Process Biochemistry 46 (2011) 1350–1357
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Biochemical characterization of a peroxidase isolated from Caribbean plant:
Euphorbia cotinifolia
Reetesh Kumar a , Kunwar Awaneesh Singh b , Vijay Kumar Singh a , Medicherla V. Jagannadham a,∗
a
b
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
a r t i c l e
i n f o
Article history:
Received 23 September 2010
Received in revised form
14 December 2010
Accepted 9 March 2011
Keywords:
Caribbean copper plant
CCPP
Euphorbiaceae
Euphorbia cotinifolia
Peroxidase
Secondary structure
a b s t r a c t
A Caribbean copper plant peroxidase (CCPP) is purified from the latex of Euphorbia cotinifolia, using anion
exchange chromatography. The molecular mass and isoelectic point of the enzyme is 43.11 kDa and pH
8.1 respectively. The peroxidase is found to be sensitive towards general phenolic substrates like guaiacol, pyrogallol, ␣-aminopterin, phloroglucinol, o-phenelenediamine and dianisidine dihydrochloride.
The substrate specificity of CCPP was distinct from that of other peroxidases, and the best substrate for
CCPP was guaiacol at pH 6.0 and 50 ◦ C. Sucrose and Ca2+ enhance the activity whereas the activity is
significantly inhibited by NaN3 and Na2 SO3 . The strong absorption at 650 nm reveals the presence of Cu
ions as a prosthetic group. Spectroscopic studies reveal that CCPP has high ␣-helicity. The enzyme was
found to be very stable at room temperature and retained more than 80% activity even after a period of
2 months and was stable for more than 6 months at 4 ◦ C without any additive or preservative. Adequate
amount of latex, easy purification method, broad substrate specificity, and high stability against pH, temperature, chaotrophs and organic solvents makes this enzyme a potential candidate in biotechnological
and industrial applications.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Peroxidases (E.C. 1.11.1.7), a ubiquitous enzymes [1] is widely
distributed in plants, microbes, fungi and vertebrates. They are
present in multiple ionic forms, and in vivo they are one of the key
enzymes controlling the plant growth and differentiation. The multiple isoperoxidase form found within the same plant source can
differ significantly with respect to molecular mass, pH and temperature optima, isoelectric point, substrate specificity, amino acid and
sugar composition and heat stability. Functionally, these enzymes
act as an oxidoreductase that catalyzes a reaction, in which H2 O2 act
as the acceptor and another compound act as the donor of hydrogen
atoms [2].
Three dimensional structure analysis and on the basis of amino
acid sequence homology, it can be broadly classified into three
major classes. Plant ascorbate peroxidase, cytochrome c peroxidase and bacterial peroxidase belong to same evolutionary branch
Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; GuHCl,
guanidine hydrochloride; H2 O2 , hydrogen peroxide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TEMED, tetramethylethylenediamine;
EDTA, ethylenediaminetetraacetic acid.
∗ Corresponding author. Tel.: +91 542 2367936; fax: +91 542 2367568.
E-mail addresses: [email protected], [email protected], [email protected]
(M.V. Jagannadham).
