the Soret absorption band at 407 nm is much lower than the protein absorption at 280 nm. The reverse is true for the fraction eluted at pH-value of 5. This results in the increased RZ-vaiue. Further chromatography o f this fraction is shown in Fig. 3, where the proteins washed through t h e column have an absorbance at 407 nm equal to that at 280 nm. The removal o f protein other than peroxidase and possibly some peroxidase isozymes other than the major cationic fraction [9] results in a fraction of high RZ. Examination of the product by polyacrylamide gel and immunoelectrophoresis showed that only one cationic peroxidase occurred {Fig. 4) compared to at least one anionic and two cationic forms [7,9]. The affinity column appeared to be also specie specific A commercial {Sigma) preparation of horseradish peroxidase in water, as well as an aqueous solution of crude peroxidase from Trametes versicolor [16] were made (5 mg each) and applied to the column with antibodies from the major cationic peanut peroxidase. Neither preparation was bound to the antibodies from major cationic peroxidase from peanuts. Therefore, it implies that the structure of peroxidase is specie-dependent [ 17]. It follows from the above that the process o f purification o f peroxidase is dependent to some degree on purity of the initial sample. However, the carrying capacity of the column does also depend on the purity of the antibodies used. Table II shows that the removal of albumin from the antibodies by Affigel increases the a m o u n t of protein coupled to the column, which is parallel to the a m o u n t of peroxidase carried by the column. In addition the carrying capacity of the column is augmented by binding antibodies to all activated sites on the Sepharose. And the carrying capacity is improved by the use of a sample of greater purity. That principle has been mentioned in relation to Table I.
43
® ~ O ~
O.D° u.~
•
O.D. 4 0 7 n m
O,D.2 8 O h m
! F
~
,° b
1,0
0.5
t /.
o \
"
!
l t
!
q
® O.D. 1.51
o
1.0,
0.5,
I ....
t/'/~''"
%,--o- ~ ,,~o 20
40
60
ml
Fig. 2. Lrnmunoaffinity chromatography of peanut peroxidase. A column of 1.2. × 7.5 cm of antibodies coupled to Sepharose was loaded with peroxidase sample (RZ = 1, 20 mg protein). After washing the column with water the peroxidase linked was eluted with 0.01 M Na acetate (pH 5). Fig, 3. Immunoaffinity chromatography of peanut peroxidase. The conditions for the chromatography are described for Fig. 2. The sample chro;xlatographed contained peroxidase with RZ = 1.5.
44
I
I
mm i
i
4-
Fig. 4. The results of one-dimensional immunoelectrophoresis in comparison to disc electrophoresis of peanut peroxidase which had been purified on antibody-Sepharose column in two consecutive steps. (See Materials and Methods). TABLE II THE IMMUNOAFFINITY ANTIBODY-SEPHAROSE CARRYING CAPACITY AS THE FUNCTION OF ANTIBODIES PURITY COUPLED TO THE MATRIX AND PEANUT PEROXIDASE SAMPLE APPLIED TO THE COLUMN Antibodies linked to 1 g Sepharose (protein) Total antiserum (25 mg) CM Affigei Blue treated (25 rag) CM Affigel Blue treated (50 mg) CM Affigel Blue treated (50 rag)
Protein (rag)
Units of peroxidase a (× 10 -s)
RZ b of sample applied
7.5
13.7
1.0
15.0
27.4
1.0
30.0
68.5
1.0
30.0
168.0
1.5
aOne unit of peroxidase equals 0.001 AA470 nm in 1 rain during assay. bRZ is ratio of absorbance at 470/280 nm.
