Purification and characterization of a wound-inducible thaumatin-like
protein from the latex of Carica papaya
Yvan Loozea, Paule Boussarda, Joëlle Hueta, Guy Vandenbusscheb, Vincent Raussensb, René Wintjensa,*
aLaboratoire de Chimie Générale (CP: 206/4), Institut de Pharmacie, Université Libre de Bruxelles, Campus de la Plaine, Boulevard du Triomphe, 1050 Brussels, Belgium
bLaboratoire de Structure et Fonction des Membranes Biologiques (CP: 206/2), Faculté des Sciences, Université Libre de Bruxelles, Campus de la Plaine,
Boulevard du Triomphe, 1050 Brussels, Belgium
a r t i c l ei n f o
Received 18 February 2009
Received in revised form 14 April 2009
Available online 13 June 2009
a b s t r a c t
A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged
papaya trees and from a commercially available papain preparation. The protein was purified up to
apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been pre-
viously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein fam-
ily, as expected for several other protein constituents of papaya latex. The protein was identified as a
thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid
sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein
were identified, where the latex constituent belongs to a well-known form, allowing the molecular mod-
eling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The pro-
tein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by
guanidium chloride, with a DG0
teinases. The physiological role of this protein is discussed.
waterof 15.2 kcal/mol and to proteolysis by the four papaya cysteine pro-
? 2009 Elsevier Ltd. All rights reserved.
Carica papaya is a soft-stemmed and un-branched tree, widely
cultivated in the tropical and subtropical regions around the world
both for its edible fruits and for its high content of cysteine pro-
teinases. The papaya latex is stored under pressure in structures
called laticifers that are displayed in all the aerial parts of the tree.
Wounding the papaya tree inevitably severs its laticifers, eliciting
an abrupt release of latex. The latex rapidly coagulates, sealing
thereby the injured area and preventing further entry of pathogens
into the plant phloem. The latex coagulation certainly constitutes
the first level of plant defence mechanisms. The process of papaya
latex coagulation remains poorly characterized (Silva et al., 1997;
Moutim et al., 1999), whereas several models for Hevea latex coag-
ulation were proposed each implicating rubber particles (Gidrol
et al., 1994; Wititsuwannakul et al., 2008). However, coagulation
for papaya trees certainly uses another mechanism as this latex
does not contain rubber particles.
In host–pathogen interactions, damage caused by the pathogen
remains restricted as a result of the plant’s defensive responses.
Among these responses, plants express various novel proteins col-
lectively referred to as ‘‘pathogenesis-related proteins” (PR pro-
teins). These proteins do not only accumulate locally but are also
induced systemically, being associated with the development of a
systemic acquired resistance against further infection by fungi,
bacteria and viruses (van Loon et al., 2006). Induction of PR-pro-
teins was found in many plant species belonging to various fami-
lies (van Loon et al., 1994), suggestive of a general role for these
proteins in adaptation to biotic stress conditions. Systemic ac-
quired resistance, likewise, is a generally occurring phenomenon,
that engenders an enhancement of the defensive capacity of plants.
PR-proteins have been grouped in up to 17 families according to
amino acid sequences, serological relationship, and/or enzymatic
or biological activity (Edreva, 2005; van Loon et al., 2006).
Revisiting the list of enzymes already identified in the laticifers
of C. papaya led to the suggestion that most of them could partic-
ipate in the plant defence mechanism. This is clearly the case of pa-
paya cysteine endoproteinases (family PR-7) (Azarkan et al.,
2006a), of a serine proteinase inhibitor (family PR-6) (Azarkan
et al., 2006b) and of a class II chitinase (family PR-3 or PR-4) (Azar-
kan et al., 2004; Huet et al., 2006). On the other hand, a glutaminyl
cyclase enzyme is also only expressed in the laticifers of papaya
plants regularly damaged for latex gathering (Azarkan et al.,
2004). This latter enzyme cyclises the N-terminal glutamine resi-
due of polypeptide chains in a pyroglutamyl moiety (Gololobov
et al., 1994; Zerhouni et al., 1998). The physiological function of pa-
paya glutaminyl enzyme remains however unexplained, as well as
its presumed presence among PR proteins (Wintjens et al., 2006).
0031-9422/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +32 26505101; fax: +32 26505929.
E-mail address: firstname.lastname@example.org (R. Wintjens).