1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.03.003
and are designated as class I, whereas secretary fungal enzymes,
ligninolytic peroxidase [3], manganese (Mn) peroxidase from fungi
[4] are classified as class II peroxidase and plant secretory peroxidases, vacuolar peroxidases such as horseradish peroxidases, that
usually contain a ferriprotoporphyrin IX prosthetic group linked
to His residue are designated as class III peroxidases [5]. Peroxidases have further been classified into anionic and cationic
group according to their electrophoretic mobility. Usually class
III peroxidase are assigned to have physiological roles in the primary and secondary metabolic processes like, catabolized phenolic
compounds for biosynthetic and catabolic functions [6], cross linking of cell wall polysaccharide, cell elongation regulation, wound
healing, abiotic stress, ethylene biosynthesis, scavenging of peroxides, oxidation of toxic compounds, defense mechanism towards
pathogens, biodegradation reactions, metabolism of plant hormone, indole acetic acid (IAA) oxidation, lignification of cell wall
[7], anthocyanin degradation [8], leaf senescence and for use in pulp
and paper industries [9]. In food industry they are associated with
development of flavor, color, texture and nutrition quality of food
[10]. Commercially, peroxidases are involved in production of alkaloid, biosensor construction, air pollution damage control [11], food
processing, food storage, biotransformation of organic compounds,
treatment of industrial waste water containing phenols and aromatic amines [12], production of oxidants, lignin degradation in
fuels, bio-bleaching process, immunoassay (ELISA kit), site directed
mutagenesis [13], production of secondary antibodies for research
R. Kumar et al. / Process Biochemistry 46 (2011) 1350–1357
and medical diagnosis, production of dimeric alkaloids, preventing
deterioration of quality of many frozen fruits, biotransformation of
organic compounds, preventing deterioration of prepared foods e.g.
beer, porridge, conscous etc. [14] and recently the combination of
peroxidase and IAA has been introduced as a novel cancer therapy
[15].
Euphorbia cotinifolia is a deciduous tropical shrub (family
Euphorbiaceae) and has many medicinal applications: primarily,
its leaves, which have molluscidal [16] and antiviral properties.
Among antiviral properties it exhibited high antiherpetic action
in its leaf/stem water methanol extracts [17]. In the course of
screening of latex for various activities, the latex of plant showed
peroxidase activity and encouraged us to look further in detail.
In this regards, in the present study, a peroxidase has been purified from the plant is being introduced and characterized. Its wide
application and stability at temperatures, pH, hydrogen peroxide,
urea and GuHCl was therefore important to explore the biochemical components of the latex of this plant in view of its applications
in biotechnology and food industries.
2. Materials and methods
5 mL of latex was collected from incisions on the leaves and stems of Euphorbia
cotinifolia in 45 mL of 10 mM Tris buffer, pH 8.4 and stored at 20 ◦ C for 72 h. Major
chemicals used in this study and their sources are given here under: sepharose,
BSA, GuHCl, urea, DMSO, glycerol, ␤-mercaptoethanol, Coomassie brilliant blue
R-250, phloroglucinol, o-phenelenediamine, avidin and horseradish (Sigma Chemical Co., USA), guaiacol (Sisco Research Lab, India), pyrogallol, ␣-aminopterin
(Spectrochem, India), dianisidine dihydrochloride (Himedia), H2 O2 (Loba Chemie
Limited), Coomassie brilliant blue G-250 (Eastman Kodak), ampholine carrier
ampholites (LKB), Molecular weight marker (Banglore Genei). All the other chemicals obtained were of highest purity commercially available.
2.1. Purification
2.1.1. Step 1. Removal of gum
The latex, collected in 10 mM Tris buffer of pH 8.4 is stored at −20 ◦ C for 72 h.
The latex was thawed at room temperature and centrifuged at 10,000 × g for 20 min
to remove any insoluble material. Clear supernatant obtained in this process is
considered as crude latex for further applications.
2.1.2. Step 2. Anion exchange chromatography on DEAE sepharose
Crude latex was applied to anion exchange chromatography on a DEAEsepharose fast flow column (5.0 cm × 5.0 cm) pre-equilibrated with Tris pH 8.4 and
eluted isocratically with 0.75 M NaCl to remove loosely bound protein with no peroxidase activity. The tightly bound proteins were eluted with a linear gradient of
0.75–1.25 M and the fractions of 3 mL volume were collected at a flow rate 3 mL/min.
The fractions were assayed for protein content, extent of homogeneity and peroxidase activity. The homogenous as well as active fractions were pooled, dialyzed and
stored in closed vials at 4 ◦ C for further biochemical characterization.