45 DISCUSSION Purification of peroxidase has been attempted by various means of affinity chromatography [16,18,19]. But invariably some difficulties arise probably due to interspecies variation such as found in this study, between peroxidase from peanut and those from horseradish and a fungus Trametes versicolor. The most specific source of affinity is the one between antigen-antibody. This has a reason for using immunoaffinity chromatography in studies of ribulose 1,5-bisphosphate carboxylase [ 20] and legumin [ 10]. However, these proteins are present in great abundance in plants. In the present study it is shown that immunoaffinity may also be applied to proteins not so prevalent in the plants. The added benefit in using peroxidase, a chromoprotein, is that the affinity and elution process can be seen on the column. The immunoaffinity procedure is also more rapid than ion exchange chromatography. Chaotropic agents such as urea [20] have been used for elution. To avoid denaturation of peroxidase the pH of the column was reduced as reported for other studies [10,23]. When crude peroxidase was purified in the first step of this process the increase in RZ was moderate. This is probably due to the interference of contaminating proteins, which include the minor cationic form and the anionic form of peanut peroxidase [7,9]. In addition the medium of the cultured cells contained probably other cell wall proteins than peroxidase. These proteins are mainly glycoproteins [21]. A non-specific affinity of some glycoprotein for the h y d r o x y l groups of the matrix has been found [22]. Higher purification of peroxidase is found when a sample already prepurified is applied. In conclusion it appears possible to purify peroxidase routinely by affinity chromatography once a sample of antibodies raised against a protein fraction -with high peroxidase activity or high RZ has been obtained. ACKNOWLEDGEMENT The authors are grateful for financial support from the Interdisciplinary Centre of Chemical Physics, University of Western Ontario, which also accorded to J. ~obarzewski, Visiting Fellow status in the Centre. In addition, financial support from N.S.E.R.C.C. to R.B. van Huystee is acknowledged. REFERENCES 1 2 3 4
R.B. van Huystee and W.L. Cairns, Bot. Rev., 46 (1980) 429. K. Hahlbrock and H. Grisebach, Annu. Rev. Plant Physiol., 30 (1979) 109. S.C. Fry, Phytochemistry, 19 (1980) 735. J. ~obarzewski, J. Trojanowski and M. Wojtas-Wasilewska,Holzforschung. (1981) in press. 5 V.C. Kossatz and R.B. van Huystee, Can. J. Bot., 54 (1976) 2089. 6 W.G. Rathmell and L. Sequiera, Plant Physiol., 53 (1974) 317.
46 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
B.M. Maldonado and R.B. van Huystee. Can. J. Bot., 58 (1980) 2280. D. Stephan and R.B. van Huystee, Can. J. Biochem., 58 (1980) 715. D. Stephan and R.B. van Huystee, Z. Pflanzenphysiol., 101 (1981) 313. R. Casey, Biochem. J., 177 (1979) 509. R.B. van Huystee, Bot. Gaz., 137 (1976b) 325. E. Cairns, R.B. van Huystee and W.L. Cairns, Physiol. Plant., 49 (1979) 78. R.A. Reisfeld, U.J. Lewis and D.E. Williams, Nature, 195 (1962) 281. O.P. Srivastava and R.B. van Huystee, Can. J. Bot., 51 (1973) 2207. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. J. ~,obarzewski, Int. J. Biol. Macromol., 3 (1981) 77. R.B. van Huystee and B. Maldonado, Physiol. Plant., 54 (1982) 88. R.B. van Huystee, Can. J. Bot., 54 (1976a) 876. L. Reimann and G.L. Schonbaum, in: S. Fleischer and L. Parker (Eds.), Methods in Enzymology, Vol. 52, Academic Press, 1978, p. 514. J.C. Gray and S.G. Wildman, Plant Sci. Lett., 6 (1976) 91. D.T.A. Lamport, Rec. Adv. Phytochem., 11 (1977) 79. G. Entlicher, M. Ticha, J.V. Kostir and J. Kocourek, Experientia, 25 (1969) 17. D.M. Livingston, in: W.B. Jakoby and M. Wilchek (Eds.), Methods in Enzymology, Vol. 34, Academic Press, 1974, p. 727.
39
Elsevier Scientific Publishers Ireland Ltd.
P U R I F I C A T I O N OF CATIONIC PEANUT PEROXIDASE BY IMMUNOAFFINITY CHROMATOGRAPHY
J. 45OBARZEWSKI and R.B. van HUYSTEE a'*
Dept. of Biochemistry, University Maria Curie-Sktodowska, 20-031 Lublin (Poland) and aDept, of Plant Sciences, University of Western Ontario, London, Ontario N6A 5B7 (Canada) {Received July 29th, 1981) (Revision received October 12th, 1981) (Accepted October 12th, 1981)
SUMMARY
Antibodies, raised against the major cationic peroxidase from cultured peanut cells, were coupled to CNBr-activated Sepharose to serve as the affinity source. The conditions for purification o f peanut peroxidase on this antibody-coupled column were studied. The a m o u n t of peroxidase that could be purified on such affinity column depends on the purity and concentration of the antibodies coupled to Sepharose. Peroxidase from horseradish or the fungus (Trametes versicolor) did n o t bind to this column. This means that the peroxidase from these sources were n o t immunologicaUy identical to that of cultured peanut cells.
