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Investigations on the multiple components of commercial horseradish peroxidase
272
BIOCHIMICAET BIOPHYSICAACTA BBA 65161
I N V E S T I G A T I O N S ON T H E M U L T I P L E COMPONENTS OF COMMERCIAL HORSERADISH PEROXIDASE
MICHAEL H. KLAPPER AND DAVID P. HACKETT Department of Biochemistry, Universi(~, of California, Berkeley, Calif. ( U.S.A .) (Received September 23rd, 1964)
SUMMARY
Investigations on the structure of horseradish peroxidase (donor:H202 oxidoreductase, EC I . I I . I . 7 ) , including its amino acid and carbohydrate composition, are described. Commercial horseradish peroxidase preparations contain multiple components which can be separated by electrophoresis on starch. The major components have been purified and their properties compared; they do not differ significantly in size, absorption spectrum, enzymatic activity, or amino acid composition. Horseradish peroxidase contains the following sugars: glucose, galactose, mannose, arabinose, xylose, fucose and hexosamine.
INTRODUCTION Peroxidase (donor :HeO 2 oxidoreductase, EC i . i i. 1.7) is very widely distributed in plants and the enzyme has been purified from such diverse sources as the fig1, horseradish2, 3, yeast 4, sweet potato 5, broad bean 6, turnip 7, Japanese radish s, lupine 9 and wheat 1°. Of these enzymes, the one from horseradish (Amoracia lapathifolia) root has been studied most extensively. Horseradish peroxidase has a molecular weight of approx. 4 ° 000 (see refs. 3, I I ) and contains I mole of protohematin IX. Following hydrolysis of the protein, THEORELL AND ~KESON li found 2 moles of histidine, 18 of arginine, and 12 of lysine per mole of horseradish peroxidase. MAEHLY AND PALI~USla subsequently reported that acid hydrolyzates of horseradish peroxidase contained all the common amino acids except tryptophan and hydroxyproline, but no further studies on the amino acid composition of horseradish peroxidase have been reported. In an early study le, horseradish peroxidase was found to contain a relatively large amount of carbohydrate, but the nature of this fraction was not determined. Meanwhile, MORITA14 has examined in some detail both the amino acid and carbohydrate composition of a Japanese radish peroxidase. We report here a qualitative identification of the sugars and a quantitative examination of the amino acids in horseradish peroxidase. Of special interest is the fact that peroxidase preparations commonly contain Biochim. Biophys. Acta, 96 (1965) 272-282
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more than one enzymatically active component. Electrophoresis on either paper 15 or starch gel 16 has revealed a multiplicity of peroxidases both in extracts of horseradish roots and in commercial preparations of horseradish peroxidase; PAI~L17 separated five horseradish peroxidase components by ion-exchange chromatography. Multiple components have also been demonstrated in the peroxidase preparations from sweet potatoS, is, Japanese radish 8, turnip 19, wheat 20, pea 21, and other leguminous plants 22. In no case has the structural basis of this heterogeneity been established. In the present study, we have investigated the possibility that the components might differ in their sizes, amino acid compositions, spectral properties or enzymatic activities. Our previous conclusion regarding the mechanism of peroxidasecatalyzed oxidations ~3 was based on the assumption that these components show the same catalytic activities. MATERIALS AND METHODS
Horseradish peroxidase was obtained from the California Corporation for Biochemical Research, Los Angeles, Calif. (U.S.A.) and from the Worthington Biochemical Corporation, Freehold, N.J. (U.S.A.).
Starch-gel electrophoresis Partially hydrolyzed starch (Connaught Medical Research Laboratories, Toronto (Canada)) was washed with a mixture containing equal volumes of n-propanol and 0. 5 N hydrochloric acid (8 1 for each 500 g of starch) and then with water to remove the acid propanol. The starch was finally washed with 95% alcohol and dried at room temperature for 2-3 days. The gel block was prepared as described by SMITH 24, using 13 g of the washed starch in IOO ml of a buffer composed of 9 parts o.o16 M Tris-o.o33 M citric acid (pH 8.1) and I part 0.02 M lithium hydroxide0.076 M boric acid (pH 8.1). The peroxidase (0.3 mg) was spotted on a small rectangle of Whatman No. 3 MM paper, which was placed in the middle of the precooled gel block. The block was connected by paper wicks to the electrode tanks, which contained fresh solutions of 0.38 M boric acid and o.I M lithium hydroxide, adjusted to pH 8.i with acetic acid. Electrophoresis was carried out for 3-4 h at 4 ° with a current of 25 mA passing through the gel bed; the potential varied from 20 to 30 V/cm during the run. After electrophoresis, the gel was cut in half. The top half was stained for protein by placing the gel in a o.05% solution of nigrosin in methanolacetic acid-water (5o:1o:4 o) for I h and then transferring it to a 0.009% nigrosin solution. The bottom half of the gel was stained for peroxidase activity by immersing it for I min in IO ml of benzidine-saturated water containing o.3% hydrogen peroxide 15 and then placing it in methanol-acetic acid-water (5o:1o:4o). The regions of the gel containing peroxidase turned blue immediately and then turned green; after a few days, discrete brown bands formed where the peroxidase components were localized.
Puri3cation of peroxidase components The starch-gel method described above was modified to permit purification of the various peroxidase components. The gel bed was made in a tray of the type described by SMITHIES25. A solution containing 15-17 mg of horseradish peroxidase Biochim. Biophys. Acta, 96 (1965) 272-282
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M. H. KLAPPER, D. P. HACKETT
in o.3-o.5 ml of water was pipetted into a slot running the width of the gel bed. Electrophoresis was conducted at 4 ° for 12 h. The current was maintained at 3o mA until the voltage increased to 39 ° V, after which the voltage was kept constant at this level ; the potential across the gel rose from 26 V/cm initially to 43 V/cm. After 12 h, the clearly separated brown bands were cut ifrom the gel bed and stored at 4 ° until processed further. In order to recover the purified peroxidase components, gel strips from three separate electrophoresis runs were ground together with 25 ml of water in the chilled cup of a Servall omnimixer. The resulting suspension was centrifuged at approx. 25 ooo × g for o. 5 h and the supernatant decanted off. The starch pellet was washed with water in the same manner two more times. The three supernatant solutions, which contained the enzyme, were pooled, concentrated by dialysis against solid sucrose, and then dialyzed against water. Some starch precipitated out during the concentration step and this was removed by centrifugation at 8o ooo × g for 1. 5 h. The clear supernatant was further concentrated by ultrafiltration 26, dialyzed against o.oi M sodium borate buffer (pH 8.6) and once again centrifuged at 8o ooo × g for 1. 5 h. Finally, to remove any contaminating starch, 1-2 ml of the enzyme in borate buffer was adsorbed onto a column (o. 9 × IO cm) of DEAE-Sephadex (Pharmacia, Uppsala (Sweden)) which had been equilibrated with the same buffer. The column was washed with 3° ml of the buffer and the peroxidase then eluted with a solution of o.oi M sodium borate and o.o 7 M sodium chloride (pH 8.6). The eluted protein was concentrated, dialyzed against water and stored in the freezer.
