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Preparation and properties of crosslinked water-insoluble catalase
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Preparation
and
136, 325-330 (1970)
Properties
Water-Insoluble ABEL Department
SCHEJTER
of Biochemistry,
Received
May
of Crosslin
ked
Catalase ATARA
AND
Tel-Aviv
BAR-ELI
University,
26, 1969; accepted
November
Tel-Aviv,
Israel
3, 1969
Catalase was crosslinked to a water-insoluble network by treatment with a bifunctional reagent, glutardialdehyde. The catalatic activity of water-insoluble catalase was 10 times less than for the soluble native enzyme. The peroxidatic activity with methanol as hydrogen donor decreased 11-fold, while the decrease was 30..fold when the donor tested was pyrogallol. The insoluble enzyme was stable for several months at 4”. Catalase inhibitors, such as cyanide, hydroxylamine, and aminotriazole, affect the insoluble and the soluble enzyme similarly. Cyanide also causes the same spectral changes in the insoluble catalase as those found in the soluble enzyme.
Rendering enzymes water-insoluble by chemical modifications has both scientific and practical appeal. The effects of insolubilization on various properties of an enzyme may be analyzed. Furthermore, water-insoluble enzymes offer a new way for activity assays or substrate transformations : the enzyme can be packed in a column through which the substrate percolates and the products are found in the effluent. The enzymic activity ceases as the solution emerges from the column; therefore, it is not necessary to add denaturing agents to stop the rea,ction. To date, many hydrolytic, proteolytic, and esterolytic water-insoluble enzymes have been prepared [reviewed by Silman and Katchalski (l)], as well as some oxidoreductases [alcohol dehydrogenase (2) and lactic dehydrogenase (3)]. The two main methods utilized for insolubilizing proteins are binding the protein covalently to a water-insoluble carrier (4, 5) and crosslinking the protein intermolecularly to a three-dimensional insoluble net. For example, employing the latter method, waterinsoluble papain was crosslinked by bisdiazobenzidine (1) and carboxypeptidase crystals were crosslinked with glutardialdehyde by Quiocho and Richards (6). Glutardialdehyde was the cross-linking 325 Copyright
@ 1970 by Academic
Press,
Ino.
agent used for preparing water-insoluble catalase. Some effects of cross-linking on the properties of catalase, such as the catalatic and peroxidatic activities and the reactions with various inhibitors, are described in this paper. EXPERIMENTAL
MATERIALS Beef liver crystalline catalase was purchased from WorthingtonBiochemicals Corp. (8.9 mg/ml). Glucose oxidase grade I was purchased from Boehringer, Germany. Sodium perborate and glutardialdehyde 25’% in water were obtained from Fluka; Hydrogen peroxide 30Yo w/v, KCN, chromotropic acid, and n-glucose were British Drug House analytical products. Pyrogallol was purchased from the Merck’s Reagents Company; 3-amino-1,2,4-triazol was a product of Eastman Organic Chemicals and was recrystallized from ethanol (melting point 154”).
METHODS Catalatic activity was determined by two titration methods: (1) the decrease of sodium perborate concentration as described by Feinstein (7); (2) the decrease in initial concentration of hydrogen peroxide according to Bonnichsen et al. (8). Peroxidatic activity of catalase was assayed with methanol and pyrogallol as hydrogen donors. Oxidation of methanol by catalase in a system
326
SCHEJTER
generating Hz02 (glucose oxidase-glucose (9)), proceeded for 1 hr at 20”. The formaldehyde formed was determined colorimetrieally with chromotropic acid reagent (10). Oxidation of pyrogallol was assayed according to Tauber (11). Assays of water-insoluble catalaee (WIC)l in the latter method were filt,ered through a glass fiber paper (GF/c Whatman) prior to absorbancy measurements. Amino acid analyses were carried out with a BeckmanUnichrome automatic amino acid analyzer with an automatic recorder, by the technique of Spackman et al. (12). Protein samples were hydrolyzed in sealed tubes with 6 N HCl at 110” for 24 hr. Protein concentration was measured by the absorbance at 405 rnp (Soret band maximum). The extinction coefficient of crystalline catalase is 340 mM-’ cm- r (13). A molecular weight of 225,000 was used for calculations. A Zeiss PM& II spectrophotometer was used for the determination of absorbance at single wavelengths. Protein concentration of WIC was calculated from the amount of alanine and phenylalanine found in the hydrolyzed protein as compared to the amount of these amino acids in hydrolyzed crystalline catalase. The molarity of WIC was calculated from the weight of the insoluble protein . Concentration of active enzyme in WIG was determined by assaying an aliquot of the waterinsoluble suspension by the method of Feinstein (7) and comparing it to the activity of a known concentration of soluble catalase. Reflectaneespectraof WIC wereobtained with a Cary Model 15 automatic recording instrument using a cell-space total diffuse reflectance accessory. Preparation oj WIG. Crystalline catalase was dissolved in 10% NaCl and diluted with 0.05 M phosphate buffer pH 7.2. Asolution of 4% glutardialdehyde (GDA) (4 ml) in the same buffer was added to 4 ml of enzyme solution (2 mg/ml) and stirred for about 1 hr at room temperature, until a green lumpy precipitate appeared. This crosslinking reaction could be performed in the cold room overnight with comparable results. The precipitate was separated by centrifugation (5 min, 4000 rpm) and washed repeatedly with 10% NaCl solution (6-8 times) until the supernatant fluid was free from catalase activity. The precipitate was homogenized in water, by a Potter homogenizer using a Teflon pestle, to a fine suspension containing 1 mg/ml of WIC. Activity r Abbreviations used: WIC--water-insoluble alase; GDA-glutardialdehyde; AT-3-amino-l, triazole.