Phytochemistry 70 (2009) 970–978
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/phytochem
In the course of a re-examination of the influence of repeated
mechanical wounds on the composition of papaya latex, a novel
constituent was identified as being expressed solely in regularly
damaged plants. On the basis of its partial amino acid sequence,
this protein was found to be homologous to thaumatin (the sweet
protein isolated from Thaumatococcus danielli) and the thaumatin-
like proteins (classified as PR-5 proteins) (Iyengar et al., 1979). This
paper describes the purification and the properties of this new con-
stituent of papaya latex.
2. Results and discussion
2.1. Purification of the latex papaya thaumatin-like protein (PTLP)
Lattices were freshly collected from full-grown but unripe pa-
paya fruits on regularly damaged trees as well as on trees that
had never been mechanically stressed.
The soluble fractions of both papaya lattices were diluted in a
50 mM sodium acetate buffer at pH 5.0 and fractionated by ion-ex-
change chromatography on a 40 ? 2.6 cm ID column of SP-Sephar-
ose Fast Flow. Elution was performed by applying a linear gradient
of KCl (from 10 to 90 mS/cm) in the 50 mM sodium acetate buffer
at pH 5.0 (total volume: 1500 ml). As shown in Fig. 1 in the case of
regularly damaged papaya trees, five fairly well-separated chro-
matographic fractions were collected. A first pool of material that
did only weakly bind to the chromatographic support was col-
lected and discarded since, on the basis of its absorbance spectrum
in the near UV (250–400 nm) region, this pool did not contain pro-
tein material. The four remaining pools were denoted I, II, III and IV
in order of their increasing elution volumes. Very similar elution
patterns were observed for both lattices in the case of pools II, III
Pool II was fractionated using hydrophobic interaction chroma-
tography. Before being separated to the hydrophobic support, the
proteins contained in this pool II was concentrated by ultrafiltra-
tion (molecular weight cut off: 10 kDa). Solid ammonium sulfate
was then added until obtaining a conductivity of 195 mS/cm. The
resulting solutions were then applied on a (20 ? 3.0 cm ID) column
of Fractogel EMD Propyl 650(S) pre-equilibrated in ammonium sul-
fate (195 mS/cm). After having eluted a first protein peak denoted
pool II-A in Fig. 2, elution was continued by applying a decreasing
linear gradient from 160 mS/cm up to 3 lS/cm at a flow-rate of
45 ml/h and at room temperature (total volumes of 1550 ml). As
shown in Fig. 2, pool II provided four chromatographic fractions
denoted II-A, II-B, II-C and II-D in the case of the latex collected
on regularly damaged trees. Fraction II-D, interestingly, was not
present in the latex collected on trees that had never been mechan-
ically stressed. Fractions II-A, II-B, II-C and II-D were analysed by
SDS–PAGE (Fig. 3). It was observed that fraction II-D, that migrated
as a 22 kDa protein, appeared to be quite homogeneous.
2.2. Evaluation of the homogeneity of the protein present in pool II-D
and preliminary characterization
Several criteria were used to evaluate the homogeneity of this
protein. It should first be mentioned that the preparation was de-
void of any detectable amidase activities (N-a-benzoyl-D,L-argi-
nine-p-nitroanilide and BOC-Ala-Ala-Gly-p-nitroanilide as the
substrates) or of any detectable proteolytic activity (azocoll as
the substrate) (Chavira et al., 1984) showing that it was substan-
tially freed from the abundant papaya cysteine proteinases. Also,
the preparation was unable to degrade chitosan showing that it
was not contaminated by papaya chitinases (Huet et al., 2006).
As above mentioned, SDS–PAGE experiments (Fig. 3) did not reveal
any contaminant although the gel was somewhat overloaded. The
chemical homogeneity of mass spectrum (Fig. 4A) suggests this
new latex constituent, characterized by a molecular mass of
22,137 Da, is not glycosylated unlike some other latex proteins
(El Moussaoui et al., 2001).