A m i n o acid analysis The amino acid compositions of the purified components were determined by standard chromatographic techniques. Approx. 2 mg of each of the components were hydrolyzed in 6 N hydrochloric acid in vacuo for 22 h at IiO °. The acid hydrolyzates were analyzed in a Beckman Model 12o amino acid analyzer by the method of SPACKMAN, STEIN AND MOORE z~.
Qualitative sugar analyses Approx. 2o mg of commercial horseradish peroxidase were hydrolyzed in I M sulfuric acid for 4 h at IOO° in vacuo. The hydrolyzate was neutralized with solid barium carbonate, and, after removing the precipitate by filtration, was applied to a column (0. 5 × 6 cm) of Dowex AG-5o W-X8, 5O-lOO mesh, hydrogen form (Bio-Rad Laboratories, Richmond, Calif. (U.S.A.)). The neutral sugars were washed through the column with 5 ml of water and the amino sugar fraction was eluted with IO ml of 0. 5 N hydrochloric acid. Both fractions were evaporated to dryness. The neutral sugar fraction was dissolved in 0.04 ml of water; 0.03 ml of this solution was streaked on Whatman No. I filter paper, and the remaining o.oi ml was spotted on the same paper as a guide. After carrying out the descending chromatography using ethyl-acetate-pyridine-water (8:2:1) as the solvent, the guide strip was sprayed with p-anisidine phosphate (2 g p-anisidine phosphate in 80 ml of water and 200 ml of 95 % ethanol) to locate the partially resolved sugars. The sugars on the unsprayed portion of the chromatogram were then eluted, and further analyzed by paper chromatography using a variety of solvent systems (Table IV). The amino sugar fraction obtained from the Dowex-5o column was analyzed for Biochim. Biophys. Acta, 96 (1965) 272-282
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Fig. i. Starch-gel electrophoresis of four horseradish peroxidase preparations. The electrophoretic p a t t e r n s on the top are, from left to right, Calbiochem lot No. 3547 ° and W o r t h i n g t o n lot No. 6253 stained with nigrosin, and W o r t h i n g t o n lot No. 6253 and Calbiochem lot No. 3547o stained with benzidine-hydrogen peroxide; on the bottom, Calbiochem lots Nos. 5o3339 and 35374 stained with nigrosin, and lots Nos. 35374 and 5o3339 stained with benzidine-hydrogen peroxide.
Biochim. Biophys. Acta, 96 (1965) 272-282
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M.H. KLAPPER, D. P. HACKETT
Fig. 2. Starch-gel electrophoresis of three purified peroxidase components. The p a t t e r n on the left represents unfractionated Calbiochem lot No. 3547 o. On the right are the three purified fractions: from left to right, horseradish peroxidase Nos. IV, l I I and II. All bands stained with nigrosin. h e x o s a m i n e b y t h e m e t h o d o f CESSI as d e s c r i b e d b y JOHANSEN et al. 2s. Sialic a c i d w a s d e t e r m i n e d b y t h e t h i o b a r b i t u r i c a s s a y o f WARREN 29 a f t e r h e a t i n g 1. 3 m g o f u n f r a c t i o n a t e d p e r o x i d a s e for I h i n 0.05 M s u l f u r i c acid.
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HORSERADISH PEROXlDASE STRUCTURE RESULTS
Heterogeneity of commercial horseradish peroxidase All of the commercial peroxidase preparations tested were found to be heterogeneous by starch-gel electrophoresis. The electrophoretic patterns obtained with four different preparations are shown in Fig. I; it is clear that they are similar, though not identical. One preparation (Calbiochem, lot No. 503339) contained nine protein-staining bands, all of which showed peroxidase activity; two additional bands showed peroxidase activity but gave no protein stain. Thus, this one preparation contained at least eleven electrophoretically distinct components with peroxidase activity. Every preparation contained appreciable amounts of the components which have been labeled horseradish peroxidase No. I, No. II, No. III and No. IV (Fig. 2). The other bands were present in much lower concentrations and were not detected in all the preparations. The major peroxidase components were purified by preparative starch-gel electrophoresis. Although the method limited the amount of material that could be processed conveniently at one time and only 4o-5o% of the applied material was recovered, the resolution was good (Fig. 2). The purified peroxidases (Nos. II, III and IV) were only slightly contaminated by their respective neighbors. These results show that the multiplicity observed in starch-gel eleetrophoresis is not an artifact of the separation method. The fact that the purified components migrated as single bands with the same mobilities as the corresponding components in the unfractionated preparation suggests that the multiplicity is not the result of reversible changes involving a single peroxidase.
Physical, chemical, and enzymatic properties of the purified peroxidase components Electrophoresis in starch gel can resolve proteins on the basis of both charge and size 30. In order to determine whether the peroxidases differ in size, the sedimentation characteristics of the unfractionated horseradish peroxidase preparation and of several of the purified components were compared in the ultracentrifuge. The whole horseradish peroxidase preparation sedimented as a single major band with a sedimentation coefficient (s20,w) of 3.42. This value agrees well with the sedimentation coefficients reported earlier by THEORELL~2 (S2o,w = 3 . 8 5 ) and by CECIL AND O G S T o N l l ($20'w - - 3 . 4 8) f o r unfractionated horseradish peroxidase. The monodisperse TABLE I SEDIMENTATION COEFFICIENTS OF PURIFIED PEROXIDASE COMPONENTS The sedimentation was followed at 4o4 m/~ in an ultracentrifuge equipped w i t h a b s o r p t i o n optics al. The purified c o m p o n e n t s were dissolved in o.i M p h o s p h a t e buffer (pH %o) at the following concentrations (mg/ml): I, o.40; I I , o.34 ; I I I , o.36; and IV, 0.45. Compone~tt
s9.o,w
I II III IV
3.43 3.7 ° 3.71 3.4 °
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M. H. KLAPPER, D. P. HACKETT
TABLE II ACTIVITIES
OF PURIFIED
PEROXIDASE
COMPONENTS
2 - M e t h y l - I , 4 - n a p h t h o h y d r o q u i n o n e oxidation was m e a s u r e d at 30°; 0.o 5 ml of oxygen-free O.Ol N HC1 solution containing o.8 mg 2 - m e t h y l - i , 4 - n a p h t h o h y d r o q u i n o n e / m l was added to a solution of horseradish peroxidase in 0.95 ml of 0.o 5 M citrate buffer (pH 6.0) and the reaction was followed b y the increase in a b s o r b a n c y at 262 m/~33. o-Dianisidine peroxidation was measured at 3 °0 b y the m e t h o d described in the catalogue of the W o r t h i n g t o n Biochemical Corp. 34, with the exception t h a t o.o 5 M citrate buffer (pH 6.0) was used. I n b o t h assays the enzyme concent r a t i o n was varied from 4 to 20 m/~g/ml. The results have been expressed as the molar oxidation per m i n u t e effected by a solution of enzyme with an a b s o r b a n c y of 1.oo at 4o3 m/~.
