- Email: [email protected]
Soluble and bound peroxidases from papaya fruit
Phytochemistry,Vol. 29, No. 4, pp. 1051-1056,1990. Printed in Great Britain.
SOLUBLE
0031 9422/90%3.00 + 0.00 0 1990Pergamon Press plc
AND BOUND PEROXIDASES
FROM PAPAYA FRUIT
ELISETE DA SILVA, EUCLIDES J. LOURENCO and VALDIR A. NEVES Department of Food Nutrition,
FCF, UNESP, 14800 Araraquara, Bras&Rodovia Araraquara-JaC Km I, Brasil (Received in reuised.form 27 September 1989)
Key Word Index-Cnrica
papaya; Caricaceae; papaya fruit; ripening; peroxidase; enzyme properties.
Abstract--Soluble and bound peroxidases were isolated from the pulp of ripening papaya fruit. During papaya ripening, soluble and bound peroxidase activities increased 2.5 and 4.2-fold, respectively. Soluble peroxidase was purified 59-fold by ammonium sulphate precipitation and chromatography on Sephadex G-25, DEAE-cellulose and Sephadex G-100. Bound peroxidase was purified 140-fold by ammonium sulphate precipitation and chromatography on Sephadex G-100 and DEAE-cellulose. Polyacrylamide gel electrophoresis of the purified preparations revealed that both enzymes were highly purified by the procedures adopted. The soluble and bound forms had a M, of 41000 and 54000, respectively. Soluble and bound peroxidases showed optimum activity at pH 6.0 and 5.5, respectively, and were inhibited by p-chloromercuribenzoate, iodoacetamide, N-ethylmaleimide, potassium cyanide and Fe’+. Soluble peroxidase was activated by ammonium sulphate and this activation was prevented by cyanide.
RESULTS AND
INTRODUCTION Papaya belongs to the climateric class of fruits. The fruit ripen while still attached to the tree, exhibiting an increase in ethylene and carbon dioxide production [l]. The characteristics feature of papaya ripening is a change in texture, colour and sweetness. Softening of the papaya fruit is brought about by changes in cell wall constituents among which pectic substance play a major role [2,3]. Peroxidase (EC 1.11.1.7) has been implicated in metabolic processes such as ethylene biogenesis, cell development and membrane integrity [4]. The properties of peroxidase and its physiological role in postharvest fruits and vegetables have been reviewed by several authors [4-61. The enzyme has been reported to occur in plant tissues in soluble and bound forms. Kahn et al. [7] have recently demonstrated the presence of soluble and bound peroxidases in potato tuber extracts. They have also examined the biochemical properties of the enzymes in both crude and partially purified preparations. Soluble and bound peroxidases isolated from green asparagus have also been studied [S]. A correlation between peroxidase activity and fruit ripening has been shown in a number of cases [9]. In mango and apples, peroxidase activity increases with ripening [lo, 111. Similarly, bound peroxidase activity isolated from the pulp of ripening banana fruit is increased three-fold at the onset of the respiration climateric [ 123, and a considerable increase in soluble activity is also observed [13]. In contrast, the soluble peroxidase form from tomato fruit reaches a maximum at mature-green stage and declines with ripening [14, 151. To our knowledge, peroxidase activity in papaya fruit during ripening has not been adequately investigated. The present investigation was designed to study the extraction of soluble and ionically bound peroxidase and the changes in the enzyme activities during ripening. Procedures for purifying the two forms of peroxidase and some physicochemical properties are also described.
Extraction
DISCUSSION
of peroxidases
Activities of soluble and ionically bound peroxidases during ripening are shown in Fig. 1. Papaya fruit stored at 20” takes about 12 days to reach the ripe (edible) stage from the mature-green stage. The activity of bound peroxidase, which was lowest in green fruit, was greatly increased with ripening but gradually fell as the fruit turned from ripe to the over-ripe stage. Soluble peroxidase activity, the main form in green fruit, also reached a peak followed by a marked decrease, returning to the level found in mature-green papaya. After 12 days storage, the activities of the bound and soluble forms were
f/
I
I
I
I
9
12
15
18
Days
after
harvest
Fig. 1. Soluble and bound peroxidase activities during the ripening of papaya fruit. (O-O) Soluble peroxidase; ( l - - - -0) bound peroxidase.
1051
E.
1052
DA SILVA
4.2- and 2.5-fold higher than the initial values, respectively. Despite the differences in activity found for soluble peroxidase from mature-green and ripe papaya, the extracts revealed a reproducible pattern when subjected to polyacrylamide gel electrophoresis (PAGE) with the presence of five active bands (Fig. 2). The mobility of these bands did not vary with ripening but the ripe extract showed two major active bands (bands d and e) and maximum activity coinciding with the maximum intensity of active bands. The reduction in activity observed when ripening progressed from the ripe to the over-ripe stage was accompanied by a reduction in the number and intensity of active bands. The PAGE patterns of bound peroxidase extracts were different from those obtained with the soluble form (Fig. 2). The spectrum of peroxidase bands revealed the presence of three active bands; the two slowest moving bands, designated a and b, were not detected in the extracts, suggesting that these are species exclusively occurring in soluble peroxidase. The soluble peroxidase of papaya fruit was readily extracted by buffer of low molarity. Because no differences in specific activity were found when using several buffer molarities ranging from 0.05 to 0.2 M, the 0.05 M concentration was used throughout the study. In contrast, the extraction of ionically bound enzyme required the addition of either calcium, magnesium or sodium chloride to the buffer to release the enzyme from cell components. An increase in specific activity with the increase in salt concentration was found (Fig. 3). Maximum yield was obtained at a concentration of0.5 M with all three salts, and activity was reduced at higher concentrations. Sodium chloride was less effective than calcium and magnesium chloride for the solubilization of bound peroxidase. The optimal pH conditions for peroxidase extraction were investigated by using phosphate buffer in the pH range 4.5.-7.0. For both enzymes, higher specific activity yields as a function of pH were obtained at pH 6.8. Soluble peroxidase activity increased three-fold as pH changed from 4.5 to 6.8. A much less pronounced pH effect was observed with the bound form, whose specific activity increased 20% in relation to the value obtained at pH 4.5. Pcroxidase
er al.