cat2,4-
AND
BAR-ELI
determinations of aliquots of this suspension gave reproducible results within 5%. Inhibition by hydroxylamine was carried out at 20” for 1.5 hr. The inhibition mixtures (1 ml) contained enzyme (0.1 mg) in 0.01 M phosphate buffer pH 6.7, and NHsOH in concentrations between 2 X 1w2 M and 2 X lO+ M. Catalatic activity was estimated by the method of Feinstein (7). Inhibition of WIC by 3-amino-1,2,4-triazole (AT) at 36” was performed as described by Margoliash and co-workers (14). The inhibition mixture (6 ml) contained 0.033 M phosphate buffer at pH 7,0.02 M AT, 0.002 M neutralized ascorbic acid, and catalase (0.1 mg/ml) or WIC (1 mg/ml). Catalatic activity was assayed by the titration method (7), and peroxidatic activity with pyrogallol as substrate (11). Inhibition by cyanide of the catalatic (8) and peroxidatic (9, 11) activities of catalase and WIC was carried out by assaying the enzymes in solutions containing both substrate and inhibitor at concentrations of 3 X l(r'~-5 X 10-6~ cyanide. A column of WIC was prepared by pouring a mixture of 1 mg of WIC and 100 mg of Geon, a polyvinyl resin, into a glass tube 0.6 cm in diameter. The column, 0.4 cm in height, was washed with 0.01 M phosphate buffer pH 6.7 before it was assayed with 0.01 M hydrogen peroxide. A flow rate of 2 ml/min was obtained by using a Desaga peristaltic pump. The decrease in the concentration of hydrogen peroxide was determined by titration with0.01 N permanganate. RESULTS
Several WIC preparations are described in Table I. Changing the concentration of catalase between 0.5 and 2 mg/ml, or the ratio of protein to GDA from 1: 1 to 1:2 in the cross-linking reaction (at 20” or 4”) had no effect on the final enzymic activity of the water-insoluble product. The catalase in the water-insoluble derivatives retained close to 12% of the activity of crystalline catalase, as determined by the Feinstein assay (7). Lowering the ratio of catalase to GDA to 1: 4 did not yield a water-insoluble enzyme. WIC, preparation I, was used in all the experiments described in this paper. A protein cross-linked with GDA releases all of its amino acids quantitatively by 6 N HCl hydrolysis except for a decrease of about 50% in the lysine content (Richards, 6, 15). The results of amino acid analysis of catalase and WIC shown in Table II confirm the conclusions of Richards (6, 15).
WATER-INSOLUBLE TABLE
I
WATER-INSOLUBLE Concentration
in the
CKXdi&iig
reaction of: Catalase (w/ml)
I II III IV
1 1 2 0.5
GDA (%I
1 2 2 0.5
CATALASE TABLE
CATALASE
II
AMINO ACID COMPOSITION OF BEEF LIVER CATALASE (residues/250,000 g (16))
Activityb of inYield of insoluble soluble enzyme in enzymea in perpercentage of centage of total wetght of cat&se in WIG’” enzyme reacted
70 85 85 9w
327
11; 10.5” 13 12 10.5
0 The weight of WIG was calculated from the amount of soluble unreacted catalase which remained in the solution. b Catalatic activity was assayed according to Feinstein (7). c The amount of catalase in this preparation was determined by analysis of its amino acids as described in Methods. d The cross-linking proceeded for 18 hr at 4“ for this sample.
