Thermal stability of peroxidase from the african oil palm treeElaeis guineensis

Anabel Rodrıguez1,*, David G. Pina1,*, Belen Yelamos2, John J. Castillo Leon3, Galina G. Zhadan1,Enrique Villar1, Francisco Gavilanes2, Manuel G. Roig4, Ivan Yu. Sakharov5 and Valery L. Shnyrov1

1Departamento de Bioquımica y Biologıa Molecular, Facultad de Biologıa, Universidad de Salamanca, Salamanca, Spain;2Departamento de Bioquımica y Biologıa Molecular, Facultad de Quımica, Universidad Complutense, Madrid, Spain;3Escuela de Quımica, Universidad Industrial de Santander, Bucaramanga, Colombia; 4Departamento de Quımica Fısica,

Facultad de Quımica, Universidad de Salamanca, Salamanca, Spain; 5Department of Chemical Enzymology,

Faculty of Chemistry, Moscow State University, Moscow, Russia

The thermal stabilityofperoxidase fromleavesof theAfricanoil palm tree Elaeis guineensis (AOPTP) at pH 3.0 wasstudied by differential scanning calorimetry (DSC), intrinsicfluorescence, CD and enzymatic assays. The spectralparameters as monitored by ellipticity changes in the far-UVCD spectrum of the enzyme as well as the increase in tryp-tophan intensity emission upon heating, together withchanges in enzymatic activity with temperature were seen tobe good complements to the highly sensitive but integralmethodofDSC. Thedataobtained in this investigationshow

that thermal denaturation of palm peroxidase is an irrevers-ible process, under kinetic control, that can be satisfactorilydescribed by the two-state kinetic scheme, N �!k D, wherek is a first-order kinetic constant that changes with tem-perature, as given by the Arrhenius equation; N is the nativestate, and D is the denatured state. On the basis of this model,the parameters of the Arrhenius equation were calculated.

Keywords: peroxidase; differential scanning calorimetry;intrinsic fluorescence; circular dichroism; protein stability.

Peroxidases (EC 1.11.1.7; donor:hydrogen-peroxide oxido-reductase) are enzymes that are widely distributed in theliving world and that are involved in many physiologicalprocesses, including abiotic and biotic stress responses.Although the function of peroxidases is often seen primarilyin terms of effecting the conversion of H2O2 to H2O, thisshould not be allowed to obscure their wider participation inother reactions, such as cell wall formation, lignification, theprotection of tissues from pathogenic microorganisms, etc.[1,2]. Several peroxidases have been isolated, sequenced andcharacterized. They have essentially been classified in threeclasses, supported in the first instance by comparison ofaminoacid sequence data and confirmed by more recentcrystal structure data (class I, intracellular prokaryoticperoxidases; class II, extracellular fungal peroxidases, andclass III, secretory plant peroxidases [2]). Peroxidase hasattracted industrial attention because of its usefulness as acatalyst in clinical biochemistry and enzyme immunoassays.Some modern applications of peroxidases include treatment

of waste water containing phenolic compounds, the synthe-sis of several different aromatic chemicals and polymericmaterials. The peroxidase most studied is the one obtainedfrom horseradish roots (HRP), which is also the mostcommercially available one. However, other plant speciesmay provide peroxidases with similar or even improvedproperties. Therefore, the availability of highly stable andactive peroxidases from sources other than horseradishroots would go a long way toward the development of acatalytic enzyme with broad commercial and environmentalpossibilities [3]. Several publications have addressed thestudy of the conformational stability of peroxidases, but todate our understanding of their folding mechanism remainscontradictory and unclear [4–11]. Factors affecting con-formational stability have been studied most intensively inproteins under reversible conditions [12,13]. However, afterdenaturation many proteins cannot refold in vitro due tomodifications such as digestion, aggregation, loss of aprosthetic group, etc. [14,15]. Thus, the thermal denatura-tion of such proteins is often discussed in terms of theLumry–Eyring model [16], in which a reversible unfoldingstep is followed by an irreversible denaturation step:N Ð U ! D, where N, U and D are the native, unfoldedor partially unfolded, and denatured states of the protein,respectively [17]. However, use of the whole Lumry–Eyringkinetic model for the quantitative description of DSC tracesis difficult because the corresponding system of differentialequations does not have an analytical solution at varyingtemperatures. Although there are computer programs thatallow the direct fitting of a system of differential equationsto experimental data, there are as yet no publications inwhich DSC data have been interpreted through the use ofthe whole Lumry–Eyring kinetic model [18]. Therefore, toanalyse the irreversible thermal denaturation of proteins,

Correspondence to V. L. Shnyrov, Departamento de Bioquımica y

Biologıa Molecular, Universidad de Salamanca, Plaza de los Doctores

de la Reina, s/n, 37007 Salamanca, Spain.