Soluble
1
2
Bound
3
4
5
6
Fig. 2. Electrophoretic patterns of soluble and bound peroxidase from papaya fruit during ripening. (gel 1,4) mature-green fruit; (gels 2,5) ripe fruit; (gels 3,6) over-ripe fruit. Electrophoresis was conducted as described in Experimental and samples containing 20 pg protein were applied on the gels. Intensity of the bands: high S; medium 8; low k!J.
t 5 .::
z L
1’
11
01
Salt concentration
(M 1
purijication
Soluble and bound peroxidases from ripe papaya were purified simultaneously by two different procedures. Because purification of soluble peroxidase was made difficult by the high concentration of pectins in the crude extract, the extract was heated for 20 min at 4.5’ for pectin removal. The use of ammonium sulphate to concentrate the protein present in the crude extracts initially caused a significant increase in enzyme activity, but this activating effect disappeared as purification proceeded. Gel filtration on Sephadex G-25 led to 8.4-fold purification and many smaller M, coloured compounds were separated from the active portion. This clean enzyme preparation was applied to a DEAE-cellulose column. The soluble peroxidase was eluted in a single activity peak (fraction 35-42) corresponding to ca 77% of the total activity applied to the column. This step brought about 21-fold purification, and the bulk of adsorbed protein was eluted in the same position as peroxidase activity. Further purification of soluble peroxidase involved gel filtration on Sephadex G-100 and resulted in 43-fold purification
Fig. 3. Extraction of bound peroxidase from ripe papaya fruit by various concentrations of salts. The salts were added to the extraction buffer. (O---O) CaCI,; (A -~A) MgCIZ; (U 0) N&l.
with 34% overall yield. The enzyme activity was eluted between fractions 16 and 30, indicating the presence of only one enzyme species or more than one species of somewhat similar N, (Fig. 4). PAGE of this enzyme preparations revealed the presence of five protein bands in the Sephadex G-100 peak (Fig. 5). The locations of these protein bands on the gel coincided with those of the bands stained for activity and it is apparent that this enzyme preparation was free of inert protein. A summary of the purification procedure is shown in Table I. Bound peroxidase was purified by a combination of 30-90 ammonium sulphate precipitation, Sephadex G100 gel filtration and DEAE-cellulose chromatography. When the bound form was eluted from the Sephadex G-
Peroxidases
Fraction
Fig. 4. Chromatography of soluble peroxidase on Sephadex G100. Enzyme solution was applied to a column of Sephadex G100 (2 x60cm) in 10 mM K-Pi buffer, pH 7.5. Elution was conducted with the same buffer. (O-O) soluble peroxidase actlvlty; (O- - - -0) A,,,.
BOWId
R
rlrl
Fig. 5. Electrohphoretic patterns of soluble and bound peroxidase. Samples of soluble peroxidase eluted from Sephadex G100 column were applied to the gels 1 and 2, and the gels were stained for activity and protein, respectively. Samples of bound peroxidase eluted from DEAE-cellulose column were applied to gels 3 and 4, and the gels were stained for activity and protein, respectively. The amount of protein applied to the gels was 20 pg. Intensity of the bands: high W; medium Q; low !&J.
Table
1053
100 column one active fraction was observed (Fig. 6). The peak eluted between fractions S&70 and the enzyme activity was reduced to 42% of the original activity present in the crude extract. Most of the protein in the preparation was eluted after the active peak. The subsequent use of DEAE-cellulose led to 140-fold purification accompanied by a 28% yield. In contrast to soluble peroxidase, the bound form was not adsorbed to the matrix under identical experimental conditions and was collected in the washings. The PAGE patterns of purified bound peroxidase showed the presence of three peroxidase components free from inert protien (Fig. 5). A summary of the overall purification procedure is shown in Table 2. The results reported here show that papaya fruit contains soluble and bound peroxidase forms and that the purification procedure adopted yielded highly purified preparations.