Moreover, the weights of WIC, preparation I, calculated from the alanine content of a hydrolyzed sample and from the amount of unreacted catalase in the cross-linking reaction, are the same, as shown in Table I. In order to establish unequivocally that the observed enzymic activity is an expression of WIC and not of any adsorbed protein, or protein cross-linked reversibly, effluents of a column of WIC were examined. No change in the extent of hydrolysis of HzOz was found even after several hours of incubation of the effluents at room temperature. Therefore, we conclude that the substrate did not desorb any catalase from the column into the solution. Stability of WIC. Samples of WIC were kept in water at 4”. A decrease of about 20 % in their initial activity was observed after 2 months. No further decrease occurred even after 5 mont,hs of storage. Enzymic activity of WIC. A linear increase in amount of decomposed Hz02 as a function of initial peroxide concentration was obtained with water-insoluble and soluble catalase (17) (Fig. 1). The first-order velocity constant, le, was obtained from the slope of the graph. The results for the insoluble and soluble enzymes are compared in Table III. The peroxidatic activity of WIC is lower
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
138 106 248 186 131 70 67 132 103 110 143 75b 82” 66 130
138” 110 244 184 131 84 70 135 56 104 133 776 82b 63 124
a The weight of WIC hydrolyzed was calculated from the alanine content. * Uncorrected for hydrolytic destruction,
than the catalatic activity (7) and it varies with the substrate used as hydrogen donor. It is 86% of its catalatic activity when assayed with methanol and 12% when assayed with pyrogallol. The reflectance spectra of WIC, reduced enzyme, and its cyanide complex are shown in Fig. 2. Reducibility by N&!&O4 indicates that the insoluble catalase is partially denatured (18, 19), although the percentage of denaturation could not be determined. Inhibition of WIC by catalase inhibitors. Hydroxylamine inhibits the catalatic activity of catalase and of WIC similarily (Fig. 3). The inhibition is independent of the time of incubation with inhibitor, as samples assayed after 15, 90, 120, and 180 min gave results similar to those depicted in the figure. Inhibition by AT was followed by comparing the inhibition of the catalatic (7) and peroxidatic (11) activities of the enzymes (Fig. 4). Both activities in each enzyme were inhibited to the same extent, but catalase is inhibited somewhat more than the insoluble catalase, 90% and 70% inhibition, respectively. Unlike the NHzOH inhibition, this is a time-dependent one
328
SCHEJTER
AND
that results from covalent binding of AT to the catalase protein (14). Cyanide inhibition of WIC is shown in Fig. 5. Percentage of inhibition was determined by assaying the enzymic activities of WIC with three substrates: Hz02 (8), methanol (9), and pyrogallol (11). Cyanide (3 X lO+ M) caused an inhibition of 70% in WIC and 85% in catalase. The inhibition was reversible, as diluting the inhibitor or removing it by dialysis against HI0 restored all the activity of WIC. I
I
DISCUSSION
When an enzyme is reacted with a crosslinking reagent, two types of changes occur. There is a chemical modification of amino acid side chains and a polymerization that results in a water-insoluble product. Thus, it is important to distinguish between the chemical and the physical effects of the cross-linking of catalase with GDA. This reagent substitutes mainly the E-amino groups of lysine (15). In our experiments, 50% of the lysine residues were substituted with a resulting lo-fold decrease in the catalatic activity. The peroxidatic activity, with methanol as hydrogen donor, decreased 11-fold, while the decrease was go-fold when the donor tested was pyrogallol. Acetylation of 50 % of the lysine groups of catalase has been found to cause a similar lo-fold decrease in the catalatic activity, together with a 3fold increase in the peroxidatic activity (18). It is known, however, that increases in the peroxidatic activity of diverse heme proteins usually accompany nonspecific, denaturative type changes (19). Although such changes may actually have happened to GDA cross-linked catalase, their effect of increasing the peroxidatic activity might have been masked by the steric hindrance imposed by a cross-linked protein network on a relatively large donor such as pyrogallol. With respect to the catalatic activity, it is to be noted that it is distinctly less affected by the cross-linking insolubilization than the enzymic activity of carboxypeptidase (6). The reflectance spectrum of the insoluble enzyme shows maxima at the same positions at which they appear in the absorption
I
LH202lM FIG. 1. Variation of the rate of decomposition of Hz02 with the concentration of peroxide. Initial rate of decrease in Hz02 concentration at 20’ was determined by the titrimetric method (8). Crystalline catalase (1.64 X 10-O M) (O-O), ordinate on right-hand side of figure; and by WIC (6.14 0) ordinate on left-hand side of x l(r9 M) (O-figure. (Molarity of WIC was calculated from the weight of the enzyme in the assay).
TABLE VELOCITY
CONSTANTS
BAR-ELI
OF CATALASE
III AND
WATER-INSOLUBLE
Cat&w k
Enzyme k’” k’d
molarity
[e]
3.75 1.M 2.17 3.0
X x X x
lOFsec-i lo-’ Ma lo7 10 set-i 10’ M-’ set-1
CATALASE Water-insoluble
cat&se
3.1 X 1O-3 set-i 5.6 X 10-O Mb 5.54 x 105 M-’ see-i -
0 Molarity of catalase was calculated from the concentration of protein in the assay. b The molarity of WIC was calculated from the weight of insoluble protein assayed. c k’ specific activity expressed as k/[e]. d This value is given in the literature (8).
WATER-INSOLUBLE
I 400
I
I 500 WVELENGTH (rnfi)
I 600
I
I
,
I
z 8~ 0 t m &o-
I
I
329
I
30
FIG. 2. Reflectance spectrum of WIC. The reflectance spectrum of asuspension of WIC, 7 mg/ml in 10% NaCl (---); WIC with a few crystals of Na2S204 (. . . .); and the spectrum of WICcyanide complex, 7 mg/ml WIC in 10% NaCl containing 1w2 M cyanide (- - - -); Reflectances measured against a blank of MgC03.
loot
CATALASE
I
I
60 90 TIME (min)
I
120
FIG. 4. Inhibition of catalase and of WIC by aminotriazole at 36’. Inhibition mixture (6 ml), consisted of 0.02 M AT, 0.092 M neutralized ascorbic acid, 0.033 M phosphate buffer, pH 7.0, plus 3.3 X 10-’ M catalase or 5.35 X 10-S M WIC. Activities of catalase (solid curve) and of WIC (dashed curve) were assayed with perborate (7) (0); and with pyrogallol (0) as substrates.