Fax: + 34 923 294579, Tel.: + 34 923 294465,

E-mail: shnyrov@usal.es

Abbreviations: ABTS, 2,2¢-azino-bis(3-ethylbenzthiazoline-6-sulfonic

acid); DSC, differential scanning calorimetry; HRP, peroxidase from

horseradish roots; AOPTP, peroxidase from the African Oil Palm Tree

Elaeis guineensis.

Enzyme: peroxidase (EC 1.11.1.7; donor:hydrogen-peroxide

oxidoreductase).

*Note: these authors contributed equally to this work.

(Received 8 February 2002, accepted 12 April 2002)

Eur. J. Biochem. 269, 2584–2590 (2002) � FEBS 2002 doi:10.1046/j.1432-1033.2002.02930.x

researchers generally look for simple models that areapproximations to the Lumry–Eyring model [17,19–21].

Recently a novel peroxidase has been isolated from theleaves of the African oil palm tree Elaeis guineensis [5]. Thisperoxidase shows a characteristic spectrum for haem-containing proteins, with a Soret maximum at 403 nm. Itsmolecular mass as estimated by SDS/PAGE is 57 000,which is higher than the values published for other plantperoxidases [1], probably because of the higher degree ofAOPTP glycosylation. It has also been found that AOPTP,similar to peroxidases earlier detected in the sweet potato,royal palm tree, tobacco, and tomato [22–24], is an anionicprotein with a pI value of 3.8. Preliminary data [25] havesuggested that AOPTP is stable over a broad pH-range,maximum stability being found at pH 7.0. Under acidic(pH 2.0) and alkaline (pH 12.0) conditions, AOPTP showsa lower stability but remains a highly stable enzyme, loosingnot more than 20% of its initial activity for 30 min at 25 �C.

In recent years there has been tremendous interest in theproduction of conducting polymers. Polyaniline is one suchcompound because it can be used in lightweight organicbatteries, in microelectronics, in optical display, in anticor-rosive protection, in bioanalysis as a sensing element, etc.[26,27]. This is because it shows good electrical and opticalproperties as well as high environmental stability. It is wellknown that peroxidases can be used in the synthesis ofpolyaniline in the presence of hydrogen peroxide as a reduc-ting substrate and sulfonated polystyrene and poly(vinyl-phosphonic acid) as polymeric templates [28], which takeplace effectively at pH values below 4.0. Consequently, forthe development of such biotechnological process, would beof interest to find and characterize peroxidases that are stableunder acidic conditions, such as the enzyme considered here(peroxidase from African oil palm tree Elaeis guineensis).

Here we describe a detailed investigation of the thermaldenaturation of AOPTP at pH 3.0. This was studied bydifferential scanning calorimetry in the combination withstructural probes, such as intrinsic fluorescence and circulardichroism, as well as enzymatic activity assays. The thermalunfolding of AOPTP was found to be irreversible andstrongly scan-rate dependent, which led us to analyse thisnonequilibrium process based on the simplest so-called two-state kinetic model:

N�!k D ð1Þ

which is a limiting case of the Lumry–Eyring model [17].This model considers only two significantly populatedmacroscopic states, the initial or native state (N) and thefinal or denatured (D) state, transition between which isdetermined by a strongly temperature-dependent first-orderrate constant (k). The data obtained demonstrate thatAOPTP is a significantly more thermostable enzyme thanother known peroxidases, that makes AOPTP an intriguingcatalyst for scientific and commercial applications wherestability at high temperatures is desirable.