Attempts to modify either the native form of the purified pro-
tein with 7 mM 2-mercaptoethanol or the denatured form with
0 300 600 900 12001500
Elution volume (ml)
I II . III IV
Fig. 1. Ion-exchange chromatography on SP-Sepharose of the soluble fraction of the
papaya latex collected on regularly damaged trees: sample: the mixture of papaya
proteins in 40 ml of 50 mM sodium acetate buffer at pH 5.00; column: 30 ? 2.6 cm
ID; fractions of 11.25 ml; flow-rate: 45 ml/h; room temperature; eluting buffer:
50 mM sodium acetate at pH 5.00 followed by a linear gradient (conductivity from
10 up to 90 mS/cm) of KCl in 50 mM sodium acetate (total volume: 1500 ml). Each
chromatographic fraction was analysed by measurements of A280nm(continuous
trace) and conductivities (dotted line).
0 300600 900 1200 1500 1800
Elution volume (ml)
Fig. 2. Hydrophobic chromatography of pool II (continuous trace: A280nm) on EMD-
propyl (S): sample: pool II from the SP-Sepharose. This pool was concentrated by
ultra-filtration (membrane cut off: 10 kDa) and solid ammonium sulfate was added
until obtaining a conductivity of 195 mS/cm. The resulting solution was then
applied on a (20 ? 3.0 cm ID) column of Fractogel EMD Propyl 650(S) pre-
equilibrated in ammonium sulfate (195 mS/cm). After having eluted a first protein
peak denoted pool II-A, elution was continued by applying a decreasing linear
gradient (dotted line) from 160 up to 3 lS/cm at a flow-rate of 45 ml/h and at room
temperature (total volumes of 1550 ml).
Y. Looze et al./Phytochemistry 70 (2009) 970–978
2.5 mM methylmethanethiolsulfonate (MMTS) did not affect the
molecular weight of the protein, thereby suggesting the absence
of free cysteine residues (Wynn and Richards, 1995). The presence
of 16 cysteine residues forming eight disulfide bonds is in fact a
hallmark of the TLPs (Iyengar et al., 1979; King et al., 1988; Takeda
et al., 1991; Malehorn et al., 1994; Fils-Lycaon et al., 1996; Barre
et al., 2000). After denaturation and reduction of the disulfide
bonds, the addition of MMTS, which converts the free thiol groups
into their S-thiomethyl derivatives (see Section 3), resulted in an
increase of the protein molecular weight by 754 Da (Fig. 4B), corre-
sponding quite well to the addition of 16 thiomethyl fragments. It
was concluded that the protein also contains 16 cysteine residues
that form eight disulfide bridges.
2.3. Three forms of PTLP and identification of latex PTLP form
The recent determination of the C. papaya genome (Ming et al.,
2008) has allowed us to perform thorough sequence analysis. By
comparison of 33 putative TPL sequences, three different forms
of TLP were found into the Carica genome, named here PTLP1
(for papaya thaumatin-like protein form 1), PTLP2 and PTLP3
(Fig. 5). As seen in Fig. 5, PTLP1 and PTLP3 are very similar (74%
of sequence similarity) while PTLP2 is more distant to the two oth-
ers (46% of sequence similarity). PTLP1 is the most well-known
form through research literature, where several X-ray structures
are available (Menu-Bouaouiche et al., 2003; Min et al., 2004;
Ghosh and Chakrabarti, 2008). It could be noted that all the three
PTLP forms contain 16 cysteine residues, in contrast to the eight
PR-5 isoforms found in barley plant (Reiss et al., 2006).
After reduction and S-thiomethylation, the latex PTLP was di-
gested with trypsin. Seven peptides have been sequenced and their
alignment onto the sequences of the three PTLP forms clearly re-
vealed that the latex PTLP belongs to PTLP1 (Fig. 6). Even if Carica
latex could contain several forms of TLP, as only one form of TLP
has been identified, this protein was then denoted as the latex pa-
paya thaumatin-like protein (PTLP).
Fig. 3. SDS–PAGE of the different chromatographic pools II-A (lane 2), II-B (lane 3),
II-C (lane 4) and II-D (lane 5). Lanes 1 and 6: molecular mass standards.
Fig. 4. Mass spectra of latex PTLP in its native form (A) and after denaturation, reduction and S-thiomethylation (B).