I
i 4
-LOG [NHflHIM
FIG. 3. Inhibition of catalase and of WIC by hydroxylamine. Inhibition proceeded for 90 min at 20”. The inhibition mixture (1 ml) contained 0.1 ml enzyme, 0.1 ml neutralized NHzOH, and 0.8 ml 0.012 M phosphate buffer, pH 6.7. Inhibition of crystalline catalase (4.45 X 1&T M) (0-O); Inhibition of WIC (4.13 X 10-r M) (O---O); Catalatio activity was assayed by the Feinstein method (7). Percent inhibition = (Control activity -Inhibited activity) X lOO/Control activity.
3.5
d
-LOG [Ci-] M
FIG. 5. Inhibition of WIC by cyanide. The enzymic activities of WIC were determined in the presence of cyanide in the concentrations indicated and 30 fig/ml WIC for the catalatic assay (8) 60 fig/ml WIC for the peroxidatic assay (0 ----a); of methanol (A---&; and 30 rg/ml WIC for the peroxidatic assay with pyrogallol (hm).
330
SCHEJTER
spectrum in solution (Fig. 2). The reaction with cyanide is reversible; it inhibits the en2ymic activity and causes identical spectral shifts (Fig. 2). Thus the heme-related properties of cross-linked catalase are very similar to those of the native enzyme in solution, with one important exception: WIC is partly reduced by dithionite. It is worthwhile to notice that active catalase is not reduced by dithionite, but becomes reducible upon denaturation (20). The catalatic reaction in the insoluble enzyme proceeds, apparently, with the same mechanism as in solution. This is evidenced by the following two facts. First, the rate of hydrogen peroxide decomposition increases linearly with its initial concentration, and does not show saturation (17); this behavior is typical of native catalase and it implies the formation of a primary enzyme-substrate complex that reacts with a second substrate molecule. Furthermore, aminotriazole inhibits the cross-linked enzyme irreversibly, at a rate and to an extent comparable to those observed for the soluble enzyme (14). Since this reaction takes place between the inhibitor and the primary complex (14), the latter must be formed during the catalatic activity of insoluble catalase. The inhibition of the catalatic activity of cross-linked catalase by hydroxylamine is quantitatively very similar to the inhibition of the soluble enzyme. This is an indication that the affinity of the enzyme heme for hydroxylamine is not altered by the GDA cross-linking. A similar situation exists with regard to cyanide, which inhibits to the same extent all the activities assayed. The most remarkable effect of cross-linking appears to be the difference in the alteration of the peroxidatic activity with different donors. The decrease in activity toward methanol is substantially the same as that of the cata-
AND
BAR-ELI
latic activity; however, pyrogallol is much more affected as a donor by the cross-linking of the enzyme. The simplest explanation for this fact is one based on steric considerations : either diminished accessibility of the active site, or hindrance of conformational changes of the enzyme necessary for the peroxidatic activity. REFERENCES 1. SILMAN, H. I., AND KATCHALSKI, E., Ann. Rev. Biochem. 36, 873 (1966). 2. MANECKE, E., Pure Appl. Chem. 4,507 (1962). 3. WILSON, R. J. H., KAY, G., AND LILLY, M. D., Biochem. J. 108,845 (1968). 4. BAR-ELI, A., AND KATCHALSKI, E., J. Viol. Chem. 238, 1690 (1963). 5. WELIKY, N., BROWN, F. S., AND DALE, E. C., Arch. Biochem. Biophys. 131, 1 (1969). 6. QUIOCHO, F. A., AND RICHARDS, F. M., Biochemistry 6, 4062 (1966). 7. FEINSTEIN, R. N., J. Biol. Chem. 180, 1197 (1949). 8. BONNICHSEN, R. K., CHANCE, B., AND THEORELL, H., Acta Chem. Scam-l. 1,685 (1947). 9. KEILIN, D., AND HARTREE, E. F., Biochem. J. 60, 310 (1955). 10. MACFAYDEN, D. A., J. Biol. Chem. 168, 107 (1945). 11. TAUBER, H., J. Bio?. Chem. M6, 395 (1953). 12. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 13. ANGER, K., Biochem. J. 32, 1702 (1938). 14. MARGOLIASH, E., NOVOGRODSKY, A., AND SCHEJTER, A., Biochem. J. 74,339 (1960). 15. RICHARDS, F. M., AND KNOWLES, J. R., J. Mol. Biol. 37, 231 (1968). 16. SCHROEDER, W. A., SAHA, A., FENNINGER, W. D., AND CUA, J. T., Biochim. Biophys. Acta 68, 611 (1962). 17. CHANCE, B., J. Biol. Chem. 194, 471 (1952). 18. HIRACA, M., ANAN, F. K., AND ABE, K., J. Biochem. Japan 66,416 (1964). 19. INADA, Y., KUROZUMI, T., AND SHIBATA, K., Arch. Biochem. Biophys. 93, 30 (1961). 20. DEISSEROTH, A., AND DOUNCE, A. L., Arch. Biochem. Biophys. 120, 671 (1967).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Preparation
and
136, 325-330 (1970)
Properties
Water-Insoluble ABEL Department
SCHEJTER
of Biochemistry,
Received
May
of Crosslin
ked
Catalase ATARA
AND
Tel-Aviv
BAR-ELI
University,
26, 1969; accepted
November
Tel-Aviv,
Israel
3, 1969
Catalase was crosslinked to a water-insoluble network by treatment with a bifunctional reagent, glutardialdehyde. The catalatic activity of water-insoluble catalase was 10 times less than for the soluble native enzyme. The peroxidatic activity with methanol as hydrogen donor decreased 11-fold, while the decrease was 30..fold when the donor tested was pyrogallol. The insoluble enzyme was stable for several months at 4”. Catalase inhibitors, such as cyanide, hydroxylamine, and aminotriazole, affect the insoluble and the soluble enzyme similarly. Cyanide also causes the same spectral changes in the insoluble catalase as those found in the soluble enzyme.