number
Soluble
from papaya
1. Purification
of soluble
Properties
ofpurified
enzymes
Soluble peroxidase activity as a function of pH using o-dianisidine as substrate showed a broad pH profile throughout the pH 5.C6.5 range. The optimum pH was pH 6.0, with the activity falling off slowly above this value and reaching half the maximum level at pH 8.8. In contrast, a sharp decrease in activity was observed as the pH was lowered below 5.0. The pH stability study showed that the enzyme lost no activity when held for 1 hr and 30” at pH 6.tL9.0, whereas on the acid side of the optimum there was a marked decrease in stability with a total loss of activity at pH 3.0. Bound peroxidase has maximum activity at pH 5.5 with a relatively steep fall on both the acid and basic side of the optimum; at pH 5.0 and 6.5 the activity was half the maximum value, and at pH 4.0 and 7.0 it was 30 and 32%, respectively. In contrast to the soluble form, bound peroxidase appears to be quite stable between pH 3.0 and unstable above 9.0. The effect of substrate concentration on soluble and bound peroxidases was investigated. Both enzymes exhibited differences in K, and V,,, values. For soluble peroxidase the K, values obtained with o-dianisidine, guaiacol and p-phenylenediamine as substrates were 0.1, 0.15 and 0.7 mM, respectively. The V,,. values were 41 144 and 76 units/ml. The K, values for bound peroxidases were 0.16,0.2 and 0.1 mM. Similarly, the corresponding V,,,., values were 25047 and 133 units/ml. The M,s of the enzymes were determined by gel filtration on Sephadex G-100. From the elution volumes of standard proteins the M, of soluble and bound peroxidase were calculated to be 41000 and 54 000, respectively. Similar M,s were established previously for per-
peroxidase
from papaya
Specific activity (units/mg protein)
Procedure
Total activity (units)
Crude extract
180400
735
(NH&SO, (3&90 % satn) Sephadex G-25 DEAE-cellulose Sephadex G-100
260400 110400 84970 61976
6771 6334 16032 32618
fruit
Purification (factor) 1 8.9 8.4 21.2 43.2
E. DA SILVA et al.
1054
Fig. 6. Chromatography of bound peroxidase on Sephadex G-100. Enzyme solution was applied to a column of Sephadex G-100 (2 x6Ocm) in 10mM K-Pi buffer, pH 7.5. Elution was conducted with the same buffer. (O----O) bound peroxidase activity; (O----O) AZsO.
Table
2. Purification
extract
(NH&SO, (3&90 % satn) Sephadex G-100 DEAE-cellulose
Table
bound
94 750
369
193 500 39 900 26910
3932 10500 51570
3. Effects of various
inhibitors
from papaya
Inhibitors p-Chloromercuribenzoate Iodacetamide N-Ethylmaleimide Na,SO, FeSO,
2.5 0.5 0.25 0.25
1
fruit
Purification (factor) 1 10.4 28.4 140.1
on peroxidase
Inhibitor concentration (mM)
from palmito [16], and red algae [17]. A wide range of M,s (3000&60000) has been reported for peroxidase from various sources [18]. Thus, the M,s obtained in this investigation are in general agreement with those reported in the literature. The effects of temperature on peroxidase activities were studied under standard assay conditions. Activation energy (E,) values of 10 300 and 8 300 kcal/mol for product formation were found for soluble and bound peroxidase, respectively. The effects of several compounds on peroxidase activities were also tested. It is worth noting that the inhibitors had different effects on peroxidases. The thiol reagents p-chloromercuribenzoate, iodacetamide and N-ethylmaleimide were more effective inhibitors of oxidase
peroxidase
Specific activity (units/mg protein)
Total activity (units)
Procedure Crude
of ionically
activities
Inhibition
(%)
Soluble
Bound
51 21 29 97 99
19 13 19 0 52
soluble peroxidase than of the bound peroxidase form. Similarly, Fe’ ’ was much more effective on soluble peroxidase, and Na+ inhibited the soluble enzyme but not bound peroxidase. The following cations had no effect on the enzymes: Cu’+, MgZt and Ca2+ (Table 3). It has been reported that the addition of ammonium salts to the peroxidase assay stimulates the peroxidation of o-dianisidine. This effect was demonstrated with horseradish peroxidase [19] but has not been shown with enzyme from other sources. Therefore, we decided to study the effect of ammonium sulphate on soluble peroxidase activity. When the salt was added to the reaction mixture as buffered solutions whose pH was adjusted to that ofthe assay mixture, the enzyme activity increased by
Peroxidases
two-fold from the control value. This activation effect was attributed to the binding of NH, or NH: to the hematin iron of the enzyme [9] and as cyanide is also capable of binding to hematin iron, we investigated the effects of cyanide on o-dianisidine peroxidation as a function of salt concentration. It was found that cyanide prevents ammonium sulphate activation. On the basis of this observation, further inhibition studies were carried out to determine the Ki values of enzyme for cyanide in the presence and in the absence of ammonium sulphate activation. The Ki values obtained from Dixon plots [20] for cyanide with or without salt were 5.8 x 10m5 and 3.5 x 10m4 M, respectively, indicating that enzyme was more sensitive to cyanide inhibition in the presence than in the absence of ammonium sulphate. According to Fridovich [19], this finding suggests the existence of two distinct sites of cyanide action, one of which results in inhibition of the enzyme and the other prevents salt activation.
