[54] Purification of plant peroxidases by affinity chromatography

514

OTHER HEMOPROTEINSYSTEMS

[54]

[54] Purification of P l a n t P e r o x i d a s e s b y A f f i n i t y Chromatography B y LARS R E I M A N N a n d GREGORY R . SCHONBAUM

In spite of extensive studies, the precise physiological function of plant peroxidase is still uncertain. On the one hand, these enzymes are credited with a central role in plant metabolism, seemingly being involved in numerous processes ranging from metabolism of hormones to host defense mechanisms. On the other hand, such wide reactivity is not readily reconcilable with the finely poised control of, say, hormone metabolism. Yet, these opposing viewpoints are not necessarily exclusive if the expression of a given activity is circumscribed by localization of the enzyme or by the properties of different isoenzymes. It is perhaps not surprising, therefore, that a concerted effort is now being made to isolate different peroxidases and their isoenzymes at various stages of cellular life. Typically, such purifications entail fractionation of plant tissue extracts with ammonium sulfate followed by repeated chromatography of the peroxidase-enriched fractions on CMand DEAE-cellulose. 1-n In this manner and, occasionally, through additional fractionations with organic solvents, 1''1-13 chromatography on hydroxyapatite, 4''3 and electrophoretic separations,12'14-'~ peroxidases have been isolated from (among other sources) pineapple, 4 fig latex, 2,9 turnip roots, 5' 14Japanese 1K. G. Paul, Acta Chem. Scand. 12, 1312 (1958). 2 S. Kon and J. R. Whitaker, J. Food Sci. 30, 977 (1965). a L. M. Shannon, E. Kay, and J. Y. Lew, J. Biol. Chem. 241, 2166 (1966). 4 C. Beaudreau and K. T. Yasunobu, Biochemistry 5, 1405 (1966). 5 E. Mazza, C. Charles, M, Bouchet, J. Ricard, and J. Reynaud, Biochim. Biophys. Acta 167, 89 (1968). 0 H. E. Kasinsky and D. P. Hackett, Phytochemistry 7, 1147 (1968). r y. Morita and S. Ida, Agric. Biol. Chem. 32, 441 (1968). s y. Morita, C. Yoshida, I. Kitamura, and S. Ida, Agric. Biol. Chem. 34, 1191 (1970). 2 M. EI-Fekih and D. Kertesz, Bull. So¢. Chim. Biol. 50, 547 (1968). 10 K. G. Paul and T. Stigbrand, Acta Chem. Scand. 24, 3607 (1970). 11 K. Asada and M. Takahashii, Plant Cell Physiol. 12, 361 (1971). 12 H. Theor¢ll, Ark. Kemi Mineral. Geol. 16A, No. 2 (1942). lz D. Keilin and E. F. Hartree, Biochem. J. 49, 88 (1951). 14 T. Hosoya, J. Biochem. (Tokyo) 47, 369 (1960). is M. H. Klapper and D. P. Hackett, Biochim. Biophys. Acta 96, 272 (1965). le j. S. Whitehead, E. Kay, J. Y. Lew, and L. M. Shannon, Anal. Biochem. 40, 287 (1971). 1~ H. Delincee and B. J. Radola, Eur. J. Bioehem. 52, 321 (1975).

[54]