Component
2-Methyl-z,4-naphthohydroquinone oxidation (moles 2-methyl-x, 4naphthohydroquinone/ liter × min/A4o 3 mu)
o-Dianisidine peroxidation (moles H202[ liter × min/A 403 mu)
I II III IV V
0.37 0.45 o.5i 0.46 0.50
1.3 1.9 2.2 1.9 2.2
TABLE Ill AMINO ACID COMPOSITION OF PURIFIED PEROXIDASE COMPONENTS Amino acid analyses were performed as described in the text. The values given h a v e been calculated b y dividing the a m o u n t of one amino acid recovered b y the a m o u n t of all the a m i n o acids recovered. The values for the three c o m p o n e n t s are each the average of three d e t e r m i n a t i o n s and are given t o g e t h e r with the calculated s t a n d a r d errors.
Amino acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine A~anine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Mole fraction × zoo Horseradish peroxidase No. I
Horseradish peroxidase No. I I
Horseradish peroxidase No. I I I
2.06 0.90 6.79 16.5 8.23 7.66 7.18 5.65 5-79 8.38 5.46 1.17 4.36 11.6 1.5o 6.70
1.75 0.80 6.12 16.8 8.35 7.66 7.13 5.67 5.74 8.11 5.42 1.17 4.25 12.1 1.39 6.76
1.89 0.84 6.86 16.8 8.36 7.72 7.13 5.81 5.79 8.03 ,5-44 1.13 4.08 12.1 1.58 6.70
4~ 444444444± ± 4± :[:
0.29 0.22 0.98 0.52 o.16 o.16 o.16 o.ii 0.24 o.31 o.13 0.04 o.12 0.32 o.17 o.io
444± 444± 4444` 44± 4-
Biochim. Biophys. Acta, 96 (1965) 272-282
0.04 0.20 0.55 0.30 o.21 0.24 o.17 o.13 o.14 o.18 o.16 o.17 o.17 0.45 0.20 o.ii
44± 4444444± 4444:t:
o.i 5 o.14 0.67 0.09 0.06 0.22 0.06 0.34 0.04 o.21 o. i8 o.23 0.o9 0.27 0.04 0-34
HORSERADISH PEROXlDASE STRUCTURE
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b e h a v i o r of t h e h o r s e r a d i s h p e r o x i d a s e p r e p a r a t i o n s u g g e s t s t h a t t h e s e v e r a l e l e c t r o p h o r e t i c a l l y s e p a r a b l e c o m p o n e n t s do n o t differ g r e a t l y in size. T h i s was t e s t e d d i r e c t l y b y d e t e r m i n i n g t h e s e d i m e n t a t i o n coefficients of f o u r p u r i f i e d c o m p o n e n t s in a n u l t r a c e n t r i f u g e e q u i p p e d w i t h a b s o r p t i o n optics al. T h e results (Table I) i n d i c a t e t h a t t h e y are all t h e s a m e size. T h e a b s o r p t i o n s p e c t r a o f t h e o x i d i z e d f o r m s of t h e f o u r p u r i f i e d p e r o x i d a s e s w e r e c o m p a r e d . T h e s p e c t r a of h o r s e r a d i s h p e r o x i d a s e Nos. I, II, n I , a n d I V are i d e n t i c a l a n d i n d i s t i n g u i s h a b l e f r o m t h e s p e c t r u m of h o r s e r a d i s h p e r o x i d a s e r e p o r t e d by KEILIN AND HARTREE a. T h e five p u r i f i e d c o m p o n e n t s w e r e t e s t e d for t h e i r e n z y m a t i c a c t i v i t i e s , as m e a s u r e d b y t h e i r abilities to c a t a l y z e t h e o x i d a t i o n of 2 - m e t h y l - I , 4 - n a p h t h o h y d r o q u i n o n e 33 a n d t h e p e r o x i d a t i o n of o - d i a n i s i d i n e a4. As s h o w n in T a b l e I I , t h e y all h a d e s s e n t i a l l y i d e n t i c a l a c t i v i t i e s in b o t h assays; t h e a c t i v i t i e s of h o r s e r a d i s h p e r o x i d a s e No. I were s l i g h t l y l o w e r t h a n t h o s e o f t h e o t h e r f o u r c o m p o n e n t s . T h e a m i n o a c i d c o m p o s i t i o n s of h o r s e r a d i s h p e r o x i d a s e Nos.I, I I a n d I I I a r e s h o w n in T a b l e I I I . T h e r e s u l t s i n d i c a t e t h a t t h e a m i n o a c i d c o m p o s i t i o n s o f t h e d i f f e r e n t p e r o x i d a s e s are v e r y similar, if n o t i d e n t i c a l . N e v e r t h e l e s s , t h e r e m a y be s m a l l differences in c o m p o s i t i o n w h i c h w e r e n o t d e t e c t e d d u e t o t h e errors i n h e r e n t in t h e procedure. The cysteine and tryptophan content were not analyzed.
Qualitative sugar analysis An unfractionated preparation of commercial horseradish peroxidase was TABLE IV SUGARS IN HORSERADISH
PEROXIDASE
All chromatograms were run by descending chromatography on Whatman No. i paper except for those in which Solvent A was used, where Whatman No. 52 was the support. Razu is the distance a sugar travelled relative to glucose, and Rxyz is the distance relative to xylose. The solvent systems are: Solvent A, ethylacetate-pyridine-water (8:2:i); Solvent B, phenol-water (3:I) ; Solvent C, butanol-ethanol-water (4 :I :I) ; Solvent D, benzene-butanol-pyridine-water (I :5:3:3) ; Solvent E, butanol-pyridine-water (io :3:3). Sugar
Solvent A RXyl
B Ra~u
C Ralu
D RGtu
E RGlu
Unknown I.OI Fucose I.OO
1.38 1.51
1.63 1.65
--
--
Unknown i.oi Xylose i.oo
1.o 7 1.12
1.42 1.44
---
---
Unknown o.71 Arabinose 0.75
1.25 1.22
1.25 1.25
---
---
Unknown Mannose Unknown Glucose
1.o6 1.o6 -i.oo
1.23 1.23 1.oi
-1.o8 0.99
-1.23 i .oo
I .oo
I.OO
I ,oo
--
o.82 0.81
o.89 o.89
0.55 o.56 ---
Unknown . Galactose - -
.
.
. --
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M. H. KLAPPER, D. P. HACKETT
hydrolyzed in acid, after which the neutral sugars were separated from amino acids, peptides, and amino sugars b y chromatography on Dowex-5o. The results of the paper chromatography of the sugars using five different solvent systems are shown in Table IV. At least six neutral sugars were identified: xylose, arabinose, fucose, mannose, galactose, and glucose. Evidence was also obtained for the presence of an amino sugar. Both the amino sugar fraction eluted from the Dowex-5o column and a whole hydrolyzate (3 N HC1 for 4 h at 95 ° in vacuo) of horseradish peroxidase gave positive tests in the CESSI assay for hexosamine. During the chromatography of the neutral and acidic amino acids in the amino acid analyzer, an unknown ninhydrin-positive peak appeared either between leucine and tyrosine or after phenylalanine, depending on the experimental conditions. This type of behavior is characteristic of glucosamine 27. In addition to these seven sugars, unidentified compounds giving positive tests with p-anisidine phosphate were seen on the paper chromatograms. A test for N-acetylneuraminic acid was negative.