M A T E R I A L S A N D M E T H O D S

Materials

2,2¢-Azino-bis(3-ethylbenzthiazoline-6-sulfonicacid)(ABTS)was purchased from Amersham International plc

(Buckinghamshire, UK). H2O2 was obtained from Merck(Darmstadt, Germany) and quantified by UV spectropho-tometry at 230 nm (e ¼ 81 M

)1Æcm)1) [29]. Phenyl-Sepharose and Sephacryl S 200 were from PharmaciaBiotech (Uppsala, Sweden), DEAE cellulose was fromServa (Heidelberg, Germany), and other reagents were fromPanreac (Barcelona, Spain). All reagents were of the highestpurity available. Double-distilled water was used through-out. All measurements were carried out in 10 mM Na-phos-phate buffer, pH 3.0.

Protein purification and determination

AOPTP was purified from African oil palm tree leaves asdescribed elsewhere [5]. Briefly, leaves were triturated andincubated with constant stirring in 10 mM phosphate buffer,pH 7.0, for 1 h at ambient temperature, and the homogen-ate obtained was filtered and centrifuged (7000 g, 15 min).For the extraction of coloured compounds, a two-phasesystem containing 14% (w/v) poly(ethylene glycol) and 20%(w/v) (NH4)2SO4 was used. Then, the aqueous phasecontaining peroxidase activity was applied to a phenyl-Sepharose column (1.5 · 30 cm) equilibrated with 100 mM

phosphate buffer, pH 6.5, containing 1.7 M (NH4)2SO4. Theenzyme was eluted by decreasing the (NH4)2SO4 concen-tration, collected and concentrated using a YM-10 mem-brane (Amicon, cut-off 10 000) and applied to a Sephacryl S200 column (2.5 · 41 cm) equilibrated with 5 mM Tris/HCl,pH 8.3. Elution was carried out in the same buffer.Fractions with enzymatic activity were collected and applieddirectly to a DEAE–cellulose column (0.9 · 9 cm) equili-brated with 5 mM Tris, pH 8.3. The peroxidase was elutedwith a linear, 0–50 mM NaCl, gradient, dialyzed againstdistilled water, freeze-dried and stored at 4 �C.

The purity of AOPTP were determined by SDS/PAGE.Electrophoresis was performed as described by Fairbrankset al. [30] on a Bio-Rad minigel apparatus, using a flat blockwith a polyacrylamide gradient of 5–25%. Gels wereprefixed and stained using the method of Merril et al. [31].Protein contents were determined by the Bradford assay[32]. The RZ (A403/A280) for the AOPTP samples used inthis work were 2.8–3.0.

Differential scanning calorimetry

DSC experiments were performed on a MicroCal MC-2Ddifferential scanning microcalorimeter (MicroCal Inc.,Northampton, MA) with cell volumes of 1.22 mL, inter-faced with a personal computer (IBM-compatible) asdescribed previously [8]. Exhaustive cleaning of the cellswas undertaken before each experiment. All protein solu-tions were dialyzed against the desired buffer, and thedialyzate was used as reference. All solutions were degassedby stirring under a vacuum prior to scanning. Different scanrates within the 0.5–1.5 KÆmin)1 range were employed andan overpressure of 2 atm of dry nitrogen was always keptover the liquids in the cells throughout the scans. Abackground scan collected with a buffer in both cells wassubtracted from each scan. The reversibility of the thermaltransitions was checked by examining the reproducibility ofthe calorimetric trace in a second heating of the sampleimmediately after cooling from the first scan. The experi-mental calorimetric traces were corrected for the effect of

� FEBS 2002 Stability of plant peroxidase (Eur. J. Biochem. 269) 2585

the instrument response time using the procedure describedpreviously [33]. The molar excess heat capacity curvesobtained by normalizing with the protein concentrationsand the known volume of the calorimeter cell weresmoothed and plotted using the Windows-based softwarepackage (ORIGIN) supplied by MicroCal. Data were ana-lyzed by the nonlinear least-squares fitting program, asreported elsewhere [19]. The correlation coefficient, r, usedas a criterion for the accuracy of fitting, was calculated bythe equation:

r ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1 �

Xn

i¼ 1

ðyi � ycalci Þ2

�Xn

i¼ 1

ðyi � ymi Þ

2

sð2Þ

where yi and ycalci are, respectively, the experimental and

calculated values of Cexp ; ymi is the mean of the experimental

values of Cexp , and n is the number of points. Typical protein

concentrations for calorimetric experiments ranged between1.0 and 2.5 mgÆmL)1. Molar transition enthalpies, DH, referto M ¼ 57 000 gÆmol)1.