EXPERIMENTAL
Plant material. Papaya fruits (Carica papaya L.) cv Solo grown under standard conditions in the state of SHo Paulo were picked at the mature-green stage, washed and stored at 20”. Exrraction. 100 g papaya pulp was homogenized in 0.05 M KPi buffer, pH 6.8 and the extract centrifuged at 25 000 g for 30 min. The supernatant was collected and immediately assayed for soluble peroxidase activity. The sediment fraction was resuspended in 40 ml of the above buffer containing 0.5 M CaCl,, stirred mechanically for 12 hr, and centrifuged as above. The supernatant was used as a source of ionically bound peroxidase. In some experiments, CaCl, was replaced with NaCl or MgCl,. All procedures were carried out at 4”. Purijication. The enzyme were precipitated from the supernatants by adding solid (NH&SO, (3&90% satn) and the suspension stirred for 2 hr and centrifuged at 25000 g. The resulting ppt. was dissolved in 10 ml K-Pi buffer, pH 7.5. The soluble enzyme was applied to a column of Sephadex G-25 (2 x 45 cm) equilibrated and eluted with 10 ml K-Pi buffer, pH 7.5. The eluate was applied to a column of DEAE-cellulose (1.5 x 23 cm) equilibrated and washed with the same buffer. The proteins were eluted with a linear gradients of NaCl (O-O.4 M) and 3 ml fractions were collected. The soluble enzyme was then applied in a vol. of 14 ml to a 2 x 60 cm column of Sephadex G100 previously equilibrated and subsequently eluted with the above buffer at a flow rate of 12 ml/hr, and 3 ml fractions were collecied. Ionically bound peroxidase was purified on Sephadex G-100 (2 x 60 cm) and DEAE-cellulose (1.5 x 23 cm). Each column was operated as described for soluble peroxidase. All procedures were carried out at 4”. Enzyme assays. Soluble and ionically bound peroxidase activities were determined by change in A at 460nm due to odianisidine oxidation in the presence of H,O, and the enzyme [Zl]. The reaction mixture consisted of 0.1 ml, 0.01 M o-dianisidine, 0.1 ml 11 M H,O*, 2.7 ml K-Pi buffer, pH 6.0 and 5.5 for the soluble and ionically bound peroxidases, respectively, in a total vol. of 3 ml. One enzyme unit is defined as the amount of enzyme producing a 0.001 A change per min under the assay conditions used. Protein concentration of the various extracts and soln was determined by the method of ref. [22] using bovine serum albumin as a standard. A at 280 nm was used to monitor protein in the column eluates. Effect oftemperature. Reactions were carried out under standard assay conditions as described above at 6 to 40”.
from papaya
1055
pH optima. Enzyme activity as a function of pH was determined using o-dianisidine, citrate-phosphate buffer (pH 3.0-6.5) and Tris-HCl buffer (pH 7.G9.0). The pH stability of the peroxidases was determined by incubating the enzymes for 1 hr at 30” in the same buffers. Assay conditions were the same as those described above. M, determination. 5 ml of purified preparations were applied to a column of Sephadex G-100 (2.5 x 50 cm) equilibrated with 0.05 M K-Pi buffer, pH 7.0. The column was calibrated with standard proteins as follows: cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin and lactate dehydrogenase. The M,s were estimated using a plot of VJV, vs log M, of standard proteins according to the method described in ref. 1231. Kinetic studies. The apparent K, and V,,, were determined from Lineweaver-Burk plots for soluble and bound pcroxidases at optimum pH conditions and concn of H,O, with o-dianisidine, guaiacol and p-phenylenediamine as substrates. PAGE was performed by the method of ref. [24] using 7% polyacrylamide gel in Tris-glycine buffer, pH 8.6, at 4” with 2.5 mA per tube. After the run, the proteins on the gels were stained with Coomassie brilliant blue R-250. For the enzyme activity, the gels were incubated in a soln containing 0.01 M odianisidine and 11 M H,O, for 1 hr. Relative mobility was determined by the migration of the bromophenol marker dye. Acknowledgements-The authors acknowledge the excellent technical assistance of Maraiza A. Silva. This work was supported by a grant from FundaqZo de Amparo a Pesquisa do Estado de SBo Paula, grant no. 80/1787-9.
REFERENCES
1. Akamine, E. K. and Theodore, G. (1979) Hart. Science 14, 138. 2. Catutani, A. T. and Lourenqo, E. J. (1982) An. Farm. Quim. Siio Paul0 22, 3. 3. Chan, H. T. Jr., Tam, S. Y. T. and Seo, S. T. (1981) J. Food Sci. 46, 190. 4. Haard, N. F. (1977) in Enzymes in Food and Beverage Processing ACS-Symposium Series 47 (Ory, R. L., ed.), p. 143. American Chemical Society, Washington. 5. Burnette, F. S. (1977) J. Food Sci. 42, 1. 6. Vgmos-Vigyb6, L. (1981) CRC Crit. Reo. Food Sci. Nutr. 15, 49. 7. Kahn, V., Goldshmidt, S. Amir, J. and Granit, R. (1981) J. Food Sci. 46, 756. 8. Wang, A. and Luh, B. S. (1983) J. Food Sci. 48, 1412. 9. Prabha, T. N. and Patwardhan, M. V. (1986) Acta Alimentaria 15, 199. 10. Mattoo, A. K., Modi, V. V. and Reddy, V. V. R. (1968) Indian J. Biochem. 5, 111. 11. Gorin, N. and Heidema, F. T. (1976) J. Agric. Food Chem. 24, 200. 12. Haard, N. F. (1973) Phytochemistry 12, 555. 13. Toraskar, M. V. and Modi, V. V. (1984) J. Agric. Food Chem. 32, 1352. 14. Kokkinakis, D. M. and Brooks, I. L. (1979) PIant Physiol. 63, 93. 15. Thomas, R. L., Jen, J. J. and Morr, C. V. (1981) J. Food Sci. 47, 158. 16. Draetta, I. S. and Ben-Shalom, N. (1984) .I. Food Biochem. 8, 109. 17. Murphy, M. J. and OhEocha, C. (1973) Phytochemistry 12, 55.
1056
E.
DA
SILVA et al.
18. Srivastava, 0. M. P. and Van Huystee, R. B. (1977) Phytochemistry 16, 1657. 19. Fridovich, I. (1963) _T.Biol. Chem. 238, 3921. 20. Dixon, M. (1953) Biochem. J. 55, 170. 21. McEwen, C. M. Jr. (1971) in Meth. Enzymol. Vol. 17B, p. 686.
22. Lowry, 0. H., Rosenbrough, N. J., Farr, A. and Randall, R. J. (1951) J. Biol. Gem. 193, 265. 23. Whitaker, J. R. (1963) Anal. Chem. 35, 1950. 24. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121. 404.