DISCUSSION
The amino acid composition of horseradish peroxidase does not show any remarkable features. THEORELL AND AKESON12 reported earlier that there are 12 lysine residues/mole of horseradish peroxidase, but our data indicate that there are only 4 lysines. This discrepancy is due to the fact that they determined the lysine content by nitrogen difference. Our results suggest indirectly that there m a y be as m a n y as 8-1o hexosamine residues in horseradish peroxidase. Another interesting point is that horseradish peroxidase contains approximately two times as m a n y acidic as basic amino acids. In spite of this, almost all of the horseradish peroxidase components migrate towards the cathode at p H 8.6. The protein almost certainly contains a large amount of glutamine and asparagine, although no quantitative determination of the amide contents was made. Horseradish peroxidase does not contain any hydroxyproline, although this amino acid does occur in Japanese radish peroxidase a 14. The latter enzyme is much richer than horseradish peroxidase in alanine, serine, and aspartic acid, but it contains relatively little glycine and arginine. All of the seven sugars identified in the horseradish peroxidase hydrolyzate have been found previously in glycoproteins: glucose, galactose, mannose, fucose, and glucosamine are present in m a n y animal proteins 3~, while the pentoses arabinose and xylose have been identified in preparations of Japanese radish peroxidase 14. The nature of the carbohydrate-protein linkage in horseradish peroxidase is not yet known. The physical basis of the multiplicity of horseradish peroxidase components has not yet been established. The heterogeneity observed in starch-gel electrophoresis is probably due only to charge differences, since the components examined appear to have the same size. The differences in charge could be the result of differences in amino acid composition, amide content, protein conformation, or sugar content. Of these possibilities, only the first has been examined in detail here. The data do not indicate any significant differences in amino acid composition between the various components, but the possibility cannot yet be absolutely ruled out. The significance of the multiplicity is not at all clear. The possibility that the components arise as Biochim. Biophys. Acta, 96 (f965) 272-282
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281
artifacts during the isolation has not yet been ruled out. Assuming that the several components are present in intact tissues, the possibility that they are related to genetic, developmental, or histological heterogeneity in the starting material should be examined. The fact that the five purified peroxidase components had identical catalytic activities in the two reactions assayed suggests that differences in their relative amounts probably have little effect on the enzymatic activities of unfractionated horseradish peroxidase preparations. We reported earlier ~z that the horseradish peroxidase-catalyzed oxidation of 2-methyl-I,4-naphthohydroquinone can be inhibited 80% by carbon monoxide under conditions where only IO to 15% of the enzyme is in the ferrous-CO form. This led us to the conclusion that the ferrous enzyme is involved in the catalysis of 2-methyl-I,4-naphthohydroquinone oxidation. If, however, the oxidation of 2-methyl-I,4-naphthohydroquinone had been catalyzed by a single component which made up only a small fraction of the horseradish peroxidase preparation, this conclusion would not have been valid. The results presented here support the previous conclusion.
ACKNOWLEDGEMEN'fS
This work was supported by a grant from the National Science Foundation. The authors are indebted to Misses F. PUTNEY and B. CHII% and Mr. D. PETERSON for assistance in the determinations of the sedimentation coefficients and the amino acid compositions.
REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25
J. B. SUMNER AND S. F. HOWELL, Enzymologia, i (1936) 133. H. THEORFLL, Enzymologia, io (1941) 25 o. D. KEILIN AND E. V. HARTREE, Biochem. J., 49 (1951) 88. R. ABRAMS, A. M. ALTSCHUL AND T. R. HOGNESS, J. Biol. Chem., 142 (1942) 303 . t(. ]4~ONDO AND Y. MORITA, Bull. Res. Inst. Food Sci. Kyoto Univ., io (1952) 33. Y. MORI'rA, Bull. Res. Inst. Food Sci. Kyoto Univ., 15 (1954) 56. I. YAMAZAKI, K. FUJINAGA, I. TAKEHARA AND H. TAKAHASHI, J. Biochem. Tokyo, 43 (1956) 377Y. MORITA AND K. I~AMEDA, Mere. Res. Inst. Food Sci. Kyoto Univ., 12 (1957) I. R. E. STUTZ, Plant Physiol., 32 (1957) 31. K. TAGAWA ANn M. SHIN, J. Biochem. Tohyo, 46 (1959) 865. R. CECIL AND A. G. OGSTON, Biochem. J., 49 (1951) lO5. H. THEORELL AND A. AKESON, Arhiv Kemi Mineral. Geol., 16 (1943) No. 8. A. G. MAEHLY AND S. PALEUS, Acta Chem. Scan&, 4 (195 o) 508. Y. MORITA AND K. KAMEDA, Bull. Agr. Chem. Soc. Japan, 23 (1959) 28. M. A. JERMVN AND R. THOMAS, Biochem. J., 56 (1954) 631. M. D. POULIK, Nature, 18o (1957) I477. K. G. PAUL, Acta Chem. Scan&, 12 (1958) 1312. N. KAWASHIMA AND I. URITANI, Agr. Biol. Chem. Tokyo, 27 (1963) 409. T. HOSOYA, J. Biochem. Tokyo, 47 (196o) 369. M. SHIN AND W. NAKAMURA, J. Biochem. Tokyo, 5 ° (I96I) 500. 19. K. MACNICOL AND J. REINERT, Z. Naturforsch., ISb (1963) 572. E. MOUSTAFA, Nature, 199 (1963) 1189. M. H. KLAPPER AND D. P. HACKETT, J. Biol. Chem., 238 (1963) 3743I. SMITH, Chromatographic and Electrophoretic Techniques, Vol. 2, Interscience, New York, t96o, p. 131. O. SMITHIES, Biochem. J., 71 (1959) 585 .
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26 E. A. PETERSON AND I-l. A. SOBER, in S. P. COLOWICK AND N. O. I~APLAN, Methods of Enzymology, Vol. 5, Academic Press, N e w York, 1962, p. 25. 27 D. H. SPACKMAN,W. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 1185. 28 P. G. JOHANSEN, R. D. MARSHALL AND A. NEUBERGER, Biochem. J., 77 (196o) 239. 29 L. WARREN, J. Biol. Chem., 234 (1959) 1961. 3 ° O. SMITHIES, Arch. Biochem. Biophys. Suppl., I (1962) 125. 31 H. K. SCHACHMAN,Biochemistry, 2 (1963) 887. 32 H. THEORELL, Arkiv Kemi Mineral. Geol., 15 (1942) No. 24. 33 M. H. I~LAPPER AND D. P. HACKETT, J. Biol. Chem., 238 (1963) 3736. 34 Worthington Biochemical Corp. Descriptive Manual, i i (1961)45. 35 R. G. SPIRO, New Engl. J. Med., 269 (1963) 566.