1351
2.4. Zymogram
25 ␮g of the purified enzyme was mixed with sample loading buffer and analyzed on 12.5% SDS–PAGE without prior boiling [21]. Gel was run at 200 V for 1 h and
soaked in 2.5% Triton X-100 for displacement of SDS. Gel was washed with water to
remove Triton X-100 and incubated for 5 min in 10 mM Tris buffer, pH 8.4 containing 20% H2 O2 (v/v). For color development 1 mL of 0.2 mM pyrogallol was added to
the above mixture followed by termination of the reaction with 1 mL of 1% EDTA.
2.5. Isoelectric focusing
The isoelectric point (pI) of purified peroxidase was determined by isoelectric focusing on polyacrylamide disk as described [22]. An electrophoretic run was
carried out with ampholine carrier ampholytes pH 7.0–9.0.
2.6. Mass spectrometry
The mass spectra were recorded on a MICRO-MASS QUATTRO II mass spectrometer (Micromass, Altricem, UK) equipped with electrospray ionization ion [23].
Protein sample (20 ␮M) was precipitated with 5% TCA, and the resulting precipitate
was washed with chilled acetone. The pellet obtained was dissolved in ultra pure
water and passed through ZIP-TIP C-18 resin before the measurement. The resulting
sample was injected into the ion source using Harvard Apparatus model 11 Syringe
pump. The source and sample was operated at 80 ◦ C and at 30 ◦ C respectively. The
electrospray capillary was set at 3.5 kV and the cone voltage at 20 V. The intensity and signal stability for ESI-MS were significantly increased by adding a small
amount of methanol (3%) to the aqueous solution of peroxidase. The scanned range
of mass spectrometer was from m/z 500 to 2200 in 6 s. The charge states and the
deconvolution of the spectra was carried out using MassLynx and MaxEnt software
respectively [24].
2.7. Antigenic properties
40 ␮g of the purified CCPP in 0.05 M Tris buffer, pH 8.4 was emulsified with Freund’s complete adjuvant and injected subcutaneously at multiple sites as described
[25]. Preimmune serum was collected prior to the immunization of the rabbit and
used as a control for immunoassays. The presence of antibodies was confirmed
by Ouchterlony’s double immunodiffusion method, described by Ouchterlony and
Nilsson [26].
2.8. pH and temperature optima
The reaction mixture, composed of 20 ␮g of peroxidase in an appropriate buffer
of desired pH and 500 ␮l of substrate (0.2 mM guaiacol) solution at the same pH,
was equilibrated for 1 min at room temperature. The enzyme was assayed using
20% H2 O2 (v/v) as substrate I and one of the hydrogen donors such as guaiacol as
reducing substrate II. The assay was carried out at 37 ◦ C using buffers such as KCl–HCl
(pH 0.5–1.5), glycine-HCl (pH 2.0–3.5), sodium acetate (pH 4.0–5.5), sodium phosphate (pH 6.0–7.5), Tris (pH 8.0–10.0) and sodium carbonate (pH 10.5–11.0), all at
molarities of 50 mM. Assay was performed at every pH without addition of enzyme
and used as control.
For temperature optimum measurement, 20 ␮g of the purified peroxidase was
incubated at 30 min in the temperature range of 20–80 ◦ C and assayed with 20% H2 O2
(v/v) and 0.2 mM guaiacol for 1 min. Activity was measured as described above.
2.9. Peroxidase activity as a function of hydrogen peroxide concentration
2.2. Peroxidase assay and protein content
Peroxidase activity was determined spectrophotometrically using H2 O2 as substrate I and one of the hydrogen donors such as guaiacol, pyrogallol, ␣-aminopterin,
phloroglucinol, o-phenelenediamine and dianisidine dihydrochloride as subsequent
reducing substrates. The absorbance was measured at 470 nm, 420 nm, 420 nm,
420 nm, 445 nm and 420 nm respectively and substrate specificity was studied under
optimal condition of pH, buffer condition, etc., as determined earlier for other plant
peroxidases [18]. One unit of activity is defined as the amount of peroxidase that
oxidizes 1 ␮mol of substrate per minute under standard conditions and its specific
activity was noted as units of activity per milligram of protein. Concentration of
protein was also determined spectrophotometrically at 280 nm as well as Bradford
assay [19] using BSA as a standard.