Intrinsic fluorescence

Fluorescence measurements were performed on a HitachiF-4010 spectrofluorimeter. Exitation was carried out at296 nm (with 5 nm excitation and emission slitwidths) inorder to avoid the contribution of tyrosine to the intrinsicfluorescence spectrum of AOPTP. The temperaturedependence of the emission fluorescence spectra wasinvestigated using thermostatically controlled water circu-lating in a hollow brass cell-holder. The temperature of thesample cell was monitored with a thermocouple immersedin the cell under observation.

Circular dichroism

CD spectra in the far-ultraviolet range (190–250 nm) wererecorded on a Jasco-715 spectropolarimeter, using a spectralband-pass of 2 nm and a cell path length of 1 mm with aprotein concentration of 0.2 mgÆmL)1. Spectra are averagesof four scans at a scan rate of 50 nmÆmin)1. All spectrawere background-corrected, smoothed, and converted to amean residue ellipticity of [H] ¼ 10 MresÆHobsÆl

)1Æp)1, whereMres ¼ 115.5 is the mean residue molar mass, Hobs is theellipticity measured (degrees) at wavelength k, l is the opticalpath-length of the cell (dm), and p is the protein concen-tration (mgÆmL)1). Spectra were analyzed using theSELCON3 software package [34]. To study the dependenceof ellipticity on temperature, the samples were heated at aconstant heating rate (� 1 KÆmin)1) using a Neslab RT-11programmable water bath.

Activity assays

AOPTP activity was assayed using ABTS as substrate [35].Aliquots of enzyme solution were added to a spectralcuvette with 1-cm optical path length containing 0.4 mM

ABTS and 5 mM H2O2 in 50 mM acetate buffer, pH 5.0 in afinal volume 2 mL. The rate of changes in absorbance at405 nm due to ABTS radical formation was measuredspectrophotometrically at 25 �C. Activities were calculatedusing a molar absorption coefficient of the ABTS oxidationproduct at 405 nm of 36.8 mM

)1Æcm)1 [36].

Kinetics of AOPTP thermal inactivation

To study the kinetics of heat denaturation by intrinsicfluorescence, 0.02 mL samples of a 0.1-mM AOPTP solu-tion were added to 1.6 mL of buffer previously thermostat-ed at the desired temperature in the fluorimeter cuvette. Themixture was stirred constantly in the cuvette and theemmision intensity at a wavelength of 340 nm was recordedat a certain time interval. In all experiments, the time fortemperture equilibrium to be reached in the cuvette aftersample introduction did not exceed 5 s. An almost identicalprocedure was applied to study the kinetics of changes inperoxidase activity with temperature. Samples of AOPTPwere incubated at the desired temperature under constantstirring. At certain times, aliquots were removed andimmediately transferred to test tubes placed in a water–icemixture to stop the inactivation process. Subsequently,enzyme activity was measured as described above. Themeasurements were made in triplicate and the data arepresented as average values.

R E S U L T S A N D D I S C U S S I O N

Differential scanning calorimetry

Figure 1 shows the calorimetric transitions of the thermaldenaturation of AOPTP at pH 3.0, at three different scanrates. The heat absorption curve apparent Tm (temperatureat the maximum of the heat capacity profile) was found tobe dependent on the scan rate and denaturation was alwayscalorimetrically irreversible, as no thermal effect wasobserved in a second heating of the enzyme solution.Inspection of the DSC curves shown in Fig. 1 furtherreveals asymmetry in the shape of the peaks, which mightarise from two overlapping transitions. This would be areasonable possibility for AOPTP, which is a fairly large

50 60 70 80 90

0

10

20

30

40

Cp

ex (

kca

l K

-1 m

ol-1

)

Temperature (oC)

Fig. 1. Temperature dependence of the excess molar heat capacity of

AOPTP at scan rates of 0.5 (circles), 1.0 (squares) and 1.5 (triangles)

KÆmin)1 at pH 3.0. Solid lines represent the best individual fit to each

experimental curve using Eqn (3). Protein concentrations were

� 2.5 mgÆmL)1 at a scan rate of 0.5 KÆmin)1, � 2 mgÆmL)1 at a scan

rates of 1.0, and � 1.0 mgÆmL)1 at a scan rate of 1.5 KÆmin)1.