SOLUBLE
0031 9422/90%3.00 + 0.00 0 1990Pergamon Press plc
AND BOUND PEROXIDASES
FROM PAPAYA FRUIT
ELISETE DA SILVA, EUCLIDES J. LOURENCO and VALDIR A. NEVES Department of Food Nutrition,
FCF, UNESP, 14800 Araraquara, Bras&Rodovia Araraquara-JaC Km I, Brasil (Received in reuised.form 27 September 1989)
Key Word Index-Cnrica
papaya; Caricaceae; papaya fruit; ripening; peroxidase; enzyme properties.
Abstract--Soluble and bound peroxidases were isolated from the pulp of ripening papaya fruit. During papaya ripening, soluble and bound peroxidase activities increased 2.5 and 4.2-fold, respectively. Soluble peroxidase was purified 59-fold by ammonium sulphate precipitation and chromatography on Sephadex G-25, DEAE-cellulose and Sephadex G-100. Bound peroxidase was purified 140-fold by ammonium sulphate precipitation and chromatography on Sephadex G-100 and DEAE-cellulose. Polyacrylamide gel electrophoresis of the purified preparations revealed that both enzymes were highly purified by the procedures adopted. The soluble and bound forms had a M, of 41000 and 54000, respectively. Soluble and bound peroxidases showed optimum activity at pH 6.0 and 5.5, respectively, and were inhibited by p-chloromercuribenzoate, iodoacetamide, N-ethylmaleimide, potassium cyanide and Fe’+. Soluble peroxidase was activated by ammonium sulphate and this activation was prevented by cyanide.
RESULTS AND
INTRODUCTION Papaya belongs to the climateric class of fruits. The fruit ripen while still attached to the tree, exhibiting an increase in ethylene and carbon dioxide production [l]. The characteristics feature of papaya ripening is a change in texture, colour and sweetness. Softening of the papaya fruit is brought about by changes in cell wall constituents among which pectic substance play a major role [2,3]. Peroxidase (EC 1.11.1.7) has been implicated in metabolic processes such as ethylene biogenesis, cell development and membrane integrity [4]. The properties of peroxidase and its physiological role in postharvest fruits and vegetables have been reviewed by several authors [4-61. The enzyme has been reported to occur in plant tissues in soluble and bound forms. Kahn et al. [7] have recently demonstrated the presence of soluble and bound peroxidases in potato tuber extracts. They have also examined the biochemical properties of the enzymes in both crude and partially purified preparations. Soluble and bound peroxidases isolated from green asparagus have also been studied [S]. A correlation between peroxidase activity and fruit ripening has been shown in a number of cases [9]. In mango and apples, peroxidase activity increases with ripening [lo, 111. Similarly, bound peroxidase activity isolated from the pulp of ripening banana fruit is increased three-fold at the onset of the respiration climateric [ 123, and a considerable increase in soluble activity is also observed [13]. In contrast, the soluble peroxidase form from tomato fruit reaches a maximum at mature-green stage and declines with ripening [14, 151. To our knowledge, peroxidase activity in papaya fruit during ripening has not been adequately investigated. The present investigation was designed to study the extraction of soluble and ionically bound peroxidase and the changes in the enzyme activities during ripening. Procedures for purifying the two forms of peroxidase and some physicochemical properties are also described.
Extraction
DISCUSSION
of peroxidases
Activities of soluble and ionically bound peroxidases during ripening are shown in Fig. 1. Papaya fruit stored at 20” takes about 12 days to reach the ripe (edible) stage from the mature-green stage. The activity of bound peroxidase, which was lowest in green fruit, was greatly increased with ripening but gradually fell as the fruit turned from ripe to the over-ripe stage. Soluble peroxidase activity, the main form in green fruit, also reached a peak followed by a marked decrease, returning to the level found in mature-green papaya. After 12 days storage, the activities of the bound and soluble forms were
f/
I
I
I
I
9
12
15
18
Days
after
harvest
Fig. 1. Soluble and bound peroxidase activities during the ripening of papaya fruit. (O-O) Soluble peroxidase; ( l - - - -0) bound peroxidase.
1051
E.
1052
DA SILVA
4.2- and 2.5-fold higher than the initial values, respectively. Despite the differences in activity found for soluble peroxidase from mature-green and ripe papaya, the extracts revealed a reproducible pattern when subjected to polyacrylamide gel electrophoresis (PAGE) with the presence of five active bands (Fig. 2). The mobility of these bands did not vary with ripening but the ripe extract showed two major active bands (bands d and e) and maximum activity coinciding with the maximum intensity of active bands. The reduction in activity observed when ripening progressed from the ripe to the over-ripe stage was accompanied by a reduction in the number and intensity of active bands. The PAGE patterns of bound peroxidase extracts were different from those obtained with the soluble form (Fig. 2). The spectrum of peroxidase bands revealed the presence of three active bands; the two slowest moving bands, designated a and b, were not detected in the extracts, suggesting that these are species exclusively occurring in soluble peroxidase. The soluble peroxidase of papaya fruit was readily extracted by buffer of low molarity. Because no differences in specific activity were found when using several buffer molarities ranging from 0.05 to 0.2 M, the 0.05 M concentration was used throughout the study. In contrast, the extraction of ionically bound enzyme required the addition of either calcium, magnesium or sodium chloride to the buffer to release the enzyme from cell components. An increase in specific activity with the increase in salt concentration was found (Fig. 3). Maximum yield was obtained at a concentration of0.5 M with all three salts, and activity was reduced at higher concentrations. Sodium chloride was less effective than calcium and magnesium chloride for the solubilization of bound peroxidase. The optimal pH conditions for peroxidase extraction were investigated by using phosphate buffer in the pH range 4.5.-7.0. For both enzymes, higher specific activity yields as a function of pH were obtained at pH 6.8. Soluble peroxidase activity increased three-fold as pH changed from 4.5 to 6.8. A much less pronounced pH effect was observed with the bound form, whose specific activity increased 20% in relation to the value obtained at pH 4.5. Pcroxidase
er al.