INTRODUCTION
Peroxidase is an enzyme of interest to a large variety of researchers. Higher plant peroxidase plays several roles [ 1] b u t its role in lignin synthesis has been emphasized primarily [2,3 ]. Fungal peroxidase is found to polymerize and depolymerize lignin [4]. Unfortunately detailed studies on peroxidase induction and structure have often been hampered due to impurities in the enzyme preparations [ 1]. The specific activity of peroxidase in the medium, supporting the growth of peanut cell, is a b o u t 20-fold greater than that in the crude cell extract [ 5]. Such high specific activity of peroxidase occurs also in the extracellular spaces in leaves [6]. The proteins were extracted from the suspension medium and fractionated by CM cellulose chromatography, which results in
*To whom correspondence should be addressed. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
40 the isolation of a single protein fraction with nearly 75% of all peroxidase activity [7]. Antibodies raised against this major cationic peroxidase subsequently were used for immunoprecipitation experiments to demonstrate in vitro peroxidase synthesis [8,9]. Antibodies in immunoaffinity chromatography have been used successfully in the purification of proteins that occur in major amounts in plants e.g. legumin [10]. The following study examines the feasibility of using antibodies for immunoaffinity chromatography of proteins, such as peroxidase, whose concentration is low. MATERIALS AND METHODS
Derivation of antibodies Peanut cultures in suspension medium were grown routinely and the medium was harvested as described [ 5]. The proteins were extracted from the medium by sequential precipitation with acetone and ammonium sulfate [11] and the major cationic peroxidase fraction was isolated by CM cellulose chromatography [ 7]. Antibodies were raised in rabbits against this peroxidase by procedures outlined [12]. The antiserum so obtained was either immediately used for immunoaffinity or was first freed from albumin and protease by passing 5 ml antiserum through 40 ml CM Affigel Blue (Biorad). In the latter case the antibodies were washed through the column with 0.01 M potassium phosphate (pH 7.25) containing 0.15 M NaC1 while the albumin and protease remained b o u n d to the column. The collected antibodies were precipitated in 45% ammonium sulfate and dialyzed against water overnight. The sample containing antibodies was then coupled to CNBr-activated Sepharose 4B (Pharmacia) by established procedures.
Immunoaffinity chromatography The CNBr-activated Sepharose was washed with 0.001 M HC1. The antibodies were coupled to the Sepharose in the presence of 0.1 M NaHCO3 containing 0.5 M NaC1 at 4°C for 16 h. The final p r o d u c t was washed with the saline coupling medium and the remaining active sites were neutralized with 1 M monoethanolamine (pH 8) for 2 h. Protein n o t covalently bound to the matrix were removed by washing three times successively in the two buffers; 0.1 M Na acetate (pH 4); and 0.1 M Na borate (pH 9). Prior to the application, peroxidase was dialyzed against water for at least 16 h. After the application of the peroxidase, the column was washed with 5 bed volumes of water. When the recorded absorption at 280 nm of the column effluent had returned to base line, the peroxidase was eluted from the column with 0.01 M Na acetate (pH 5).
Electrophoresis Disc electrophoresis was carried out for cationic proteins under nondenaturaing conditions according to procedures by Reisfeld et al. [ 13 ] and
41
the gels stained for peroxidase as reported [ 14]. Two-dimensional immunoelectrophoresis was carried out as described [ 12].
Determination of protein and peroxidase activity The protein concentration was measured either for absorption at 280 nm or by the technique of Lowry et al. [15]. Peroxidase presence was measured at 407 n m absorption and its purity determined by the 407/280 nm absorption values called RZ. Peroxidase activity was measured by incubating 0.1 ml of sample in a medium of 3 ml 0.05 M phosphate (pH 7.0), 1 ml 0.3% H202 and 1 ml 1% of guaiacol and measured as change of absorbance at 470 nm after 1 min [ 14]. One unit o f peroxidase activity was considered to be equal to AA470nm of 0.001 per rain [16]. RESULTS
The purity of antibodies for peroxidase in the serum used to couple to Sepharose was examined by two-dimensional immunoelectrophoresis (Fig. 1). The single reaction arc shows satisfactory purity in terms of peroxidase. It was noted that when antiserum containing 50 mg protein was coupled to 1 g CNBr-activated Sepharose at least 90% protein remained with the column during subsequent washing. The action of the column to purify peroxidase is expressed in terms of increase in RZ-values, as used before [7]. The values in Table I indicate a stepwise purification process. The upper limit for purification is set by the
Fig. 1. Two-dimensional immunoelectrophoresis o f all peanut peroxidase forms against s e r u m containing antibodies raised against pure major cationic fraction. The gel was w a s h e d extensively in saline, dried a n d t h e n s t a i n e d as b e f o r e [ 1 2 ] .