2586 A. Rodrıguez et al. (Eur. J. Biochem. 269) � FEBS 2002

protein and may, in principle, comprise several domains[37]. We analyzed this possibility by applying the successiveannealing procedure [38]. Thus, AOPTP was first heated ata scan rate of 60 KÆh)1 in the microcalorimeter cell to atemperature of 69 �C, which would be close to themaximum for a putative first transition. The sample wascooled and then heated to 90 �C at the same scan rate. Thereheating scan revealed that the only effect of the first scanwas to decrease the peak intensity by a scale factordetermined by the difference in the amounts of proteinundergoing denaturation, and that there was no change inTm or any effect on the shape of the curve (not shown).These experiments rule out the possibility of overlappingindependent transitions. The effect of the scan rate on thecalorimetric profiles clearly indicated that they correspon-ded to irreversible, kinetically controlled transitions. Forthis reason the analysis of DSC transitions on the basis ofequilibrium thermodynamics was ruled out [39] and wasaccomplished using the simple two-state irreversible model(Eqn 1), in which only the native (N) and final (irreversiblydenatured) (D) states are significantly populated and inwhich the conversion from N to D is determined by astrongly temperature-dependent, first order rate constant (k)that changes with temperature, as given by the Arrheniusequation. In this case, the excess heat capacity Cex

p is givenby the following equation [19]:

Cexp ¼ 1

vDH exp

EA

R

1

T� �1

T

� �� �

exp � 1

v

ZT

T0

expEA

R

1

T� �1

T

� � �dT

8<:

9=; ð3Þ

where v ¼ dT/dt (KÆmin)1) is a scan rate value; DH is theenthalpy difference between the denatured and native states;EA is the activation energy of the denaturation process; R isa gas constant, and T* is temperature, where k is equal to1 min)1.

The excess heat capacity functions obtained for AOPTPwere analysed by fitting the data to the two-state irreversiblemodel (Eqn 3), either individually or by fitting this theor-etical expression simoultaneously to all the experimentalcurves, using the scan rate as an additional variable. Thehighest likelihood values for EA and T* obtained with thenonlinear least squares minimization procedure are shownin Table 1. It may be seen that the calculated andexperimental curves are in good agreement. Also, theparameters obtained from individual fits were in reasonableagreement with those obtained from the global fit, indica-ting that the two-state irreversible model offers a goodexplanation of the AOPTP denaturation process. Addition-

ally it should be noted that no dependence of the shape ofthe DSC contour on the AOPTP concentration was foundat a scan rate of 60 KÆh)1 in the 0.7–3.8 mgÆmL)1 range. Nopronounced dependence of the denaturation enthalpy onscan rate was observed (see Table 1). These data argueagainst an effect of intermolecular aggregation on the DSCtraces obtained.

Fluorescence and enzymatic activity

Conformational changes in the surroundings of AOPTParomatic side chains were detected by intrinsic fluorescencespectroscopy. The emission spectra from 300 to 400 nm ofintact and thermally denatured AOPTP are represented inFig. 2. Intact AOPTP displayed a low emission intensitydue to energy transfer to haem, which, as can be seen inFig. 3, significantly increased in the denatured enzymeowing to a change in the relative orientation or distancebetween the haem and tryptophan residue(s) [40]. Therefore,the intrinsic fluorescence of AOPTP was monitored at340 nm for thermal denaturation. Figure 3A shows thekinetic data on AOPTP denaturation as observed bychanges in the fluorescence intensity obtained at fivedifferent temperatures. This figure shows that althoughthe denaturation rate does increase with temperature, the

Table 1. Arrhenius equation parameter estimates for the two-state irreversible model of the thermal denaturation of AOPTP at pH 3.0.

Parameter

Temperature scan rate (KÆmin)1)

0.5 1.0 1.5 Global fitting

DH, kcalÆmol)1 251 ± 9 257 ± 7 256 ± 7

T*, K 347.6 ± 0.2 347.6 ± 0.2 347.3 ± 0.3 347.5 ± 0.3

EA, kcalÆmol)1 99.7 ± 1.2 98.8 ± 1.4 101.1 ± 0.9 102.1 ± 1.4

r 0.9990 0.9987 0.9989 0.9959

300 320 340 360 380 4000

5

10

15

20

25

30

Wavelength (nm)

Flu

ore

sce

nce

in

ten

sity (

rela

tive

un

its)

Fig. 2. Fluorescence spectra of intact at 25 �C (solid line) and thermally

denatured at 80 �C (dashed line) 1 lM AOPTP at pH 3.0. Excitation

wavelength, 296 nm.