Soluble
1
2
Bound
3
4
5
6
Fig. 2. Electrophoretic patterns of soluble and bound peroxidase from papaya fruit during ripening. (gel 1,4) mature-green fruit; (gels 2,5) ripe fruit; (gels 3,6) over-ripe fruit. Electrophoresis was conducted as described in Experimental and samples containing 20 pg protein were applied on the gels. Intensity of the bands: high S; medium 8; low k!J.
t 5 .::
z L
1’
11
01
Salt concentration
(M 1
purijication
Soluble and bound peroxidases from ripe papaya were purified simultaneously by two different procedures. Because purification of soluble peroxidase was made difficult by the high concentration of pectins in the crude extract, the extract was heated for 20 min at 4.5’ for pectin removal. The use of ammonium sulphate to concentrate the protein present in the crude extracts initially caused a significant increase in enzyme activity, but this activating effect disappeared as purification proceeded. Gel filtration on Sephadex G-25 led to 8.4-fold purification and many smaller M, coloured compounds were separated from the active portion. This clean enzyme preparation was applied to a DEAE-cellulose column. The soluble peroxidase was eluted in a single activity peak (fraction 35-42) corresponding to ca 77% of the total activity applied to the column. This step brought about 21-fold purification, and the bulk of adsorbed protein was eluted in the same position as peroxidase activity. Further purification of soluble peroxidase involved gel filtration on Sephadex G-100 and resulted in 43-fold purification
Fig. 3. Extraction of bound peroxidase from ripe papaya fruit by various concentrations of salts. The salts were added to the extraction buffer. (O---O) CaCI,; (A -~A) MgCIZ; (U 0) N&l.
with 34% overall yield. The enzyme activity was eluted between fractions 16 and 30, indicating the presence of only one enzyme species or more than one species of somewhat similar N, (Fig. 4). PAGE of this enzyme preparations revealed the presence of five protein bands in the Sephadex G-100 peak (Fig. 5). The locations of these protein bands on the gel coincided with those of the bands stained for activity and it is apparent that this enzyme preparation was free of inert protein. A summary of the purification procedure is shown in Table I. Bound peroxidase was purified by a combination of 30-90 ammonium sulphate precipitation, Sephadex G100 gel filtration and DEAE-cellulose chromatography. When the bound form was eluted from the Sephadex G-
Peroxidases
Fraction
Fig. 4. Chromatography of soluble peroxidase on Sephadex G100. Enzyme solution was applied to a column of Sephadex G100 (2 x60cm) in 10 mM K-Pi buffer, pH 7.5. Elution was conducted with the same buffer. (O-O) soluble peroxidase actlvlty; (O- - - -0) A,,,.
BOWId
R
rlrl
Fig. 5. Electrohphoretic patterns of soluble and bound peroxidase. Samples of soluble peroxidase eluted from Sephadex G100 column were applied to the gels 1 and 2, and the gels were stained for activity and protein, respectively. Samples of bound peroxidase eluted from DEAE-cellulose column were applied to gels 3 and 4, and the gels were stained for activity and protein, respectively. The amount of protein applied to the gels was 20 pg. Intensity of the bands: high W; medium Q; low !&J.
Table
1053
100 column one active fraction was observed (Fig. 6). The peak eluted between fractions S&70 and the enzyme activity was reduced to 42% of the original activity present in the crude extract. Most of the protein in the preparation was eluted after the active peak. The subsequent use of DEAE-cellulose led to 140-fold purification accompanied by a 28% yield. In contrast to soluble peroxidase, the bound form was not adsorbed to the matrix under identical experimental conditions and was collected in the washings. The PAGE patterns of purified bound peroxidase showed the presence of three peroxidase components free from inert protien (Fig. 5). A summary of the overall purification procedure is shown in Table 2. The results reported here show that papaya fruit contains soluble and bound peroxidase forms and that the purification procedure adopted yielded highly purified preparations.
number
Soluble
from papaya
1. Purification
of soluble
Properties
ofpurified
enzymes
Soluble peroxidase activity as a function of pH using o-dianisidine as substrate showed a broad pH profile throughout the pH 5.C6.5 range. The optimum pH was pH 6.0, with the activity falling off slowly above this value and reaching half the maximum level at pH 8.8. In contrast, a sharp decrease in activity was observed as the pH was lowered below 5.0. The pH stability study showed that the enzyme lost no activity when held for 1 hr and 30” at pH 6.tL9.0, whereas on the acid side of the optimum there was a marked decrease in stability with a total loss of activity at pH 3.0. Bound peroxidase has maximum activity at pH 5.5 with a relatively steep fall on both the acid and basic side of the optimum; at pH 5.0 and 6.5 the activity was half the maximum value, and at pH 4.0 and 7.0 it was 30 and 32%, respectively. In contrast to the soluble form, bound peroxidase appears to be quite stable between pH 3.0 and unstable above 9.0. The effect of substrate concentration on soluble and bound peroxidases was investigated. Both enzymes exhibited differences in K, and V,,, values. For soluble peroxidase the K, values obtained with o-dianisidine, guaiacol and p-phenylenediamine as substrates were 0.1, 0.15 and 0.7 mM, respectively. The V,,. values were 41 144 and 76 units/ml. The K, values for bound peroxidases were 0.16,0.2 and 0.1 mM. Similarly, the corresponding V,,,., values were 25047 and 133 units/ml. The M,s of the enzymes were determined by gel filtration on Sephadex G-100. From the elution volumes of standard proteins the M, of soluble and bound peroxidase were calculated to be 41000 and 54 000, respectively. Similar M,s were established previously for per-
peroxidase
from papaya
Specific activity (units/mg protein)
Procedure
Total activity (units)