2.3. Electrophoresis
SDS–PAGE was used to assess the homogeneity of enzymes preparation as well
as estimation of molecular mass. Purified enzyme (25 ␮g) from the homogenous
pool fractions (30–45) were mixed with sample loading buffer and loaded on 12.5%
SDS–PAGE under both non-reducing and reducing conditions [20]. The gels were
stained with 0.2% Coomassie brilliant blue R-250. Molecular mass standard used
were phosphorylase b (93.7 kDa), BSA (66.0 kDa), ovalbumin (43.0 kDa), carbonic
anhydrase (29.0 kDa), soybean trypsin inhibitor (20.1 kDa), and chicken egg white
lysozyme (14.3 kDa).
The effect of initial hydrogen peroxide concentration on the peroxidase activity
of purified peroxidase was investigated at pH 8.4 and 25 ◦ C with different concentration of hydrogen peroxide. The absorbance was measured at 470 nm with 0.2 mM
guaiacol used as reducing substrate.
2.10. Effect of additives on peroxidase
20 ␮g of the purified peroxidase was incubated with different additives such
as EDTA (1 mM), sucrose (10%, w/v), sodium azide (1 mM) and DMSO (2%, v/v) for
30 min and assayed as described above.
2.11. Effect of salts on peroxidase
To study the effect of salts, the enzyme was incubated with increasing concentration of salts in the range of 10–100 mM for 30 min and assayed as mentioned
above. The salts used for the present study were NaCl, CaCl2 , MgCl2 , and Na2 SO3 .
2.12. Spectroscopic studies: absorbance, fluorescence, and circular dichroism
Absorbance measurements were carried out on a Beckman DU-640B spectrophotometer. Absorbance spectra were recorded between 300 and 900 nm.
Protein concentration for all absorbance measurements was 0.05 mg/mL. To assess
the presence of copper ions, the enzyme was reduced with ascorbic acid followed
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R. Kumar et al. / Process Biochemistry 46 (2011) 1350–1357
Fig. 1. (A) Chromatography of peroxidase on fast flow Sepharose column. The eluted fractions were assayed for activity () and protein content (䊉). The fractions from 30
to 45 were pooled and are indicated by a horizontal line. (B) Electrophoretic analysis of the purified proteins. (1) Polyacrylamide gel electrophoresis of purified peroxidase
(25 ␮g) was electrophoresed in 12.5% polyacrylamide gel: line M, protein marker; line I, crude in non-reducing condition; line II, peroxidase in non-reducing condition; line
III, peroxidase in reducing condition. The proteins were stained using Coomassie brilliant blue R-250. (2) Zymogram of peroxidase. (3) Isoelectric focusing of peroxidase.
(C) Mass determination of the peroxidase by ESI-MS (electrospray ionization mass spectrometry). (D) Ouchterlony’s double immunodiffusion was carried out in 1% agarose
dissolved in phosphate-buffered saline. Antiserum (100 ␮l) was added in the central well, and 40 ␮g of four purified (Avidin, MGP and horseradish was added in peripheral
wells. The precipitin band was observed after 24 h of incubation.
by oxidation with H2 O2 and the color was monitored. Fluorescence measurements
were carried out on a Perkin-Elmer LS-50B spectrofluorometer. The protein concentration used was 0.01 mg/mL for all fluorescence measurements. Tryptophan was
selectively excited at 292 nm, whereas for both the tryptophan and tyrosine fluorescence of CCPP, the excitation wavelength was 278 nm. The emission was recorded
from 300 to 400 nm with 5 and 10 nm slit widths for emission and excitation, respectively.
The circular dichroism (CD) spectra of CCPP under native and denatured conditions (6 M GuHCl and 8 M urea) were taken on a JASCO J 500A spectropolarimeter.