PURIFICATION OF PLANT PEROXIDASES

515

radish, s rice, 7 spinach leaves, ~1 wheat germ, is'x9 and horseradish roots. 1"3"1°'~2"~3Yet, as noted by Whitehead et al.~6 the efficacy of ionexchange chromatography is occasionally lacking, owing to protein losses during rechromatography of various isoenzyme fractions and the attendant, partial irreversible protein adsorptions. These disadvantages are less pronounced in affinity chromatography with either Sepharose-bound concanavalin A (Con A) 2°-2~ or--as outlined in this account--with an aromatic hydroxamic acid bound to an agarose derivative. However, other limitations must be taken into account. For example, lectins such as Con A have a general affinity for mannose-containing carbohydrates. 2a This necessarily precludes the separation of peroxidases from other glycoproteins. By the same token, the Sepharose-Con A method does not lend itself to a resolution of the peroxidase isoenzymes. 22 Yet, it remains the method of choice in studies on the de n o v o synthesis of peroxidases, especially in investigations bearing on the elucidation of events preceding glycosidation of the protein core. 2°'21 In contrast to the "Con A" method, the "hydroxamate" method is more selective and, at least for enzymes of some members of the mustard family, it facilitates: (a) total separation of cationic from anionic isoenzymes; (b) removal of contaminating proteins from cationic peroxidases; and (c) quantitative recoveries of cationic isoenzymes from the ammonium sulfate precipitates of plant extracts. The selectivity of the hydroxamate method stems from the preferential affinity of aromatic hydroxamic acids for the substrate binding site of peroxidases, particularly for isoenzymes typified by isozyme C of horseradish peroxidase (HRP) 24 (see the table). Synthesis of the Hydroxamic Acid-Affinity Matrix As outlined in the following scheme, the desired derivative, pacylamidobenzohydroxamic acid (IV), is obtained with N-hydroxysuccinimide ester of succinylated Bio-Gel A (Bio-Rad, Affi-Gel 10) used as the initial reactant. The three-stage synthesis involves: (a) reaction of Nhydroxysuccinimide ester (I) with p-aminobenzoic acid (PABA); (b) Ls K. Tagawa and M. Shin, J. Biochem. (Tokyo) 46, 865 (1959). 19 M. Shin and W. Nakamura, J. Biochem. (Tokyo) 50, 500 (1961). zo B. Darbyshire, Physiol. Plant. 29, 293 (1973). 21 R. B. van Huystee, Can. J. Bot. 54, 876 (1976). 22 M. G. Brittain, M. E. Marks, and T. G. Pretlow, Anal. Biochem. 72, 346 (1976). 2z N. Sharon and H. Lis, Science 177, 949 (1972). 24 G. R. Schonbaum, J. Biol. Chem. 248, 502 (1973).

516

OTHER HEMOPROTEIN SYSTEMS

[54]

APPARENT DISSOCIATION CONSTANTS (K1) OF PEROXIDASE-HYDROXAMIC ACID COMPLEXES IN 10 ~ POTASSIUM PHOSPHATE BUFFER, PH 7, AT 25 °

Peroxidase

2-Naphthohydroxamic acid 106KI (M)

Benzohydroxamic acid 106K1 (M)

Horseradish (isoenzyme C) Horseradish (isoenzyme A) Mustard-green leaf Cotton leaf Wheat germ

0.2 10.5 0.1~ 11.3 --

2.3 2480 9.7 1100 1600

e s t e r i f i c a t i o n o f t h e r e s u l t i n g d e r i v a t i v e (II) w i t h p - n i t r o p h e n o l ( P N P ) in the p r e s e n c e o f N-ethyl-N'-(3-dimethyl a m i n o p r o p y l ) c a r b o d i i m i d e ( E D A C ) ; a n d (c) h y d r o x y l a m i n o l y s i s o f e s t e r ( l i D .

O AI~

0

,..~.11 ~ R C~.O j N ~ II

(T)

o

0

,A,A

~/c'~O

R,v..I C I . . ~ N / ~ f J H

~

(TT)

EDAC

o II

0 ,,..11

o II

~/Cxo l( )l [

11'1'I")

0 II

I

NO,

H

~/C"NIO l(

)l

H

('I~T)

R = agarose-O-(CH2),-NH-CO-(CHe)2SCHEME

Synthesis ofp-Acylamidobenzoic

Acid (II)

S u s p e n d a p p r o x i m a t e l y 0.8 m m o l o f N - h y d r o x y s u c c i n i m i d e e s t e r 0 ) 25 ( 5 - 6 g o f B i o - R a d A f f i - G ¢ l 10) in 200 ml o f 50 m M s u c c i n a t e b u f f e r , 25 To evaluate the N-hydroxysuccinimide ester content of Affi-G¢l 10: (a) wash ~100 mg of gel with dry acetonitrile; (b) hydrolyze the ester in 25 mM borate, pH 8.5 for 2 hr; and (c) assay the gel-free supernatant at 260 nm (e2~08960 M -z cm -1) using as reference its absorbance at pH 4.

[54]

PURIFICATION OF PLANT PEROXIDASES

517

PHao, 4.5, containing 50% (v/v) dimethyl formamide (DMF) and 0.3 mol o f p - a m i n o b e n z o i c acid. Gently rotate the suspension for 10-15 hr at 22 °, then wash the gel thoroughly--first, with 50% DMF/succinate buffer, to remove unbound p-aminobenzoic acid, and then with water until free of DMF. To assess the degree of derivatization the gel is solubilized by heating for 2 rain at 95 ° - 5 ° in the presence of 0.3 M methane sulfonic acid. The resulting solution is assayed spectrophotometrically at 270 nm (e270 1.78 × 104 M-1 cm-~). The yield is 90 - 10%.