Y. Looze et al./Phytochemistry 70 (2009) 970–978
2.4. Evaluation of the resistance of the latex PTLP to proteolysis
In order to evaluate the resistance of latex PTLP to proteolysis,
the protein was purified from a commercial liquid refined papain
sample, which is known to contain up to 1 mM fully active cysteine
proteinases. Indeed, it must be known that the commercial sample
used here was prepared from papain flakes obtained after drying
the freshly collected papaya latex in an oven, at 45 ?C. These flakes
were re-suspended in water and filtrated. The resulting solution
was then sterilized by filtration and concentrated by ultra-filtra-
tion. The preparation was finally stabilized with glycerol before
its commercialisation. Altogether, refinement of papaya latex is
an operation that goes on for one week, during which time, the la-
tex PTLP cohabits with the catalytically competent cysteine endo-
peptidases present in the commercial papain sample. Under such
harsh conditions (the endoproteinases papain, chymopapain, caric-
ain and glycyl endopeptidase constitute around 85% of the proteins
that are present in the sample), the half-live of proteins susceptible
to proteolysis would be expected to be very short (El Moussaoui
et al., 2001).
The purification procedure used for that purpose was identical
to that used for the freshly collected latex. PTLP was not only pres-
ent in the commercial preparation but the yield of obtainment was
quite comparable (PTLP represents some 2% of the total protein
content). The molecular mass of the purified protein was identical
to the one obtained from the freshly collected papaya latex, there-
by indicating the absence of any polypeptidic cleavage. The results
of this experiment strongly suggest that the latex PTLP is highly
resistant to proteolysis.
2.5. Evaluation of the resistance of the latex PTLP to chemical
The effect of pH on the latex PTLP fold was first examined. The
protein (1.85 lM) was incubated for 72 h, at 25 ?C, in buffers of
various pHs. Buffers used were glycine–HCl for pH 2.0, sodium ace-
tate for pH 4.5, potassium phosphate for pH 6.5, Tris–HCl for pH 8.5
and sodium hydrogenocarbonate–sodium carbonate for pH 10.0.
All buffers were 50 mM and contained 150 mM KCl. Intrinsic emis-
sion fluorescence spectra were then recorded. It was observed that
the kmax value (342 nm) remained unchanged in the broad pH
interval from 2.0 to 10.0. Such a kmaxis characteristic of a complex
situation where the tryptophyl residues are partly buried and
partly exposed. On the other hand, the fluorescence intensities at
342 nm of the PTLP only slightly differed as a function of pH. Acid-
ification of the protein solution was associated to a quenching of
the fluorescence emission (some 10% of variation) between the ex-
tremes of pH. Such an observation could be explained by the pro-
tonation of amino and of carboxylic functions since both
protonations are known to be accompanied by a quenching of tryp-
tophan fluorescence (Brand and Witholt, 1967) and, most proba-
bly, does not reflect a conformational change.
8-Anilinonaphtalene-1-sulfonic acid (ANS) was used to confirm
this hypothesis. In aqueous solutions, the hydrophobic dye ANS has
a low quantum yield and exhibits a kmaxemission at 519 nm. Bind-
ing of this probe to hydrophobic sites of proteins leads to both an
enhancement of its fluorescence intensity and a blue shifting of its
maximum of emission. On the other hand, ANS is well known to
only bind to partially denatured proteins but not to native or to-
tally unfolded ones (Ptitsyn, 1995). The fluorescence emission
spectra of ANS (50 lM) in the presence of the latex PTLP
(1.85 lM) at pH 2.0, 4.5, 6.5, 8.5 and 10.0 were recorded. The re-
sults clearly indicated the absence of binding in the pH range 2–
10, thereby strongly suggesting that the PTLP did not undergo con-
formational transition. In its native state, the latex PTLP exhibited a
kmaxat 342 nm and, at pH 6.5, a relative quantum yield of 0.047
assuming a value of 0.14 for the quantum yield of N-acetyl-L-tryp-
tophanamide in water.
In its denatured state (in 50 mM Hepes buffer containing 5 M
Gu?HCl at pH: 6.5, 25 ?C after 72 h), the latex PTLP was character-
Fig. 5. A similarity tree on 33 PTLP sequences found in Carica papaya cDNA data, which shows three clusters (PTLP-1 to PTLP-3). Each sequence is named by its accession
number in EMBL data bank and by the open reading frame of translate. The tree was generated with Clustal (Tompson et al., 1994) and depicted with program FigTree.
Y. Looze et al./Phytochemistry 70 (2009) 970–978
ized by a kmaxat 357.5 nm and a relative quantum yield of 0.063
indicating that the indole side chains were completely exposed
to the external milieu as a result of unfolding.