� FEBS 2002 Stability of plant peroxidase (Eur. J. Biochem. 269) 2587

final level of intrinsic fluorescence is independent of thedenaturation temperature. This supports the idea that thethermal denaturation of AOPTP is not a reversible equilib-rium process between the native and denatured enzymebecause if this was the case the relative amounts of nativeand denatured states would be expected to show a definitetemperature dependence. Therefore, this appears to be akinetic phenomenon involving an irreversible process.

The same experimental approach was applied to theenzymatic activity assays, as the denaturation of anyenzyme is expected to abolish its biological activity, allowingus to monitor thermally induced conformational changes inthe catalytic surroundings by measuring the loss of enzy-matic activity vs. time at different temperatures (Fig. 3B).

The best fit of the experimental data, represented ascontinuous lines in Fig. 3, was achieved with an exponentialfunction:

F ¼ F1 þ ðF0 � F1Þ exp ð�ktÞ ð4Þ

where F is the function value at a given time (t) and F0 andF1 are normalization parameters (at t ¼ 0, F ¼ F0, and att ¼ 1, F ¼ F1), indicating a first-order kinetic process.

The temperature dependence of the rate constantsobtained from the data shown in Fig. 3 was expressed bythe Arrhenius equation:

k ¼ expEA

R

1

T� �1

T

� � �ð5Þ

and is represented in Fig. 4. Thus, the activation energy andT* can be calculated from the linear fit of both the

fluorescence and enzymatic assay data. The value thusobtained (EA ¼ 110.8 ± 3.2 kcalÆmol)1) and (T* ¼345.9 ± 1.8 K), were in satisfactory agreement with thevalues obtained from the DSC experiments (Table 1).

Circular dichroism

CD is one of the most sensitive physical technique fordetermining structures and monitoring the structural

15

20

25

30F

luo

resce

nce

inte

nsity a

t 3

40

nm

a

0 20 40 60 80

0.0

0.2

0.4

0.6

0.8

1.0

Re

lative

activity

Time (min)

b

0 20 40 60 80 100

0.01

100

0.1

1Log (

activity)

Time (min)

Fig. 3. Temperature dependence of the thermal denaturation kinetics of

AOPTP at pH 3.0 as monitored by intrinsic fluorescence (a) and per-

oxidase activity shown at normal (b) and semilog scale (b, insert).

Symbols refer to the experimental data at different temperatures:

73.6 �C (s), 70.9 �C (d), 69.2 �C (n), 68.7 �C (m), and 65.9 �C (,) in

(a); 71.0 �C (s), 68.0 �C (d), 66.5 �C (n), and 65.2 �C (m) in (b).

2.90 2.92 2.94 2.96

-3

-2

-1

0

ln k

103/T in K

Fig. 4. Dependence of the logarithm of the inactivation rate constant

(min)1) on the reciprocal value of the absolute temperature as monitored

by intrinsic fluorescence (solid symbols) and enzymatic activity assays

(open symbols) for AOPTP at pH 3.0. The line was fitted by linear

regression.

200 220 240-10000

-5000

0

5000

10000

15000

[Θ] (

deg

cm2 d

mol

-1)

Wavelength (nm)

40 50 60 70 80 90

-7000

-6000

-5000

-4000

Temperature (oC)

[Θ] (

deg

cm2 d

mol

-1)

Fig. 5. CD spectra in the far-ultraviolet spectral region of intact (solid

line) and irreversible thermally denatured (dashed line) 2 lM AOPTP at

pH 3.0 and 25 �C. (Inset) Temperature dependences of ellipticity at

222 nm for AOPTP at pH 3.0 obtained upon heating with a constant

scan rate of � 1 KÆmin)1. Solid line is best fit obtained using Eqn (7).