Crude extract
180400
735
(NH&SO, (3&90 % satn) Sephadex G-25 DEAE-cellulose Sephadex G-100
260400 110400 84970 61976
6771 6334 16032 32618
fruit
Purification (factor) 1 8.9 8.4 21.2 43.2
E. DA SILVA et al.
1054
Fig. 6. Chromatography of bound peroxidase on Sephadex G-100. Enzyme solution was applied to a column of Sephadex G-100 (2 x6Ocm) in 10mM K-Pi buffer, pH 7.5. Elution was conducted with the same buffer. (O----O) bound peroxidase activity; (O----O) AZsO.
Table
2. Purification
extract
(NH&SO, (3&90 % satn) Sephadex G-100 DEAE-cellulose
Table
bound
94 750
369
193 500 39 900 26910
3932 10500 51570
3. Effects of various
inhibitors
from papaya
Inhibitors p-Chloromercuribenzoate Iodacetamide N-Ethylmaleimide Na,SO, FeSO,
2.5 0.5 0.25 0.25
1
fruit
Purification (factor) 1 10.4 28.4 140.1
on peroxidase
Inhibitor concentration (mM)
from palmito [16], and red algae [17]. A wide range of M,s (3000&60000) has been reported for peroxidase from various sources [18]. Thus, the M,s obtained in this investigation are in general agreement with those reported in the literature. The effects of temperature on peroxidase activities were studied under standard assay conditions. Activation energy (E,) values of 10 300 and 8 300 kcal/mol for product formation were found for soluble and bound peroxidase, respectively. The effects of several compounds on peroxidase activities were also tested. It is worth noting that the inhibitors had different effects on peroxidases. The thiol reagents p-chloromercuribenzoate, iodacetamide and N-ethylmaleimide were more effective inhibitors of oxidase
peroxidase
Specific activity (units/mg protein)
Total activity (units)
Procedure Crude
of ionically
activities
Inhibition
(%)
Soluble
Bound
51 21 29 97 99
19 13 19 0 52
soluble peroxidase than of the bound peroxidase form. Similarly, Fe’ ’ was much more effective on soluble peroxidase, and Na+ inhibited the soluble enzyme but not bound peroxidase. The following cations had no effect on the enzymes: Cu’+, MgZt and Ca2+ (Table 3). It has been reported that the addition of ammonium salts to the peroxidase assay stimulates the peroxidation of o-dianisidine. This effect was demonstrated with horseradish peroxidase [19] but has not been shown with enzyme from other sources. Therefore, we decided to study the effect of ammonium sulphate on soluble peroxidase activity. When the salt was added to the reaction mixture as buffered solutions whose pH was adjusted to that ofthe assay mixture, the enzyme activity increased by
Peroxidases
two-fold from the control value. This activation effect was attributed to the binding of NH, or NH: to the hematin iron of the enzyme [9] and as cyanide is also capable of binding to hematin iron, we investigated the effects of cyanide on o-dianisidine peroxidation as a function of salt concentration. It was found that cyanide prevents ammonium sulphate activation. On the basis of this observation, further inhibition studies were carried out to determine the Ki values of enzyme for cyanide in the presence and in the absence of ammonium sulphate activation. The Ki values obtained from Dixon plots [20] for cyanide with or without salt were 5.8 x 10m5 and 3.5 x 10m4 M, respectively, indicating that enzyme was more sensitive to cyanide inhibition in the presence than in the absence of ammonium sulphate. According to Fridovich [19], this finding suggests the existence of two distinct sites of cyanide action, one of which results in inhibition of the enzyme and the other prevents salt activation.