Esterification o f p - A c y l a m i d o b e n z o i c Acid with p - N i t r o p h e n o l To 200 ml of 25% (v/v) aqueous acetone containing 0.8 mmol of (lI) and 40 mmol of p-nitrophenol, add, at 22 °, 12.6 mmol of EDAC dissolved in 100 ml of water. Maintain the reaction mixture at pH 5 for l hr by dropwise addition of 1 M HCI. Using ice-cold, weakly acidified water, wash the gel on a sintered-glass funnel until no p-nitrophenol can be detected in the filtrate alkalinized with sodium hydroxide. Immediately afterward, withdraw 0.2 - 0.05 g samples of the swollen gel; hydrolyze the p-nitrophenyl ester (III) at pH 13, and assay the released p-nitrophenoi spectrophotometrically at 400 nm (e4o0 20 x 103 M -1 cm -~ at pH 13). The yield is 70 +- 5%.

H y d r o x y l a m i n o l y s i s of E s t e r (III) Dissolve 41 + 1 g o f hydroxylamine hydrochloride in 300 ml of 1 M NaOH; if necessary, adjust to pH 6 and add the resulting solution to ester (III) suspended in 300 ml of water. After a 2-hr reaction at 22 °, the gel is thoroughly washed on a sintered-glass funnel, first with water and then with 50 mM succinate buffer, pH 5.5. To determine the hydroxamic acid content in (IV), the gels are solubilized (see above) and assayed via a modification z6 of the standard L i p m a n n - T u t t l e test for hydroxamates. 2r The yield is 75 + 5%. 26W. P. Jencks and M. Gilchrist, J. Am. Chem. Soc. 86, 5616 (1964). z7 F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945).

518

OTHERHEMOPROTEINSYSTEMS

[54]

t) tx

f\ o

I \ I \ I

\\

O O FIG. 1. Purification of crude horseradish peroxidase at 4 °. Crude enzyme (1.5 g, RZ 1) was applied to 1.5 × 35 cm column of affinity matrix (IV) and washed with 200 ml of 50 mM succinate buffer, pH 5.5; the retained peroxidases were eluted (8 mllhr) with 30 mM phosphate buffer, pH 7, containing 0.1 M boric acid (arrow). The properties of eluents were monitored spectrophotometrically at 280 n m ( ) and 403 nm and 412 nm ( - - - ) , using a light path of 0. I cm. Note: crude enzyme contains a significant fraction of low-spin cyanoperoxidase whose absorbance is isosbestic with that of the high-spin peroxidase at 4125 ,~. RZ (A4o3/Azso) of fractions eluting between 340 and 420 ml was 3.25 - 0.1.

Chromatography of Crude Horseradish Peroxidase (I-IRP) Dissolve 50-500 mg of lyophilized horseradish root extract (RZ -> 0.3) in 1-2 ml of 50 mM succinate buffer, pH 5.5. Apply the resulting solution to a 1.5 × 35 cm column of affinity matrix (IV), previously equilibrated with the same buffer, at 4 °. After removal of proteins 2s that do not bind to benzohydroxamic acid (Fig. 1), elute the peroxidase(s), at a flow rate not exceeding 15 ml/cm ~ per hour, with a ligand having an affinity either for the hydroxamate group (e.g., borate, 0.1-0.2 M) or for the enzyme (free hydroxamic acid, 2 mM). Borate is a preferable eluent since, unlike hydroxamate, it is readily removed by simple dialysis. a8 Among proteins not retarded on the affinity matrix are: a copper protein, possibly umecyanin; ~° a cyanoperoxidase;29 and HRP isoenzymes A, and A2. ~'a 2a I. Yamazaki, R. Nakajima, H, Honma, and M. Tamura, Biochem, Biophys. Res. Commun. 27, 53 (1967).

[54]

PURIFICATION OF PLANT PEROXIDASES

5 19

Such single-pass chromatography is remarkably effective in separating the cationic peroxidase isozyme both from the anionic isoenzymes and from other components of crude horseradish root isolates; it gives a peroxidase with purity equivalent to that obtained conventionally through three- to four-cycle ion-exchange chromatography. 3'1° Such an enzyme is characterized by R Z - 3 . 2 5 +_ 0.1 (Figs. 1 and 3) and, judging from gel isoelectric focusing, it appears to be homogeneous (Fig. 2).

-Ia

b

FIG. 2. Gel isoelectrofocusing of (a) crude horseradish peroxidase (30/xg, R Z - 1 ) , and (b) sample purified on the affinity column (23 txg, RZ-3.32 -+ 0.02) on 5% acrylamide containing I% Ampholine, pH 2-10. The isoelectric point of the isoenzyme in (b) is 9.0 ± 0.2. [K. G. Paul and T. Stigbrand, Acta Chem. Scand. 24, 3607 (1970); K. G. Welinder, L. B. Smillie, and G. R. Schonbaum, Can. J. Biochem. 50, 44 (1972).]