42 TABLE I PROGRESS OF PURIFICATION OF PEANUT PEROXIDASE ON IMMUNOAFFINITY COLUMN A n t i b o d i e s c o u p l e d to Sepharose were raised against a single c a t i o n i c protein with peroxidase activity [ 7,8 ]. The antiserum was purified b y passage through CM Affigel Blue. The 50 mg o f so o b t a i n e d protein was c o u p l e d to 1 g CNBr-activated S e p h a r o s e 4B. Sample
RZ (A407/~80 nm ) Before a
Aftera
A
1
B
1.5
1.5 2.5
aBefore or after chromatography. purity of the antigen used to elicit the antibodies, which serve for immunoaffinity. A more detailed view of the purification process is shown in Fig. 2. In this representative experiment most o f the non-heme proteins are washed through the column in the first peak, since the Soret absorption band at 407 nm is much lower than the protein absorption at 280 nm. The reverse is true for the fraction eluted at pH-value of 5. This results in the increased RZ-vaiue. Further chromatography o f this fraction is shown in Fig. 3, where the proteins washed through t h e column have an absorbance at 407 nm equal to that at 280 nm. The removal o f protein other than peroxidase and possibly some peroxidase isozymes other than the major cationic fraction [9] results in a fraction of high RZ. Examination of the product by polyacrylamide gel and immunoelectrophoresis showed that only one cationic peroxidase occurred {Fig. 4) compared to at least one anionic and two cationic forms [7,9]. The affinity column appeared to be also specie specific A commercial {Sigma) preparation of horseradish peroxidase in water, as well as an aqueous solution of crude peroxidase from Trametes versicolor [16] were made (5 mg each) and applied to the column with antibodies from the major cationic peanut peroxidase. Neither preparation was bound to the antibodies from major cationic peroxidase from peanuts. Therefore, it implies that the structure of peroxidase is specie-dependent [ 17]. It follows from the above that the process o f purification o f peroxidase is dependent to some degree on purity of the initial sample. However, the carrying capacity of the column does also depend on the purity of the antibodies used. Table II shows that the removal of albumin from the antibodies by Affigel increases the a m o u n t of protein coupled to the column, which is parallel to the a m o u n t of peroxidase carried by the column. In addition the carrying capacity of the column is augmented by binding antibodies to all activated sites on the Sepharose. And the carrying capacity is improved by the use of a sample of greater purity. That principle has been mentioned in relation to Table I.
43
® ~ O ~
O.D° u.~
•
O.D. 4 0 7 n m
O,D.2 8 O h m
! F
~
,° b
1,0
0.5
t /.
o \
"
!
l t
!
q
® O.D. 1.51
o
1.0,
0.5,
I ....
t/'/~''"
%,--o- ~ ,,~o 20
40
60
ml
Fig. 2. Lrnmunoaffinity chromatography of peanut peroxidase. A column of 1.2. × 7.5 cm of antibodies coupled to Sepharose was loaded with peroxidase sample (RZ = 1, 20 mg protein). After washing the column with water the peroxidase linked was eluted with 0.01 M Na acetate (pH 5). Fig, 3. Immunoaffinity chromatography of peanut peroxidase. The conditions for the chromatography are described for Fig. 2. The sample chro;xlatographed contained peroxidase with RZ = 1.5.
44
I
I
mm i
i
4-
Fig. 4. The results of one-dimensional immunoelectrophoresis in comparison to disc electrophoresis of peanut peroxidase which had been purified on antibody-Sepharose column in two consecutive steps. (See Materials and Methods). TABLE II THE IMMUNOAFFINITY ANTIBODY-SEPHAROSE CARRYING CAPACITY AS THE FUNCTION OF ANTIBODIES PURITY COUPLED TO THE MATRIX AND PEANUT PEROXIDASE SAMPLE APPLIED TO THE COLUMN Antibodies linked to 1 g Sepharose (protein) Total antiserum (25 mg) CM Affigei Blue treated (25 rag) CM Affigel Blue treated (50 mg) CM Affigel Blue treated (50 rag)
Protein (rag)
Units of peroxidase a (× 10 -s)
RZ b of sample applied
7.5
13.7
1.0
15.0
27.4
1.0
30.0
68.5
1.0
30.0
168.0
1.5
aOne unit of peroxidase equals 0.001 AA470 nm in 1 rain during assay. bRZ is ratio of absorbance at 470/280 nm.