The instrument was calibrated using ammonium (+)-10-camphorsulfonate. Conformational changes in the secondary structure of the protein were monitored in the
region between 200 and 260 nm with a protein concentration of 0.1 mg/mL in a
1 mm path length cuvette. The results were expressed as mean residue ellipticity
[]MRW , using the equation
[]MRW =
obs MRW
10cl
where obs , c, and l represent the observed ellipticity in degrees, the protein concentration in mg/mL, and the path length in cm, respectively. The mean weight of
amino acid residues (MRW) was taken as 110 for the calculations.
2.13. Atomic absorbance spectroscopy study
The peroxidase was inferred from absorbance spectra in the wavelength range of
200–800 nm, and the copper and Iron content was estimated by atomic absorption
spectroscopy. 1 mg of peroxidase was taken in 10 mL of nitric acid and perchloric
acid in the ratio of 6:1 (v/v) in a conical flask free of any metals and digested slowly
at 90 ◦ C on a hot plate until white fumes appeared. Five milliliters of 1 N nitric acid
was added, and the resulting solution was used for atomic absorption spectroscopy.
Blank also prepared by similar procedure without protein. The sample was analyzed
on a Shimadzu atomic absorbance spectrum, and the copper content was deduced
from the standard Cu plot.
3. Results and discussion
3.1. Purification of Peroxidase, CCPP
A peroxidase was purified to homogeneity by using anion
exchange chromatography. The unbound, buffer wash and fractions
eluted isocratically with 0.75 M NaCl did not show any peroxidase
activity. The tightly bound protein eluted with 0.75–1.25 M NaCl
showed a major peak with high peroxidase activity (Fig. 1A). The
major peak was not chosen because most of the fractions were nonhomogeneous in nature due to close isoelectric point among these
proteins. Therefore, only the pure fractions from DEAE-Sepharose
were chosen for further studies. After homogeneity and activity analysis the pure fractions were pooled, dialyzed and named
CCPP following standard peroxidase nomenclature. The purification protocol is, however, highly reproducible and the results are
summarized in Table 1. The yield of purified CCPP is 3.5 ± 0.5%
R. Kumar et al. / Process Biochemistry 46 (2011) 1350–1357
1353
Table 1
Purification of peroxidase form the latex of Euphorbia cotinifolia.
Steps
Total protein (mg)
Total activitya (units)
Specific activity (units/mg)
Yield (%)
Crude extract
Sepharose fast flow
315
10.5
3455
294
11
28
100
3.3
a
One unit of enzyme activity is defined as the amount of enzyme that, under conditions described, gives rise to an increase of one unit of absorbance at 470 nm per 60 s
of incubation.
of the total protein in the crude latex with a high specific activity 28 ± 0.25 units/mg. Substrate specificity of CCPP was carried
out under different substrate concentration with 20% of H2 O2 sub
stock solution (30% H2 O2 solution). A comparison of physiochemical properties of peroxidase, CCPP with other plant peroxidase is
summarized in Table 2. The enzyme is highly active at pH 6, similar
to other known plant peroxidase, suggesting CCPP to be a classical
plant peroxidase The estimated molecular mass of CCPP (43.11 kDa)
was also in the range of molecular mass (34–51 kDa) reported for
the majority of plant peroxidase.
3.2. Homogeneity and physical properties of purified peroxidase
Homogeneity of enzyme and also its activity were checked by
SDS–PAGE (Fig. 1B.1) and Zymogram respectively (Fig. 1B.2). Peroxidase migrates as single band on SDS–PAGE under non-reducing
conditions and is twice that of the peroxidase in reducing conditions. This indicates that the protein is a dimer and the monomers
are held together by disulfide bond.