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BIOCHIMICAET BIOPHYSICAACTA BBA 65161
I N V E S T I G A T I O N S ON T H E M U L T I P L E COMPONENTS OF COMMERCIAL HORSERADISH PEROXIDASE
MICHAEL H. KLAPPER AND DAVID P. HACKETT Department of Biochemistry, Universi(~, of California, Berkeley, Calif. ( U.S.A .) (Received September 23rd, 1964)
SUMMARY
Investigations on the structure of horseradish peroxidase (donor:H202 oxidoreductase, EC I . I I . I . 7 ) , including its amino acid and carbohydrate composition, are described. Commercial horseradish peroxidase preparations contain multiple components which can be separated by electrophoresis on starch. The major components have been purified and their properties compared; they do not differ significantly in size, absorption spectrum, enzymatic activity, or amino acid composition. Horseradish peroxidase contains the following sugars: glucose, galactose, mannose, arabinose, xylose, fucose and hexosamine.
INTRODUCTION Peroxidase (donor :HeO 2 oxidoreductase, EC i . i i. 1.7) is very widely distributed in plants and the enzyme has been purified from such diverse sources as the fig1, horseradish2, 3, yeast 4, sweet potato 5, broad bean 6, turnip 7, Japanese radish s, lupine 9 and wheat 1°. Of these enzymes, the one from horseradish (Amoracia lapathifolia) root has been studied most extensively. Horseradish peroxidase has a molecular weight of approx. 4 ° 000 (see refs. 3, I I ) and contains I mole of protohematin IX. Following hydrolysis of the protein, THEORELL AND ~KESON li found 2 moles of histidine, 18 of arginine, and 12 of lysine per mole of horseradish peroxidase. MAEHLY AND PALI~USla subsequently reported that acid hydrolyzates of horseradish peroxidase contained all the common amino acids except tryptophan and hydroxyproline, but no further studies on the amino acid composition of horseradish peroxidase have been reported. In an early study le, horseradish peroxidase was found to contain a relatively large amount of carbohydrate, but the nature of this fraction was not determined. Meanwhile, MORITA14 has examined in some detail both the amino acid and carbohydrate composition of a Japanese radish peroxidase. We report here a qualitative identification of the sugars and a quantitative examination of the amino acids in horseradish peroxidase. Of special interest is the fact that peroxidase preparations commonly contain Biochim. Biophys. Acta, 96 (1965) 272-282
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more than one enzymatically active component. Electrophoresis on either paper 15 or starch gel 16 has revealed a multiplicity of peroxidases both in extracts of horseradish roots and in commercial preparations of horseradish peroxidase; PAI~L17 separated five horseradish peroxidase components by ion-exchange chromatography. Multiple components have also been demonstrated in the peroxidase preparations from sweet potatoS, is, Japanese radish 8, turnip 19, wheat 20, pea 21, and other leguminous plants 22. In no case has the structural basis of this heterogeneity been established. In the present study, we have investigated the possibility that the components might differ in their sizes, amino acid compositions, spectral properties or enzymatic activities. Our previous conclusion regarding the mechanism of peroxidasecatalyzed oxidations ~3 was based on the assumption that these components show the same catalytic activities. MATERIALS AND METHODS
Horseradish peroxidase was obtained from the California Corporation for Biochemical Research, Los Angeles, Calif. (U.S.A.) and from the Worthington Biochemical Corporation, Freehold, N.J. (U.S.A.).
Starch-gel electrophoresis Partially hydrolyzed starch (Connaught Medical Research Laboratories, Toronto (Canada)) was washed with a mixture containing equal volumes of n-propanol and 0. 5 N hydrochloric acid (8 1 for each 500 g of starch) and then with water to remove the acid propanol. The starch was finally washed with 95% alcohol and dried at room temperature for 2-3 days. The gel block was prepared as described by SMITH 24, using 13 g of the washed starch in IOO ml of a buffer composed of 9 parts o.o16 M Tris-o.o33 M citric acid (pH 8.1) and I part 0.02 M lithium hydroxide0.076 M boric acid (pH 8.1). The peroxidase (0.3 mg) was spotted on a small rectangle of Whatman No. 3 MM paper, which was placed in the middle of the precooled gel block. The block was connected by paper wicks to the electrode tanks, which contained fresh solutions of 0.38 M boric acid and o.I M lithium hydroxide, adjusted to pH 8.i with acetic acid. Electrophoresis was carried out for 3-4 h at 4 ° with a current of 25 mA passing through the gel bed; the potential varied from 20 to 30 V/cm during the run. After electrophoresis, the gel was cut in half. The top half was stained for protein by placing the gel in a o.05% solution of nigrosin in methanolacetic acid-water (5o:1o:4 o) for I h and then transferring it to a 0.009% nigrosin solution. The bottom half of the gel was stained for peroxidase activity by immersing it for I min in IO ml of benzidine-saturated water containing o.3% hydrogen peroxide 15 and then placing it in methanol-acetic acid-water (5o:1o:4o). The regions of the gel containing peroxidase turned blue immediately and then turned green; after a few days, discrete brown bands formed where the peroxidase components were localized.
Puri3cation of peroxidase components The starch-gel method described above was modified to permit purification of the various peroxidase components. The gel bed was made in a tray of the type described by SMITHIES25. A solution containing 15-17 mg of horseradish peroxidase Biochim. Biophys. Acta, 96 (1965) 272-282
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M. H. KLAPPER, D. P. HACKETT
in o.3-o.5 ml of water was pipetted into a slot running the width of the gel bed. Electrophoresis was conducted at 4 ° for 12 h. The current was maintained at 3o mA until the voltage increased to 39 ° V, after which the voltage was kept constant at this level ; the potential across the gel rose from 26 V/cm initially to 43 V/cm. After 12 h, the clearly separated brown bands were cut ifrom the gel bed and stored at 4 ° until processed further. In order to recover the purified peroxidase components, gel strips from three separate electrophoresis runs were ground together with 25 ml of water in the chilled cup of a Servall omnimixer. The resulting suspension was centrifuged at approx. 25 ooo × g for o. 5 h and the supernatant decanted off. The starch pellet was washed with water in the same manner two more times. The three supernatant solutions, which contained the enzyme, were pooled, concentrated by dialysis against solid sucrose, and then dialyzed against water. Some starch precipitated out during the concentration step and this was removed by centrifugation at 8o ooo × g for 1. 5 h. The clear supernatant was further concentrated by ultrafiltration 26, dialyzed against o.oi M sodium borate buffer (pH 8.6) and once again centrifuged at 8o ooo × g for 1. 5 h. Finally, to remove any contaminating starch, 1-2 ml of the enzyme in borate buffer was adsorbed onto a column (o. 9 × IO cm) of DEAE-Sephadex (Pharmacia, Uppsala (Sweden)) which had been equilibrated with the same buffer. The column was washed with 3° ml of the buffer and the peroxidase then eluted with a solution of o.oi M sodium borate and o.o 7 M sodium chloride (pH 8.6). The eluted protein was concentrated, dialyzed against water and stored in the freezer.