45 DISCUSSION Purification of peroxidase has been attempted by various means of affinity chromatography [16,18,19]. But invariably some difficulties arise probably due to interspecies variation such as found in this study, between peroxidase from peanut and those from horseradish and a fungus Trametes versicolor. The most specific source of affinity is the one between antigen-antibody. This has a reason for using immunoaffinity chromatography in studies of ribulose 1,5-bisphosphate carboxylase [ 20] and legumin [ 10]. However, these proteins are present in great abundance in plants. In the present study it is shown that immunoaffinity may also be applied to proteins not so prevalent in the plants. The added benefit in using peroxidase, a chromoprotein, is that the affinity and elution process can be seen on the column. The immunoaffinity procedure is also more rapid than ion exchange chromatography. Chaotropic agents such as urea [20] have been used for elution. To avoid denaturation of peroxidase the pH of the column was reduced as reported for other studies [10,23]. When crude peroxidase was purified in the first step of this process the increase in RZ was moderate. This is probably due to the interference of contaminating proteins, which include the minor cationic form and the anionic form of peanut peroxidase [7,9]. In addition the medium of the cultured cells contained probably other cell wall proteins than peroxidase. These proteins are mainly glycoproteins [21]. A non-specific affinity of some glycoprotein for the h y d r o x y l groups of the matrix has been found [22]. Higher purification of peroxidase is found when a sample already prepurified is applied. In conclusion it appears possible to purify peroxidase routinely by affinity chromatography once a sample of antibodies raised against a protein fraction -with high peroxidase activity or high RZ has been obtained. ACKNOWLEDGEMENT The authors are grateful for financial support from the Interdisciplinary Centre of Chemical Physics, University of Western Ontario, which also accorded to J. ~obarzewski, Visiting Fellow status in the Centre. In addition, financial support from N.S.E.R.C.C. to R.B. van Huystee is acknowledged. REFERENCES 1 2 3 4
R.B. van Huystee and W.L. Cairns, Bot. Rev., 46 (1980) 429. K. Hahlbrock and H. Grisebach, Annu. Rev. Plant Physiol., 30 (1979) 109. S.C. Fry, Phytochemistry, 19 (1980) 735. J. ~obarzewski, J. Trojanowski and M. Wojtas-Wasilewska,Holzforschung. (1981) in press. 5 V.C. Kossatz and R.B. van Huystee, Can. J. Bot., 54 (1976) 2089. 6 W.G. Rathmell and L. Sequiera, Plant Physiol., 53 (1974) 317.
46 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
B.M. Maldonado and R.B. van Huystee. Can. J. Bot., 58 (1980) 2280. D. Stephan and R.B. van Huystee, Can. J. Biochem., 58 (1980) 715. D. Stephan and R.B. van Huystee, Z. Pflanzenphysiol., 101 (1981) 313. R. Casey, Biochem. J., 177 (1979) 509. R.B. van Huystee, Bot. Gaz., 137 (1976b) 325. E. Cairns, R.B. van Huystee and W.L. Cairns, Physiol. Plant., 49 (1979) 78. R.A. Reisfeld, U.J. Lewis and D.E. Williams, Nature, 195 (1962) 281. O.P. Srivastava and R.B. van Huystee, Can. J. Bot., 51 (1973) 2207. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. J. ~,obarzewski, Int. J. Biol. Macromol., 3 (1981) 77. R.B. van Huystee and B. Maldonado, Physiol. Plant., 54 (1982) 88. R.B. van Huystee, Can. J. Bot., 54 (1976a) 876. L. Reimann and G.L. Schonbaum, in: S. Fleischer and L. Parker (Eds.), Methods in Enzymology, Vol. 52, Academic Press, 1978, p. 514. J.C. Gray and S.G. Wildman, Plant Sci. Lett., 6 (1976) 91. D.T.A. Lamport, Rec. Adv. Phytochem., 11 (1977) 79. G. Entlicher, M. Ticha, J.V. Kostir and J. Kocourek, Experientia, 25 (1969) 17. D.M. Livingston, in: W.B. Jakoby and M. Wilchek (Eds.), Methods in Enzymology, Vol. 34, Academic Press, 1974, p. 727.