Rendering enzymes water-insoluble by chemical modifications has both scientific and practical appeal. The effects of insolubilization on various properties of an enzyme may be analyzed. Furthermore, water-insoluble enzymes offer a new way for activity assays or substrate transformations : the enzyme can be packed in a column through which the substrate percolates and the products are found in the effluent. The enzymic activity ceases as the solution emerges from the column; therefore, it is not necessary to add denaturing agents to stop the rea,ction. To date, many hydrolytic, proteolytic, and esterolytic water-insoluble enzymes have been prepared [reviewed by Silman and Katchalski (l)], as well as some oxidoreductases [alcohol dehydrogenase (2) and lactic dehydrogenase (3)]. The two main methods utilized for insolubilizing proteins are binding the protein covalently to a water-insoluble carrier (4, 5) and crosslinking the protein intermolecularly to a three-dimensional insoluble net. For example, employing the latter method, waterinsoluble papain was crosslinked by bisdiazobenzidine (1) and carboxypeptidase crystals were crosslinked with glutardialdehyde by Quiocho and Richards (6). Glutardialdehyde was the cross-linking 325 Copyright
@ 1970 by Academic
Press,
Ino.
agent used for preparing water-insoluble catalase. Some effects of cross-linking on the properties of catalase, such as the catalatic and peroxidatic activities and the reactions with various inhibitors, are described in this paper. EXPERIMENTAL
MATERIALS Beef liver crystalline catalase was purchased from WorthingtonBiochemicals Corp. (8.9 mg/ml). Glucose oxidase grade I was purchased from Boehringer, Germany. Sodium perborate and glutardialdehyde 25’% in water were obtained from Fluka; Hydrogen peroxide 30Yo w/v, KCN, chromotropic acid, and n-glucose were British Drug House analytical products. Pyrogallol was purchased from the Merck’s Reagents Company; 3-amino-1,2,4-triazol was a product of Eastman Organic Chemicals and was recrystallized from ethanol (melting point 154”).
METHODS Catalatic activity was determined by two titration methods: (1) the decrease of sodium perborate concentration as described by Feinstein (7); (2) the decrease in initial concentration of hydrogen peroxide according to Bonnichsen et al. (8). Peroxidatic activity of catalase was assayed with methanol and pyrogallol as hydrogen donors. Oxidation of methanol by catalase in a system
326
SCHEJTER
generating Hz02 (glucose oxidase-glucose (9)), proceeded for 1 hr at 20”. The formaldehyde formed was determined colorimetrieally with chromotropic acid reagent (10). Oxidation of pyrogallol was assayed according to Tauber (11). Assays of water-insoluble catalaee (WIC)l in the latter method were filt,ered through a glass fiber paper (GF/c Whatman) prior to absorbancy measurements. Amino acid analyses were carried out with a BeckmanUnichrome automatic amino acid analyzer with an automatic recorder, by the technique of Spackman et al. (12). Protein samples were hydrolyzed in sealed tubes with 6 N HCl at 110” for 24 hr. Protein concentration was measured by the absorbance at 405 rnp (Soret band maximum). The extinction coefficient of crystalline catalase is 340 mM-’ cm- r (13). A molecular weight of 225,000 was used for calculations. A Zeiss PM& II spectrophotometer was used for the determination of absorbance at single wavelengths. Protein concentration of WIC was calculated from the amount of alanine and phenylalanine found in the hydrolyzed protein as compared to the amount of these amino acids in hydrolyzed crystalline catalase. The molarity of WIC was calculated from the weight of the insoluble protein . Concentration of active enzyme in WIG was determined by assaying an aliquot of the waterinsoluble suspension by the method of Feinstein (7) and comparing it to the activity of a known concentration of soluble catalase. Reflectaneespectraof WIC wereobtained with a Cary Model 15 automatic recording instrument using a cell-space total diffuse reflectance accessory. Preparation oj WIG. Crystalline catalase was dissolved in 10% NaCl and diluted with 0.05 M phosphate buffer pH 7.2. Asolution of 4% glutardialdehyde (GDA) (4 ml) in the same buffer was added to 4 ml of enzyme solution (2 mg/ml) and stirred for about 1 hr at room temperature, until a green lumpy precipitate appeared. This crosslinking reaction could be performed in the cold room overnight with comparable results. The precipitate was separated by centrifugation (5 min, 4000 rpm) and washed repeatedly with 10% NaCl solution (6-8 times) until the supernatant fluid was free from catalase activity. The precipitate was homogenized in water, by a Potter homogenizer using a Teflon pestle, to a fine suspension containing 1 mg/ml of WIC. Activity r Abbreviations used: WIC--water-insoluble alase; GDA-glutardialdehyde; AT-3-amino-l, triazole.