A m i n o acid analysis The amino acid compositions of the purified components were determined by standard chromatographic techniques. Approx. 2 mg of each of the components were hydrolyzed in 6 N hydrochloric acid in vacuo for 22 h at IiO °. The acid hydrolyzates were analyzed in a Beckman Model 12o amino acid analyzer by the method of SPACKMAN, STEIN AND MOORE z~.
Qualitative sugar analyses Approx. 2o mg of commercial horseradish peroxidase were hydrolyzed in I M sulfuric acid for 4 h at IOO° in vacuo. The hydrolyzate was neutralized with solid barium carbonate, and, after removing the precipitate by filtration, was applied to a column (0. 5 × 6 cm) of Dowex AG-5o W-X8, 5O-lOO mesh, hydrogen form (Bio-Rad Laboratories, Richmond, Calif. (U.S.A.)). The neutral sugars were washed through the column with 5 ml of water and the amino sugar fraction was eluted with IO ml of 0. 5 N hydrochloric acid. Both fractions were evaporated to dryness. The neutral sugar fraction was dissolved in 0.04 ml of water; 0.03 ml of this solution was streaked on Whatman No. I filter paper, and the remaining o.oi ml was spotted on the same paper as a guide. After carrying out the descending chromatography using ethyl-acetate-pyridine-water (8:2:1) as the solvent, the guide strip was sprayed with p-anisidine phosphate (2 g p-anisidine phosphate in 80 ml of water and 200 ml of 95 % ethanol) to locate the partially resolved sugars. The sugars on the unsprayed portion of the chromatogram were then eluted, and further analyzed by paper chromatography using a variety of solvent systems (Table IV). The amino sugar fraction obtained from the Dowex-5o column was analyzed for Biochim. Biophys. Acta, 96 (1965) 272-282
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Fig. i. Starch-gel electrophoresis of four horseradish peroxidase preparations. The electrophoretic p a t t e r n s on the top are, from left to right, Calbiochem lot No. 3547 ° and W o r t h i n g t o n lot No. 6253 stained with nigrosin, and W o r t h i n g t o n lot No. 6253 and Calbiochem lot No. 3547o stained with benzidine-hydrogen peroxide; on the bottom, Calbiochem lots Nos. 5o3339 and 35374 stained with nigrosin, and lots Nos. 35374 and 5o3339 stained with benzidine-hydrogen peroxide.
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M.H. KLAPPER, D. P. HACKETT
Fig. 2. Starch-gel electrophoresis of three purified peroxidase components. The p a t t e r n on the left represents unfractionated Calbiochem lot No. 3547 o. On the right are the three purified fractions: from left to right, horseradish peroxidase Nos. IV, l I I and II. All bands stained with nigrosin. h e x o s a m i n e b y t h e m e t h o d o f CESSI as d e s c r i b e d b y JOHANSEN et al. 2s. Sialic a c i d w a s d e t e r m i n e d b y t h e t h i o b a r b i t u r i c a s s a y o f WARREN 29 a f t e r h e a t i n g 1. 3 m g o f u n f r a c t i o n a t e d p e r o x i d a s e for I h i n 0.05 M s u l f u r i c acid.
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HORSERADISH PEROXlDASE STRUCTURE RESULTS
Heterogeneity of commercial horseradish peroxidase All of the commercial peroxidase preparations tested were found to be heterogeneous by starch-gel electrophoresis. The electrophoretic patterns obtained with four different preparations are shown in Fig. I; it is clear that they are similar, though not identical. One preparation (Calbiochem, lot No. 503339) contained nine protein-staining bands, all of which showed peroxidase activity; two additional bands showed peroxidase activity but gave no protein stain. Thus, this one preparation contained at least eleven electrophoretically distinct components with peroxidase activity. Every preparation contained appreciable amounts of the components which have been labeled horseradish peroxidase No. I, No. II, No. III and No. IV (Fig. 2). The other bands were present in much lower concentrations and were not detected in all the preparations. The major peroxidase components were purified by preparative starch-gel electrophoresis. Although the method limited the amount of material that could be processed conveniently at one time and only 4o-5o% of the applied material was recovered, the resolution was good (Fig. 2). The purified peroxidases (Nos. II, III and IV) were only slightly contaminated by their respective neighbors. These results show that the multiplicity observed in starch-gel eleetrophoresis is not an artifact of the separation method. The fact that the purified components migrated as single bands with the same mobilities as the corresponding components in the unfractionated preparation suggests that the multiplicity is not the result of reversible changes involving a single peroxidase.
Physical, chemical, and enzymatic properties of the purified peroxidase components Electrophoresis in starch gel can resolve proteins on the basis of both charge and size 30. In order to determine whether the peroxidases differ in size, the sedimentation characteristics of the unfractionated horseradish peroxidase preparation and of several of the purified components were compared in the ultracentrifuge. The whole horseradish peroxidase preparation sedimented as a single major band with a sedimentation coefficient (s20,w) of 3.42. This value agrees well with the sedimentation coefficients reported earlier by THEORELL~2 (S2o,w = 3 . 8 5 ) and by CECIL AND O G S T o N l l ($20'w - - 3 . 4 8) f o r unfractionated horseradish peroxidase. The monodisperse TABLE I SEDIMENTATION COEFFICIENTS OF PURIFIED PEROXIDASE COMPONENTS The sedimentation was followed at 4o4 m/~ in an ultracentrifuge equipped w i t h a b s o r p t i o n optics al. The purified c o m p o n e n t s were dissolved in o.i M p h o s p h a t e buffer (pH %o) at the following concentrations (mg/ml): I, o.40; I I , o.34 ; I I I , o.36; and IV, 0.45. Compone~tt
s9.o,w
I II III IV
3.43 3.7 ° 3.71 3.4 °
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M. H. KLAPPER, D. P. HACKETT
TABLE II ACTIVITIES
OF PURIFIED
PEROXIDASE
COMPONENTS
2 - M e t h y l - I , 4 - n a p h t h o h y d r o q u i n o n e oxidation was m e a s u r e d at 30°; 0.o 5 ml of oxygen-free O.Ol N HC1 solution containing o.8 mg 2 - m e t h y l - i , 4 - n a p h t h o h y d r o q u i n o n e / m l was added to a solution of horseradish peroxidase in 0.95 ml of 0.o 5 M citrate buffer (pH 6.0) and the reaction was followed b y the increase in a b s o r b a n c y at 262 m/~33. o-Dianisidine peroxidation was measured at 3 °0 b y the m e t h o d described in the catalogue of the W o r t h i n g t o n Biochemical Corp. 34, with the exception t h a t o.o 5 M citrate buffer (pH 6.0) was used. I n b o t h assays the enzyme concent r a t i o n was varied from 4 to 20 m/~g/ml. The results have been expressed as the molar oxidation per m i n u t e effected by a solution of enzyme with an a b s o r b a n c y of 1.oo at 4o3 m/~.