520

OTHER HEMOPROTEINSYSTEMS

[54]

3

oE co' ..Q

o

2N

o

t

loo

260

(ml

o

FIG. 3. Isolation of horseradish peroxidase isozyme C at 4 °. Enzyme (100 rag, RZ-2.8

- 0.2) was applied to l x 30 cm column of(IV) and washed with 70 ml of 50 mM succinate buffer, pH 5.5; the retained peroxidases were eluted with 30 mM phosphate buffer, pH 7, containing 0.2 M boric acid (arrow). The properties of the eluents were monitored spectrophotometrically at 403 nm ( ) and 280 nm, RZ = A4oJA28o (L-~--e). There are two reasons, however, against sole reliance on the affinity method for isolating peroxidases from crude plant extracts: first, ion exchange is decidedly faster; and second, the affinity ligand is slowly degraded by some plant component(s). Thus, for the isolation of the highest-purity e n z y m e (Fig. 3), yet at the same time avoiding a partial degradation o f the affinity ligand, it is advantageous to prepurify the crude peroxidase by one-cycle chromatography on DEAE-cellulose. Used in this manner, the affinity matrix retains its properties for extended periods (months) when kept at p H < 7.5. cf'3° Moreover, after elution with borate, or hydroxamate, the affinity support can be "regene r a t e d " upon thorough washing with buffers of p H < 7.5. When not in use, the gel should be preserved at approximately pH 6 in a medium containing a bacteriostatic agent. Isozyme Patterns of Plant Peroxidases In view o f the preferential interaction o f hydroxamic acids with isoenzymes comparable to H R P isozyme C, affinity chromatography a0G. I. Tesser, H. U. Fisch, and R. Schwyzer, Heir. Chim. Acta 57, 1718 (1974).

[55]

PURIFICATION OF CHLOROPEROXIDASE

521

allows a rapid evaluation of isoenzyme patterns in various plant tissues and the purification of "high affinity" components. For example, the activity of peroxidases in freshly harvested mustard-green leaves is predominantly attributable to isoenzymes that avidly bind benzohydroxamic acid (see the table). They can therefore be readily purified by the affinity method. 32 By contrast, the dominant peroxidases in wheat germ, sweet potato, and "aged" cotton leaves, like isoenzyme A of HRP, interact only weakly with benzohydroxamic acid (see the table). As might be expected, in these cases affinity chromatography is not the preferred method for purification of the dominant enzymes. 31 :u L. Reimann, unpublished observations, 1976.

[55] P u r i f i c a t i o n o f C h l o r o p e r o x i d a s e

from

Caldariomyces

fumago By PAUL F. HALLENBERGand LOWELL P. HAGER A H + X - + H202+ H+---~ AX + 2H20

(1)

Chloroperoxidase (chloride:hydrogen peroxide oxidoreductase, EC 1.11.1.10) is a monomeric heme protein (molecular weight -42,000) which was isolated in our laboratory during the course of studies on the mechanisms of the biological halogenation reactions involved in the biosynthesis of caldariomycin.l-7 Chloroperoxidase is a glycoprotein and is excreted into the growth medium by Caldariomyces fumago during the final stages of growth, s Chloroperoxidase concentrations in the culture medium reach levels as high as 100 mg per liter of culture medium, and therefore this organism is an excellent source for this enzyme. Chioroperoxidase catalyzes three different types of reactions; all of which use hydrogen peroxide as the oxidant. The most studied of these P. D. Shaw and L. P. Hager, J. Am. Chem. Soc. 81, 1011 (1959). 2 p. D. Shaw, J. Beckwith, and L. P. Hager, J. Biol. Chem. 234, 2560 (1959). P. D. Shaw and L. P. Hager, J. Biol. Chem. 234, 2565 (1959). 4 p. D. Shaw and L. P. Hager, J. Am. Chem. Soc. 81, 6527 (1959). 5 j. Beckwith and L. P. Hager, J. Org. Chem. 26, 5206 (1961). P. D. Shaw and L. P. Hager, J. Biol. Chem. 236, 1626 (1961). 7 j. R. Beckwith, R. Clark, and L. P. Hager, J. Biol. Chem. 238, 3086 (1963). s D, R. Morris and L. P. Hager, d. Biol. Chem. 241, 1763 (1966).