cat2,4-
AND
BAR-ELI
determinations of aliquots of this suspension gave reproducible results within 5%. Inhibition by hydroxylamine was carried out at 20” for 1.5 hr. The inhibition mixtures (1 ml) contained enzyme (0.1 mg) in 0.01 M phosphate buffer pH 6.7, and NHsOH in concentrations between 2 X 1w2 M and 2 X lO+ M. Catalatic activity was estimated by the method of Feinstein (7). Inhibition of WIC by 3-amino-1,2,4-triazole (AT) at 36” was performed as described by Margoliash and co-workers (14). The inhibition mixture (6 ml) contained 0.033 M phosphate buffer at pH 7,0.02 M AT, 0.002 M neutralized ascorbic acid, and catalase (0.1 mg/ml) or WIC (1 mg/ml). Catalatic activity was assayed by the titration method (7), and peroxidatic activity with pyrogallol as substrate (11). Inhibition by cyanide of the catalatic (8) and peroxidatic (9, 11) activities of catalase and WIC was carried out by assaying the enzymes in solutions containing both substrate and inhibitor at concentrations of 3 X l(r'~-5 X 10-6~ cyanide. A column of WIC was prepared by pouring a mixture of 1 mg of WIC and 100 mg of Geon, a polyvinyl resin, into a glass tube 0.6 cm in diameter. The column, 0.4 cm in height, was washed with 0.01 M phosphate buffer pH 6.7 before it was assayed with 0.01 M hydrogen peroxide. A flow rate of 2 ml/min was obtained by using a Desaga peristaltic pump. The decrease in the concentration of hydrogen peroxide was determined by titration with0.01 N permanganate. RESULTS
Several WIC preparations are described in Table I. Changing the concentration of catalase between 0.5 and 2 mg/ml, or the ratio of protein to GDA from 1: 1 to 1:2 in the cross-linking reaction (at 20” or 4”) had no effect on the final enzymic activity of the water-insoluble product. The catalase in the water-insoluble derivatives retained close to 12% of the activity of crystalline catalase, as determined by the Feinstein assay (7). Lowering the ratio of catalase to GDA to 1: 4 did not yield a water-insoluble enzyme. WIC, preparation I, was used in all the experiments described in this paper. A protein cross-linked with GDA releases all of its amino acids quantitatively by 6 N HCl hydrolysis except for a decrease of about 50% in the lysine content (Richards, 6, 15). The results of amino acid analysis of catalase and WIC shown in Table II confirm the conclusions of Richards (6, 15).
WATER-INSOLUBLE TABLE
I
WATER-INSOLUBLE Concentration
in the
CKXdi&iig
reaction of: Catalase (w/ml)
I II III IV
1 1 2 0.5
GDA (%I
1 2 2 0.5
CATALASE TABLE
CATALASE
II
AMINO ACID COMPOSITION OF BEEF LIVER CATALASE (residues/250,000 g (16))
Activityb of inYield of insoluble soluble enzyme in enzymea in perpercentage of centage of total wetght of cat&se in WIG’” enzyme reacted
70 85 85 9w
327
11; 10.5” 13 12 10.5
0 The weight of WIG was calculated from the amount of soluble unreacted catalase which remained in the solution. b Catalatic activity was assayed according to Feinstein (7). c The amount of catalase in this preparation was determined by analysis of its amino acids as described in Methods. d The cross-linking proceeded for 18 hr at 4“ for this sample.