Component
2-Methyl-z,4-naphthohydroquinone oxidation (moles 2-methyl-x, 4naphthohydroquinone/ liter × min/A4o 3 mu)
o-Dianisidine peroxidation (moles H202[ liter × min/A 403 mu)
I II III IV V
0.37 0.45 o.5i 0.46 0.50
1.3 1.9 2.2 1.9 2.2
TABLE Ill AMINO ACID COMPOSITION OF PURIFIED PEROXIDASE COMPONENTS Amino acid analyses were performed as described in the text. The values given h a v e been calculated b y dividing the a m o u n t of one amino acid recovered b y the a m o u n t of all the a m i n o acids recovered. The values for the three c o m p o n e n t s are each the average of three d e t e r m i n a t i o n s and are given t o g e t h e r with the calculated s t a n d a r d errors.
Amino acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine A~anine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Mole fraction × zoo Horseradish peroxidase No. I
Horseradish peroxidase No. I I
Horseradish peroxidase No. I I I
2.06 0.90 6.79 16.5 8.23 7.66 7.18 5.65 5-79 8.38 5.46 1.17 4.36 11.6 1.5o 6.70
1.75 0.80 6.12 16.8 8.35 7.66 7.13 5.67 5.74 8.11 5.42 1.17 4.25 12.1 1.39 6.76
1.89 0.84 6.86 16.8 8.36 7.72 7.13 5.81 5.79 8.03 ,5-44 1.13 4.08 12.1 1.58 6.70
4~ 444444444± ± 4± :[:
0.29 0.22 0.98 0.52 o.16 o.16 o.16 o.ii 0.24 o.31 o.13 0.04 o.12 0.32 o.17 o.io
444± 444± 4444` 44± 4-
Biochim. Biophys. Acta, 96 (1965) 272-282
0.04 0.20 0.55 0.30 o.21 0.24 o.17 o.13 o.14 o.18 o.16 o.17 o.17 0.45 0.20 o.ii
44± 4444444± 4444:t:
o.i 5 o.14 0.67 0.09 0.06 0.22 0.06 0.34 0.04 o.21 o. i8 o.23 0.o9 0.27 0.04 0-34
HORSERADISH PEROXlDASE STRUCTURE
279
b e h a v i o r of t h e h o r s e r a d i s h p e r o x i d a s e p r e p a r a t i o n s u g g e s t s t h a t t h e s e v e r a l e l e c t r o p h o r e t i c a l l y s e p a r a b l e c o m p o n e n t s do n o t differ g r e a t l y in size. T h i s was t e s t e d d i r e c t l y b y d e t e r m i n i n g t h e s e d i m e n t a t i o n coefficients of f o u r p u r i f i e d c o m p o n e n t s in a n u l t r a c e n t r i f u g e e q u i p p e d w i t h a b s o r p t i o n optics al. T h e results (Table I) i n d i c a t e t h a t t h e y are all t h e s a m e size. T h e a b s o r p t i o n s p e c t r a o f t h e o x i d i z e d f o r m s of t h e f o u r p u r i f i e d p e r o x i d a s e s w e r e c o m p a r e d . T h e s p e c t r a of h o r s e r a d i s h p e r o x i d a s e Nos. I, II, n I , a n d I V are i d e n t i c a l a n d i n d i s t i n g u i s h a b l e f r o m t h e s p e c t r u m of h o r s e r a d i s h p e r o x i d a s e r e p o r t e d by KEILIN AND HARTREE a. T h e five p u r i f i e d c o m p o n e n t s w e r e t e s t e d for t h e i r e n z y m a t i c a c t i v i t i e s , as m e a s u r e d b y t h e i r abilities to c a t a l y z e t h e o x i d a t i o n of 2 - m e t h y l - I , 4 - n a p h t h o h y d r o q u i n o n e 33 a n d t h e p e r o x i d a t i o n of o - d i a n i s i d i n e a4. As s h o w n in T a b l e I I , t h e y all h a d e s s e n t i a l l y i d e n t i c a l a c t i v i t i e s in b o t h assays; t h e a c t i v i t i e s of h o r s e r a d i s h p e r o x i d a s e No. I were s l i g h t l y l o w e r t h a n t h o s e o f t h e o t h e r f o u r c o m p o n e n t s . T h e a m i n o a c i d c o m p o s i t i o n s of h o r s e r a d i s h p e r o x i d a s e Nos.I, I I a n d I I I a r e s h o w n in T a b l e I I I . T h e r e s u l t s i n d i c a t e t h a t t h e a m i n o a c i d c o m p o s i t i o n s o f t h e d i f f e r e n t p e r o x i d a s e s are v e r y similar, if n o t i d e n t i c a l . N e v e r t h e l e s s , t h e r e m a y be s m a l l differences in c o m p o s i t i o n w h i c h w e r e n o t d e t e c t e d d u e t o t h e errors i n h e r e n t in t h e procedure. The cysteine and tryptophan content were not analyzed.
Qualitative sugar analysis An unfractionated preparation of commercial horseradish peroxidase was TABLE IV SUGARS IN HORSERADISH
PEROXIDASE
All chromatograms were run by descending chromatography on Whatman No. i paper except for those in which Solvent A was used, where Whatman No. 52 was the support. Razu is the distance a sugar travelled relative to glucose, and Rxyz is the distance relative to xylose. The solvent systems are: Solvent A, ethylacetate-pyridine-water (8:2:i); Solvent B, phenol-water (3:I) ; Solvent C, butanol-ethanol-water (4 :I :I) ; Solvent D, benzene-butanol-pyridine-water (I :5:3:3) ; Solvent E, butanol-pyridine-water (io :3:3). Sugar
Solvent A RXyl
B Ra~u
C Ralu
D RGtu
E RGlu
Unknown I.OI Fucose I.OO
1.38 1.51
1.63 1.65
--
--
Unknown i.oi Xylose i.oo
1.o 7 1.12
1.42 1.44
---
---
Unknown o.71 Arabinose 0.75
1.25 1.22
1.25 1.25
---
---
Unknown Mannose Unknown Glucose
1.o6 1.o6 -i.oo
1.23 1.23 1.oi
-1.o8 0.99
-1.23 i .oo
I .oo
I.OO
I ,oo
--
o.82 0.81
o.89 o.89
0.55 o.56 ---
Unknown . Galactose - -
.
.
. --
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M. H. KLAPPER, D. P. HACKETT
hydrolyzed in acid, after which the neutral sugars were separated from amino acids, peptides, and amino sugars b y chromatography on Dowex-5o. The results of the paper chromatography of the sugars using five different solvent systems are shown in Table IV. At least six neutral sugars were identified: xylose, arabinose, fucose, mannose, galactose, and glucose. Evidence was also obtained for the presence of an amino sugar. Both the amino sugar fraction eluted from the Dowex-5o column and a whole hydrolyzate (3 N HC1 for 4 h at 95 ° in vacuo) of horseradish peroxidase gave positive tests in the CESSI assay for hexosamine. During the chromatography of the neutral and acidic amino acids in the amino acid analyzer, an unknown ninhydrin-positive peak appeared either between leucine and tyrosine or after phenylalanine, depending on the experimental conditions. This type of behavior is characteristic of glucosamine 27. In addition to these seven sugars, unidentified compounds giving positive tests with p-anisidine phosphate were seen on the paper chromatograms. A test for N-acetylneuraminic acid was negative.