EXPERIMENTAL
Plant material. Papaya fruits (Carica papaya L.) cv Solo grown under standard conditions in the state of SHo Paulo were picked at the mature-green stage, washed and stored at 20”. Exrraction. 100 g papaya pulp was homogenized in 0.05 M KPi buffer, pH 6.8 and the extract centrifuged at 25 000 g for 30 min. The supernatant was collected and immediately assayed for soluble peroxidase activity. The sediment fraction was resuspended in 40 ml of the above buffer containing 0.5 M CaCl,, stirred mechanically for 12 hr, and centrifuged as above. The supernatant was used as a source of ionically bound peroxidase. In some experiments, CaCl, was replaced with NaCl or MgCl,. All procedures were carried out at 4”. Purijication. The enzyme were precipitated from the supernatants by adding solid (NH&SO, (3&90% satn) and the suspension stirred for 2 hr and centrifuged at 25000 g. The resulting ppt. was dissolved in 10 ml K-Pi buffer, pH 7.5. The soluble enzyme was applied to a column of Sephadex G-25 (2 x 45 cm) equilibrated and eluted with 10 ml K-Pi buffer, pH 7.5. The eluate was applied to a column of DEAE-cellulose (1.5 x 23 cm) equilibrated and washed with the same buffer. The proteins were eluted with a linear gradients of NaCl (O-O.4 M) and 3 ml fractions were collected. The soluble enzyme was then applied in a vol. of 14 ml to a 2 x 60 cm column of Sephadex G100 previously equilibrated and subsequently eluted with the above buffer at a flow rate of 12 ml/hr, and 3 ml fractions were collecied. Ionically bound peroxidase was purified on Sephadex G-100 (2 x 60 cm) and DEAE-cellulose (1.5 x 23 cm). Each column was operated as described for soluble peroxidase. All procedures were carried out at 4”. Enzyme assays. Soluble and ionically bound peroxidase activities were determined by change in A at 460nm due to odianisidine oxidation in the presence of H,O, and the enzyme [Zl]. The reaction mixture consisted of 0.1 ml, 0.01 M o-dianisidine, 0.1 ml 11 M H,O*, 2.7 ml K-Pi buffer, pH 6.0 and 5.5 for the soluble and ionically bound peroxidases, respectively, in a total vol. of 3 ml. One enzyme unit is defined as the amount of enzyme producing a 0.001 A change per min under the assay conditions used. Protein concentration of the various extracts and soln was determined by the method of ref. [22] using bovine serum albumin as a standard. A at 280 nm was used to monitor protein in the column eluates. Effect oftemperature. Reactions were carried out under standard assay conditions as described above at 6 to 40”.
from papaya
1055
pH optima. Enzyme activity as a function of pH was determined using o-dianisidine, citrate-phosphate buffer (pH 3.0-6.5) and Tris-HCl buffer (pH 7.G9.0). The pH stability of the peroxidases was determined by incubating the enzymes for 1 hr at 30” in the same buffers. Assay conditions were the same as those described above. M, determination. 5 ml of purified preparations were applied to a column of Sephadex G-100 (2.5 x 50 cm) equilibrated with 0.05 M K-Pi buffer, pH 7.0. The column was calibrated with standard proteins as follows: cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin and lactate dehydrogenase. The M,s were estimated using a plot of VJV, vs log M, of standard proteins according to the method described in ref. 1231. Kinetic studies. The apparent K, and V,,, were determined from Lineweaver-Burk plots for soluble and bound pcroxidases at optimum pH conditions and concn of H,O, with o-dianisidine, guaiacol and p-phenylenediamine as substrates. PAGE was performed by the method of ref. [24] using 7% polyacrylamide gel in Tris-glycine buffer, pH 8.6, at 4” with 2.5 mA per tube. After the run, the proteins on the gels were stained with Coomassie brilliant blue R-250. For the enzyme activity, the gels were incubated in a soln containing 0.01 M odianisidine and 11 M H,O, for 1 hr. Relative mobility was determined by the migration of the bromophenol marker dye. Acknowledgements-The authors acknowledge the excellent technical assistance of Maraiza A. Silva. This work was supported by a grant from FundaqZo de Amparo a Pesquisa do Estado de SBo Paula, grant no. 80/1787-9.
REFERENCES
1. Akamine, E. K. and Theodore, G. (1979) Hart. Science 14, 138. 2. Catutani, A. T. and Lourenqo, E. J. (1982) An. Farm. Quim. Siio Paul0 22, 3. 3. Chan, H. T. Jr., Tam, S. Y. T. and Seo, S. T. (1981) J. Food Sci. 46, 190. 4. Haard, N. F. (1977) in Enzymes in Food and Beverage Processing ACS-Symposium Series 47 (Ory, R. L., ed.), p. 143. American Chemical Society, Washington. 5. Burnette, F. S. (1977) J. Food Sci. 42, 1. 6. Vgmos-Vigyb6, L. (1981) CRC Crit. Reo. Food Sci. Nutr. 15, 49. 7. Kahn, V., Goldshmidt, S. Amir, J. and Granit, R. (1981) J. Food Sci. 46, 756. 8. Wang, A. and Luh, B. S. (1983) J. Food Sci. 48, 1412. 9. Prabha, T. N. and Patwardhan, M. V. (1986) Acta Alimentaria 15, 199. 10. Mattoo, A. K., Modi, V. V. and Reddy, V. V. R. (1968) Indian J. Biochem. 5, 111. 11. Gorin, N. and Heidema, F. T. (1976) J. Agric. Food Chem. 24, 200. 12. Haard, N. F. (1973) Phytochemistry 12, 555. 13. Toraskar, M. V. and Modi, V. V. (1984) J. Agric. Food Chem. 32, 1352. 14. Kokkinakis, D. M. and Brooks, I. L. (1979) PIant Physiol. 63, 93. 15. Thomas, R. L., Jen, J. J. and Morr, C. V. (1981) J. Food Sci. 47, 158. 16. Draetta, I. S. and Ben-Shalom, N. (1984) .I. Food Biochem. 8, 109. 17. Murphy, M. J. and OhEocha, C. (1973) Phytochemistry 12, 55.
1056
E.
DA
SILVA et al.
18. Srivastava, 0. M. P. and Van Huystee, R. B. (1977) Phytochemistry 16, 1657. 19. Fridovich, I. (1963) _T.Biol. Chem. 238, 3921. 20. Dixon, M. (1953) Biochem. J. 55, 170. 21. McEwen, C. M. Jr. (1971) in Meth. Enzymol. Vol. 17B, p. 686.
22. Lowry, 0. H., Rosenbrough, N. J., Farr, A. and Randall, R. J. (1951) J. Biol. Gem. 193, 265. 23. Whitaker, J. R. (1963) Anal. Chem. 35, 1950. 24. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121. 404.