The pI was determined by isoelectric focusing (IEF) on polyacrylamide gels containing ampholytes in range of pH 7.0–9.0, and
found to be 8.1 (Fig. 1B.3). Basic peroxidase is also found in turnip
roots [27]. The molecular weight obtained with mass spectrometry
of CCPP was found to be 43.11 kDa (Fig. 1C) which is almost similar
to natural and basic peroxidase of Brassica oleracea [28].
3.3. Antigenic determinants
Polyclonal antibodies against CCPP were raised in the male
albino rabbit. The presence of antibodies in the anti-rabbit serum
was checked by Ouchterlony’s immunodiffusion method. The precipitin line is formed as a result of precipitation of antigen–antibody
complexes near the equivalence zone; it indicates purity as well
as specificity of the antigenic determinant of protein. Control
experiments with pre immune serum did not show any crossreactivity. Precipitin lines start appearing after about 10–12 h of
incubation at room temperature and are distinctly visible by about
24–30 h (Fig. 1D). A distinct precipitin line was observed with CCPP,
whereas, it did not cross-react with avidin, horseradish peroxidase
and MGP [29] suggesting that the antigenic determinants of CCPP
are unique. The raised anti-CCPP may be important in identification,
localization and other binding studies.
3.4. pH and temperature optima
In case of peroxidase, activity was observed in pH range of
3.0–8.0 at 30 ◦ C with optima at pH 6.0 (Fig. 2A). The temperature
for optimum activity was observed at 50 ◦ C at pH 7.0, similar to
peroxidase from W. somnifera by [21]. It is found that the activity was lost at a temperature above 70 ◦ C (Fig. 2B). These results
reveal that peroxidases are stable in broad range of pH and temperature. Under this optimum condition, peroxidase has maximum
activity with guaiacol as reducing substrate. CCPP also showed
activity with pyrogallol, dianisidine-dihydrochloride, o-phenelene
diamine, ␣-aminopterin and phloroglucinol. Typical results of relative activity measurements using different substrates are shown
in Fig. 3.
3.5. Spectroscopic studies: absorbance, fluorescence and circular
dichroism
The absorbance spectra of CCPP at neutral pH shows peak at
280 nm indicating significant contribution from tryptophan and
tyrosine residues. A small inflection point on the declining shoulder of the main peak around 290 nm originates from tryptophan
residues (data not shown). The increase in the absorbance as the
concentration increase at 650 nm reveals the presence of Cu ions
in the peroxidase as shown in Fig. 4. Peroxidase in the eluted
fraction is greenish in color and has high absorbance at 650 nm
reveals to have copper ions in its native structure. These copper
ions are reduced by ascorbic acid and the color of the peroxidase disappears. Cu (II) is colored in the native condition of the
peroxidase, while after reduction it changes into Cu (I), which is
in colorless state. This is a reversible process as well and with
some stirring with H2 O2 the color returns. Copper in its various
roles in biological systems displays different spectroscopic and
chemical properties presumably because of the different ligand
environments and coordination numbers. It is one of the essen-
Table 2
Comparison of physiochemical properties of peroxidase, CCPP with other plant peroxidase.
Source
Peroxidase
Mol. wt. [31]
pH optima
pI
Km against H2 O2 (mM)
Euphorbia cotinifolia
Olea europaeaa
Brassica oleracea Var. Italicab
CCPP
Nd
Acidic
Neutral
Basic
mPOD-I mPOD-II
43.11
18–20
48
43
43
51.2
43.8
34
48
45
6
7
4
6
6
6.0
5.5
5
5
6
8.4
4.4
4
5
8
Nd
Nd
3.6
4.8
Nd
13.3
0.53
0.305
0.711
8.789
0.08
0.06
Nd
Nd
0.1
Metroxylon saguc
Withania somniferad
Beta vulgarise
Nd in the table shown data not detected.
a
[1].
b
[28].
c
[5].
d
[21].
e
[12].
WS1
WS2
Nd
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R. Kumar et al. / Process Biochemistry 46 (2011) 1350–1357
Fig. 4. Visible absorption spectra of CCPP: 2 ␮M (dashed line), 5 ␮M (solid line) and
10 ␮M (dotted line) in 50 mM Tris buffer, pH 8.4.