2588 A. Rodrıguez et al. (Eur. J. Biochem. 269) � FEBS 2002

changes occurring in biomacromolecules [41], affording adirect interpretation of the changes in protein secondarystructure. Figure 5 shows the far-UV CD spectra of intact(solid line) and thermally denatured (dashed line) AOPTP atpH 3.0. The fractions of ahelix, a strand, turns, andunordered secondary structures obtained following theSELCON3 self-consistent method [34] are given in Table 2.It is clear that AOPTP is significantly different from otherhaem peroxidases from plants for which, despite the lowlevel of sequence homology (often less than 20%), the overallfolding and the organization of the secondary structure isconserved [42]. The structure of haem peroxidases fromplants is formed by 10–11 ahelices (c. 40%), linked by loopsand turns, whilea structures are essentially absent or are onlya minor component [43]. By contrast, intact AOPTPcontains a considerable amount of a-structure (� 38%)and only 15% of ahelices, at pH 3.0. This probably makesthis enzyme more stable in comparison with horseradishperoxidase which under the same experimental conditionshas 42% of ahelices and only 11% of a structure [8].

Upon heating AOPTP to the denaturation temperature,the shape of the CD spectrum changes, showing an increasein unordered structure from � 30%, for the intact enzyme,up to � 50% for the denatured one (see Table 2). Theprocess of thermal denaturation of AOPTP was monitoreddirectly by following the changes in molar ellipticity at222 nm as at this wavelength the changes in ellipticity aresignificant upon heating. On increasing temperature (Fig. 5,insert), irreversible cooperative transitions to the denaturedstate occurred, which were analyzed using a nonlinear leastsquares fitting (see lines through the data points in Fig. 5,insert). In this case, the fraction of denatured AOPTP, FU

was calculated from the spectral parameter used to followdenaturation (y) prior to the minimization procedure,according to the expression:

FU ¼ ðy� yNÞ=ðyU � yNÞ ð6Þ

where yN ¼ a1 + a2T and yU ¼ b1 + b2T represents themean values of the y characteristic of the native anddenatured conformations, respectively, obtained by linearregressions of pre- and post-transitional baselines; T is thetemperature. In this case, the parameter used to followdenaturation, y, can be expressed as a function of the kineticparameters by equation [19]:

y ¼ yU � ½yU � yN exp1

v

ðTT0

expEA

R

1

T� �1

T

� � �dT

8<:

9=;ð7Þ

Fitting of the experimental data to this equation affordedthe T* parameter and the activation energy for AOPTP.These results were 347.2 ± 1.6 K and 106.0 ± 1.4 kcalÆ

mol)1, respectively, which are similar to the values for thesame parameters obtained by the other methods used in thiswork. Thus, all these independent experimental approachessupport the conclusion that AOPTP thermal denaturationcan be interpreted in terms of the irreversible two-statekinetic model, and that only two states, native anddenatured, are populated in its denaturation process.

Finally, it is interesting to compare the thermal stabilityof AOPTP with that of other peroxidases. In our previouspublication [8] we reported the results of a detailedinvestigation of the thermal denaturation of horseradishperoxidase isoenzyme c under the same experimentalconditions as those used here. It is clear that AOPTP issubstantially more thermostable than HRPc. Thus, the Tm

for AOPTP at a scan rate of 60 KÆh)1 is 72.3 ± 0.2 �Cwhile for HRPc this value is only 60.2 ± 0.2 �C.TheArrhenius denaturation energy of AOPTP obtained bydifferent methods, 103 ± 6 kcalÆmol)1, is a high value incomparison not only with value for HRPc (38 ± 1 kcalÆmol)1) but also in comparison with those found for otherplant peroxidases [4]. Coupled with its high catalyticpotential [44], the unique high thermostability of AOPTPpromises good perspectives for this peroxidase in biotech-nological applications.

A C K N O W L E D G E M E N T S

This work was supported by NATO Linkage Grant LST.CLG 975189

(to M. G. R., I. Y. S. and V. L. S.). D.G.P. is a fellowship holder from

Fundacao para a Ciencia e a Tecnologia, Portugal (Ref. SFRH/BD/

1067/2000). We thank N. S. D. Skinner for proof-reading the manu-

script.

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Press, Boca Raton, FL, USA.

2. Welinder, K.G. (1992) Superfamily of plant, fungal and bacterial

peroxidases. Curr. Opin. Struct. Biol. 2, 388–393.

3. Krell, H.-W. (1991) Peroxidase. An important enzyme for diag-

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