Moreover, the weights of WIC, preparation I, calculated from the alanine content of a hydrolyzed sample and from the amount of unreacted catalase in the cross-linking reaction, are the same, as shown in Table I. In order to establish unequivocally that the observed enzymic activity is an expression of WIC and not of any adsorbed protein, or protein cross-linked reversibly, effluents of a column of WIC were examined. No change in the extent of hydrolysis of HzOz was found even after several hours of incubation of the effluents at room temperature. Therefore, we conclude that the substrate did not desorb any catalase from the column into the solution. Stability of WIC. Samples of WIC were kept in water at 4”. A decrease of about 20 % in their initial activity was observed after 2 months. No further decrease occurred even after 5 mont,hs of storage. Enzymic activity of WIC. A linear increase in amount of decomposed Hz02 as a function of initial peroxide concentration was obtained with water-insoluble and soluble catalase (17) (Fig. 1). The first-order velocity constant, le, was obtained from the slope of the graph. The results for the insoluble and soluble enzymes are compared in Table III. The peroxidatic activity of WIC is lower
Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
138 106 248 186 131 70 67 132 103 110 143 75b 82” 66 130
138” 110 244 184 131 84 70 135 56 104 133 776 82b 63 124
a The weight of WIC hydrolyzed was calculated from the alanine content. * Uncorrected for hydrolytic destruction,
than the catalatic activity (7) and it varies with the substrate used as hydrogen donor. It is 86% of its catalatic activity when assayed with methanol and 12% when assayed with pyrogallol. The reflectance spectra of WIC, reduced enzyme, and its cyanide complex are shown in Fig. 2. Reducibility by N&!&O4 indicates that the insoluble catalase is partially denatured (18, 19), although the percentage of denaturation could not be determined. Inhibition of WIC by catalase inhibitors. Hydroxylamine inhibits the catalatic activity of catalase and of WIC similarily (Fig. 3). The inhibition is independent of the time of incubation with inhibitor, as samples assayed after 15, 90, 120, and 180 min gave results similar to those depicted in the figure. Inhibition by AT was followed by comparing the inhibition of the catalatic (7) and peroxidatic (11) activities of the enzymes (Fig. 4). Both activities in each enzyme were inhibited to the same extent, but catalase is inhibited somewhat more than the insoluble catalase, 90% and 70% inhibition, respectively. Unlike the NHzOH inhibition, this is a time-dependent one
328
SCHEJTER
AND
that results from covalent binding of AT to the catalase protein (14). Cyanide inhibition of WIC is shown in Fig. 5. Percentage of inhibition was determined by assaying the enzymic activities of WIC with three substrates: Hz02 (8), methanol (9), and pyrogallol (11). Cyanide (3 X lO+ M) caused an inhibition of 70% in WIC and 85% in catalase. The inhibition was reversible, as diluting the inhibitor or removing it by dialysis against HI0 restored all the activity of WIC. I
I
DISCUSSION
When an enzyme is reacted with a crosslinking reagent, two types of changes occur. There is a chemical modification of amino acid side chains and a polymerization that results in a water-insoluble product. Thus, it is important to distinguish between the chemical and the physical effects of the cross-linking of catalase with GDA. This reagent substitutes mainly the E-amino groups of lysine (15). In our experiments, 50% of the lysine residues were substituted with a resulting lo-fold decrease in the catalatic activity. The peroxidatic activity, with methanol as hydrogen donor, decreased 11-fold, while the decrease was go-fold when the donor tested was pyrogallol. Acetylation of 50 % of the lysine groups of catalase has been found to cause a similar lo-fold decrease in the catalatic activity, together with a 3fold increase in the peroxidatic activity (18). It is known, however, that increases in the peroxidatic activity of diverse heme proteins usually accompany nonspecific, denaturative type changes (19). Although such changes may actually have happened to GDA cross-linked catalase, their effect of increasing the peroxidatic activity might have been masked by the steric hindrance imposed by a cross-linked protein network on a relatively large donor such as pyrogallol. With respect to the catalatic activity, it is to be noted that it is distinctly less affected by the cross-linking insolubilization than the enzymic activity of carboxypeptidase (6). The reflectance spectrum of the insoluble enzyme shows maxima at the same positions at which they appear in the absorption
I
LH202lM FIG. 1. Variation of the rate of decomposition of Hz02 with the concentration of peroxide. Initial rate of decrease in Hz02 concentration at 20’ was determined by the titrimetric method (8). Crystalline catalase (1.64 X 10-O M) (O-O), ordinate on right-hand side of figure; and by WIC (6.14 0) ordinate on left-hand side of x l(r9 M) (O-figure. (Molarity of WIC was calculated from the weight of the enzyme in the assay).
TABLE VELOCITY
CONSTANTS
BAR-ELI
OF CATALASE
III AND
WATER-INSOLUBLE
Cat&w k
Enzyme k’” k’d
molarity
[e]
3.75 1.M 2.17 3.0
X x X x
lOFsec-i lo-’ Ma lo7 10 set-i 10’ M-’ set-1
CATALASE Water-insoluble
cat&se
3.1 X 1O-3 set-i 5.6 X 10-O Mb 5.54 x 105 M-’ see-i -
0 Molarity of catalase was calculated from the concentration of protein in the assay. b The molarity of WIC was calculated from the weight of insoluble protein assayed. c k’ specific activity expressed as k/[e]. d This value is given in the literature (8).
WATER-INSOLUBLE
I 400
I
I 500 WVELENGTH (rnfi)
I 600
I
I
,
I
z 8~ 0 t m &o-
I
I
329
I
30
FIG. 2. Reflectance spectrum of WIC. The reflectance spectrum of asuspension of WIC, 7 mg/ml in 10% NaCl (---); WIC with a few crystals of Na2S204 (. . . .); and the spectrum of WICcyanide complex, 7 mg/ml WIC in 10% NaCl containing 1w2 M cyanide (- - - -); Reflectances measured against a blank of MgC03.
loot
CATALASE
I
I
60 90 TIME (min)
I
120
FIG. 4. Inhibition of catalase and of WIC by aminotriazole at 36’. Inhibition mixture (6 ml), consisted of 0.02 M AT, 0.092 M neutralized ascorbic acid, 0.033 M phosphate buffer, pH 7.0, plus 3.3 X 10-’ M catalase or 5.35 X 10-S M WIC. Activities of catalase (solid curve) and of WIC (dashed curve) were assayed with perborate (7) (0); and with pyrogallol (0) as substrates.