Fig. 2. Effect of pH and temperature on the activity of the proteins (A) Effect of
pH on the activity of the CCPP (䊉) was studied by caring out the activity measurement of different pH in the range of 1.0–10.0. (B) Thermal stability of CCPP () was
equilibrated at particular temperature for 5 min with guaiacol as substrate.
Fig. 3. Effect of substrates on the activity of CCPP. The enzyme was incubated with
each substrate for 5 min at room temperature, and the relative activity was measured.
tial micronutrient for higher plants, but at high concentration, it
can induce alterations in plant tissues [30]. Copper content was
also monitored by Atomic absorption spectrometry (AAS) and the
result reveals that it has approximately 20–25 ppm/mg of protein.
Though, peroxidase activity was observed high in the exposed portion to environmental injury such as abiotic and biotic stress and
it also play key role in defense mechanisms that are induced to
the numerous mechanisms plant possess to reduce damages from
exposure of metal ions. Among the essential metal ions, copper is
known to be as micronutrient for higher plants and its high concentration, induce many physiological alternations in plant cells
e.g. catalyze the formation of harmful free radicals, which causes
an oxidative burst and this effect can be improved by anti oxidative
systems including peroxidase, e.g. sunflower seeding treated with
CuSO4 [31]. The conformational changes of CCPP under native and
denatured GuHCl and urea conditions were studied by fluorescence
and circular dichroism.
Fluorescence spectra provide a sensitive means of characterizing proteins and their conformation. The spectrum is determined
chiefly by the polarity of the environment of the tryptophan and
tyrosine residues and by their specific interactions. The spectral
parameters of tryptophan fluorescence like position, shape and
intensity are dependent on the electronic and dynamic properties of the chromophore environment. The fluorescence emission
maximum suffers a red shift when chromophores become more
exposed to solvent and the quantum yield of fluorescence decreases
when the chromophores interact with quenching agents either
in a solvent or in the protein itself. Intrinsic fluorescence spectra of CCPP in native and denatured conditions are shown in
(Fig. 5A). In the native state CCPP exhibits a fluorescence emission
maximum of 347 nm. The fluorescence intensity of the fluorescence decreases by 29% at 347 nm, whereas a wavelength shift
of 8 nm in emission maximum from 347 to 355 nm is seen, as
the protein is denatured in 3 M GuHCl while it decreases by 16%
at 347 nm, whereas a wavelength shift of 8.5 nm in emission
maximum from 347 to 355.5 nm in the case of 6 M GuHCl, indicating a nonpolar environment around tryptophan residues. On
the other hand red shift also happens in case of 4 M and 8 M urea.
With 4 M intensity was decreased by 27% and wavelength shift of
3 nm from 347 to 350 nm, while 9% and 6 nm for 8 M urea was
observed respectively. Such a red shift in the wavelength maximum indicates that more tryptophan residues of the protein are
exposed to a polar environment which is characteristic of unfolding.
In the far-UV region of 200–260 nm, the CD spectra of proteins are particularly sensitive to protein secondary structure. The
R. Kumar et al. / Process Biochemistry 46 (2011) 1350–1357
1355
Fig. 5. Spectroscopic studies of CCPP peroxidase. For denaturation the protein was incubated under 6 M GuHCl in 50 mM, pH 7.0, buffers for 24 h at room temperature before
the experiments. (A) Intrinsic fluorescence spectrum of CCPP in native and denatured conditions. The protein concentration used was 0.01 mg/mL; excited at wavelength
292 nm and scanned in the 300–400 nm range for the native (solid line) as well as the denatured state, 3 M GuHCl (dash dot dot dash); 6 M GuHCl (dotted); 4 M urea (dashed);
8 M urea (dash dot dash). (B) Circular cichroism spectrum of CCPP in native and denatured conditions. The protein concentration used was 0.10 mg/mL; the spectrum has
been taken in the near-UV region of 260–200 nm for the native (solid line) and denatured states, 6 M GuHCl (dashed); 8 M Urea (dotted).