DISCUSSION
The amino acid composition of horseradish peroxidase does not show any remarkable features. THEORELL AND AKESON12 reported earlier that there are 12 lysine residues/mole of horseradish peroxidase, but our data indicate that there are only 4 lysines. This discrepancy is due to the fact that they determined the lysine content by nitrogen difference. Our results suggest indirectly that there m a y be as m a n y as 8-1o hexosamine residues in horseradish peroxidase. Another interesting point is that horseradish peroxidase contains approximately two times as m a n y acidic as basic amino acids. In spite of this, almost all of the horseradish peroxidase components migrate towards the cathode at p H 8.6. The protein almost certainly contains a large amount of glutamine and asparagine, although no quantitative determination of the amide contents was made. Horseradish peroxidase does not contain any hydroxyproline, although this amino acid does occur in Japanese radish peroxidase a 14. The latter enzyme is much richer than horseradish peroxidase in alanine, serine, and aspartic acid, but it contains relatively little glycine and arginine. All of the seven sugars identified in the horseradish peroxidase hydrolyzate have been found previously in glycoproteins: glucose, galactose, mannose, fucose, and glucosamine are present in m a n y animal proteins 3~, while the pentoses arabinose and xylose have been identified in preparations of Japanese radish peroxidase 14. The nature of the carbohydrate-protein linkage in horseradish peroxidase is not yet known. The physical basis of the multiplicity of horseradish peroxidase components has not yet been established. The heterogeneity observed in starch-gel electrophoresis is probably due only to charge differences, since the components examined appear to have the same size. The differences in charge could be the result of differences in amino acid composition, amide content, protein conformation, or sugar content. Of these possibilities, only the first has been examined in detail here. The data do not indicate any significant differences in amino acid composition between the various components, but the possibility cannot yet be absolutely ruled out. The significance of the multiplicity is not at all clear. The possibility that the components arise as Biochim. Biophys. Acta, 96 (f965) 272-282
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281
artifacts during the isolation has not yet been ruled out. Assuming that the several components are present in intact tissues, the possibility that they are related to genetic, developmental, or histological heterogeneity in the starting material should be examined. The fact that the five purified peroxidase components had identical catalytic activities in the two reactions assayed suggests that differences in their relative amounts probably have little effect on the enzymatic activities of unfractionated horseradish peroxidase preparations. We reported earlier ~z that the horseradish peroxidase-catalyzed oxidation of 2-methyl-I,4-naphthohydroquinone can be inhibited 80% by carbon monoxide under conditions where only IO to 15% of the enzyme is in the ferrous-CO form. This led us to the conclusion that the ferrous enzyme is involved in the catalysis of 2-methyl-I,4-naphthohydroquinone oxidation. If, however, the oxidation of 2-methyl-I,4-naphthohydroquinone had been catalyzed by a single component which made up only a small fraction of the horseradish peroxidase preparation, this conclusion would not have been valid. The results presented here support the previous conclusion.
ACKNOWLEDGEMEN'fS
This work was supported by a grant from the National Science Foundation. The authors are indebted to Misses F. PUTNEY and B. CHII% and Mr. D. PETERSON for assistance in the determinations of the sedimentation coefficients and the amino acid compositions.
REFERENCES i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25
J. B. SUMNER AND S. F. HOWELL, Enzymologia, i (1936) 133. H. THEORFLL, Enzymologia, io (1941) 25 o. D. KEILIN AND E. V. HARTREE, Biochem. J., 49 (1951) 88. R. ABRAMS, A. M. ALTSCHUL AND T. R. HOGNESS, J. Biol. Chem., 142 (1942) 303 . t(. ]4~ONDO AND Y. MORITA, Bull. Res. Inst. Food Sci. Kyoto Univ., io (1952) 33. Y. MORI'rA, Bull. Res. Inst. Food Sci. Kyoto Univ., 15 (1954) 56. I. YAMAZAKI, K. FUJINAGA, I. TAKEHARA AND H. TAKAHASHI, J. Biochem. Tokyo, 43 (1956) 377Y. MORITA AND K. I~AMEDA, Mere. Res. Inst. Food Sci. Kyoto Univ., 12 (1957) I. R. E. STUTZ, Plant Physiol., 32 (1957) 31. K. TAGAWA ANn M. SHIN, J. Biochem. Tohyo, 46 (1959) 865. R. CECIL AND A. G. OGSTON, Biochem. J., 49 (1951) lO5. H. THEORELL AND A. AKESON, Arhiv Kemi Mineral. Geol., 16 (1943) No. 8. A. G. MAEHLY AND S. PALEUS, Acta Chem. Scan&, 4 (195 o) 508. Y. MORITA AND K. KAMEDA, Bull. Agr. Chem. Soc. Japan, 23 (1959) 28. M. A. JERMVN AND R. THOMAS, Biochem. J., 56 (1954) 631. M. D. POULIK, Nature, 18o (1957) I477. K. G. PAUL, Acta Chem. Scan&, 12 (1958) 1312. N. KAWASHIMA AND I. URITANI, Agr. Biol. Chem. Tokyo, 27 (1963) 409. T. HOSOYA, J. Biochem. Tokyo, 47 (196o) 369. M. SHIN AND W. NAKAMURA, J. Biochem. Tokyo, 5 ° (I96I) 500. 19. K. MACNICOL AND J. REINERT, Z. Naturforsch., ISb (1963) 572. E. MOUSTAFA, Nature, 199 (1963) 1189. M. H. KLAPPER AND D. P. HACKETT, J. Biol. Chem., 238 (1963) 3743I. SMITH, Chromatographic and Electrophoretic Techniques, Vol. 2, Interscience, New York, t96o, p. 131. O. SMITHIES, Biochem. J., 71 (1959) 585 .
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26 E. A. PETERSON AND I-l. A. SOBER, in S. P. COLOWICK AND N. O. I~APLAN, Methods of Enzymology, Vol. 5, Academic Press, N e w York, 1962, p. 25. 27 D. H. SPACKMAN,W. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 1185. 28 P. G. JOHANSEN, R. D. MARSHALL AND A. NEUBERGER, Biochem. J., 77 (196o) 239. 29 L. WARREN, J. Biol. Chem., 234 (1959) 1961. 3 ° O. SMITHIES, Arch. Biochem. Biophys. Suppl., I (1962) 125. 31 H. K. SCHACHMAN,Biochemistry, 2 (1963) 887. 32 H. THEORELL, Arkiv Kemi Mineral. Geol., 15 (1942) No. 24. 33 M. H. I~LAPPER AND D. P. HACKETT, J. Biol. Chem., 238 (1963) 3736. 34 Worthington Biochemical Corp. Descriptive Manual, i i (1961)45. 35 R. G. SPIRO, New Engl. J. Med., 269 (1963) 566.
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