I
i 4
-LOG [NHflHIM
FIG. 3. Inhibition of catalase and of WIC by hydroxylamine. Inhibition proceeded for 90 min at 20”. The inhibition mixture (1 ml) contained 0.1 ml enzyme, 0.1 ml neutralized NHzOH, and 0.8 ml 0.012 M phosphate buffer, pH 6.7. Inhibition of crystalline catalase (4.45 X 1&T M) (0-O); Inhibition of WIC (4.13 X 10-r M) (O---O); Catalatio activity was assayed by the Feinstein method (7). Percent inhibition = (Control activity -Inhibited activity) X lOO/Control activity.
3.5
d
-LOG [Ci-] M
FIG. 5. Inhibition of WIC by cyanide. The enzymic activities of WIC were determined in the presence of cyanide in the concentrations indicated and 30 fig/ml WIC for the catalatic assay (8) 60 fig/ml WIC for the peroxidatic assay (0 ----a); of methanol (A---&; and 30 rg/ml WIC for the peroxidatic assay with pyrogallol (hm).
330
SCHEJTER
spectrum in solution (Fig. 2). The reaction with cyanide is reversible; it inhibits the en2ymic activity and causes identical spectral shifts (Fig. 2). Thus the heme-related properties of cross-linked catalase are very similar to those of the native enzyme in solution, with one important exception: WIC is partly reduced by dithionite. It is worthwhile to notice that active catalase is not reduced by dithionite, but becomes reducible upon denaturation (20). The catalatic reaction in the insoluble enzyme proceeds, apparently, with the same mechanism as in solution. This is evidenced by the following two facts. First, the rate of hydrogen peroxide decomposition increases linearly with its initial concentration, and does not show saturation (17); this behavior is typical of native catalase and it implies the formation of a primary enzyme-substrate complex that reacts with a second substrate molecule. Furthermore, aminotriazole inhibits the cross-linked enzyme irreversibly, at a rate and to an extent comparable to those observed for the soluble enzyme (14). Since this reaction takes place between the inhibitor and the primary complex (14), the latter must be formed during the catalatic activity of insoluble catalase. The inhibition of the catalatic activity of cross-linked catalase by hydroxylamine is quantitatively very similar to the inhibition of the soluble enzyme. This is an indication that the affinity of the enzyme heme for hydroxylamine is not altered by the GDA cross-linking. A similar situation exists with regard to cyanide, which inhibits to the same extent all the activities assayed. The most remarkable effect of cross-linking appears to be the difference in the alteration of the peroxidatic activity with different donors. The decrease in activity toward methanol is substantially the same as that of the cata-
AND
BAR-ELI
latic activity; however, pyrogallol is much more affected as a donor by the cross-linking of the enzyme. The simplest explanation for this fact is one based on steric considerations : either diminished accessibility of the active site, or hindrance of conformational changes of the enzyme necessary for the peroxidatic activity. REFERENCES 1. SILMAN, H. I., AND KATCHALSKI, E., Ann. Rev. Biochem. 36, 873 (1966). 2. MANECKE, E., Pure Appl. Chem. 4,507 (1962). 3. WILSON, R. J. H., KAY, G., AND LILLY, M. D., Biochem. J. 108,845 (1968). 4. BAR-ELI, A., AND KATCHALSKI, E., J. Viol. Chem. 238, 1690 (1963). 5. WELIKY, N., BROWN, F. S., AND DALE, E. C., Arch. Biochem. Biophys. 131, 1 (1969). 6. QUIOCHO, F. A., AND RICHARDS, F. M., Biochemistry 6, 4062 (1966). 7. FEINSTEIN, R. N., J. Biol. Chem. 180, 1197 (1949). 8. BONNICHSEN, R. K., CHANCE, B., AND THEORELL, H., Acta Chem. Scam-l. 1,685 (1947). 9. KEILIN, D., AND HARTREE, E. F., Biochem. J. 60, 310 (1955). 10. MACFAYDEN, D. A., J. Biol. Chem. 168, 107 (1945). 11. TAUBER, H., J. Bio?. Chem. M6, 395 (1953). 12. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 13. ANGER, K., Biochem. J. 32, 1702 (1938). 14. MARGOLIASH, E., NOVOGRODSKY, A., AND SCHEJTER, A., Biochem. J. 74,339 (1960). 15. RICHARDS, F. M., AND KNOWLES, J. R., J. Mol. Biol. 37, 231 (1968). 16. SCHROEDER, W. A., SAHA, A., FENNINGER, W. D., AND CUA, J. T., Biochim. Biophys. Acta 68, 611 (1962). 17. CHANCE, B., J. Biol. Chem. 194, 471 (1952). 18. HIRACA, M., ANAN, F. K., AND ABE, K., J. Biochem. Japan 66,416 (1964). 19. INADA, Y., KUROZUMI, T., AND SHIBATA, K., Arch. Biochem. Biophys. 93, 30 (1961). 20. DEISSEROTH, A., AND DOUNCE, A. L., Arch. Biochem. Biophys. 120, 671 (1967).