Fig. 6. (A) Effect of H2 O2 concentration on peroxidase activity. Activity measurement () were calculated at different H2 O2 concentration in the range of 0.1–1.0 M with
guaiacol as substrate. (B) Effect of additives on peroxidase activity. 20 ␮g of the purified peroxidase was incubated with different additives different additives for 30 min at
room temperature, and the relative activity was measured with guaiacol as substrate.
secondary structural features of CCPP in native and denatured
conditions are shown in (Fig. 5B). CCPP revealed well-resolved
negative peaks around 222 nm and at 215 nm with greater ellipticity at 215 nm. The mean residue ellipticity at 215 nm was
around 2.7 × 103 deg cm2 dmol−1 , while at 208 and 222 nm were
1.85 × 103 deg cm2 dmol−1 and 1.62 × 103 deg cm2 dmol−1 respectively. CCPP in the native state retains all secondary features,
suggesting ␣-class of proteins. However, the secondary structural features of CCPP have been completely lost with the
disappearance of all prominent peaks in the presence of 6 M
GuHCl, while in case of 8 M GuHCl, ␣ class of proteins bit
remains.
3.6. Peroxidase activity as a function of hydrogen peroxide
concentration
The effect of initial hydrogen peroxide concentration on the
peroxidase activity was investigated at the pH 8.4 and 25 ◦ C with
different enzyme concentrations. The maximum peroxidase activity was observed at the initial hydrogen peroxide concentration of
about 0.2 mM, followed by decline as the concentration increases
as shown in Fig. 6A. Similar pattern of H2 O2 inhibition is detected
in case of DyP [32] and corpinus peroxidase [2]. The peroxidase
is first oxidized by two electrons from H2 O2 to form compound
I then one electron is removed by a reducing substrate changing
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R. Kumar et al. / Process Biochemistry 46 (2011) 1350–1357
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2011.03.003.
References
Fig. 7. Effect of concentration of salts on CCPP activity measured towards guaiacol:
() CaCl2 ; () MgCl2 ; (䊉) NaCl; () Na2 SO3 .
compound I to compound II. Compound II is further reduced to a
resting enzyme by another electron from the reducing substrate. At
higher H2 O2 concentration, H2 O2 react with compound II to form
relatively inactive compound III decreasing its peroxidase activity.
3.7. Effect of additives
Sodium azide, a typical peroxidase inhibitors, led to complete
loss of the peroxidase activity [33] and on the other, sucrose, DMSO,
and EDTA were able to enhance the peroxidase as shown in Fig. 6B.
Similar results were found for other known plant peroxidase [29].
3.8. Effect of salts
It is well established that peroxidase activity increases with
increase in ionic concentration of salts [34]. The “activity” vs “ionic
concentration”, plot with different salts shows an almost linear
curve (Fig. 7) with different magnitude of activation of the peroxidase activity. Maximum enhancement was observed with CaCl2
followed by MgCl2, NaCl and Na2 SO3. This indicated that the inhibiting effect was not specific and depended only on ionic strength. The
Ca2+ are found to be important for both the activity and thermal
stability of the enzyme [14].
4. Conclusion
This is the first report of a CCPP, peroxidase from the latex of
the plant weed E. cotinifolia. Adequate amount of latex, easy and
economic purification, stability in different conditions, activity over
a broad range of temperature and pH, identification of physiological
substrates, biophysical and structural studies will give insight into
the utility of the enzyme in food and biotechnological industries
and also an excellent model system to study the structure–function
relationship of plant peroxidase, as well as protein folding.
Acknowledgements
The financial assistance from CSIR/UGC/UGC, in the form of
Senior/Junior/Senior Research Fellowships to RK, KAS and VK is
acknowledged. Financial assistance from DBT and UGC for the
infrastructure is also acknowledged.
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