The Gu?HCl induced unfolding of the PTLP at pH 6.5 and 25 ?C is
shown in Fig. 7. The transition was monitored by measuring the
intensities of the fluorescence emission spectra at 357.5 and
342 nm and plotting the intensities ratios as a function of denatur-
ant concentrations. This transition was quite reversible as shown
by kinetic studies. In addition, elimination of the denaturant by
diafiltration resulted in a PTLP preparation that exhibited spectral
properties that did not differ from the spectral properties of the na-
tive protein. As a consequence, the experimental points reported in
Fig. 7 have been validly used to measure the relative populations of
the sole native (N) and unfolded (U) forms that were in equilibrium
at a given Gu?HCl concentration and therefore to calculate equilib-
rium constants KU
sion of the N state into the U state DG0as ?RT lnKU
dependence of the free energy of unfolding of the latex PTLP upon
Gu?HCl concentration was observed to be linear in the region
where the transition occurred (r2= 0.9778) (insert to Fig. 7). Hence,
Nas [U]/[N] and free energy changes for conver-
Fig. 6. Ribbon view of latex PTLP model (upper part) and alignment of sequenced peptides of latex PTLP onto the three forms of PTLP (lower part). a-Helices are in orange and
b-strands in pale green. The 16 cysteine residues are showed in CPK representation, with carbon, nitrogen, oxygen and sulfur atoms in grey, blue, red and yellow, respectively.
Sequences of the three forms of PTLP are EX276621_3, EX262199_2 and EX255680_1, respectively (see Fig. 5 and Supplemental Fig. S1). The amino acid colour scheme is
taken from BioEdit program (http://www.mbio.ncsu.edu/bioedit/bioedit.html). (For interpretation of the references in colour in this figure legend, the reader is referred to the
web version of this article.)
Y. Looze et al./Phytochemistry 70 (2009) 970–978
it was analysed as previously described by Myers et al. (1995),
leading to an extrapolated value of 63.06 kJ/mol (15.2 kcal/mol)
for the slope (m).
waterand a value of 22.4 kJ mol?1M?1(5.4 kcal mol?1M?1)
2.6. Molecular modelling and circular dichroism studies
As several crystal structures were determined for TLPs of form
1, the spatial structure of the latex PTLP was modelled by compar-
ative modelling using the sequence EMBL:EX276621 and three
homologous template structures. Considering the high level of se-
quence similarities between the templates and latex PTLP, the
model was obviously evaluated to be a good quality structure
using ProQ, Verify3D and ProSA web servers. It shows an equiva-
lent resolution of 1.7 Å according to hydrogen bond energy criteria,
with 90% residues in most favoured regions of Ramachandran plot
and no residue found in the disallowed regions. The sequence of la-
tex PTLP was then structurally compatible with the fold of form 1
TLPs. Similar to other TLPs, latex PTLP structure was composed of
three structural domains, encompassing 10% of a-helices and 34%
of b-strands as secondary structure elements (Fig. 6).
The CD spectrum of the latex PTLP in the far-UV (190-240 nm)
region, recorded in water at pH 5.25, experimentally confirmed
this result (Fig. 8). CD spectrum, which was atypical with its posi-
tive CD band at 230 nm, was analysed using the CONTINLL method
and the reference set SP175 (Lees et al., 2006). This analysis indi-
cated that a-helices (8%), b-sheets (41%) and turns (12%) contrib-
ute to the secondary structure of this papaya protein. These
values fit rather well with the structural model of latex PTLP. The
reconstructed spectrum is also found to superimpose reasonably
well to the experimental one (Fig. 8).
2.7. What is the biological function of latex PTLP?
The physiological role of latex PTLP has yet to be established.
Thaumatin-like proteins generally display a high degree of se-
quence similarity with thaumatin, a sweet-tasting protein from
the ripe fruits of Thaumatococcus daniellii. TLPs, also called proteins
of the PR-5 family, are associated with diverse functions like anti-
fungal activity (Hejgaard et al., 1991), b-1,3-glucanase (Menu-
Bouaouiche et al., 2003; Sakamoto et al., 2006), carbohydrate-bind-
ing properties (Osmond et al., 2001), protection against osmotic
stress (Takeda et al., 1991) or freezing tolerance (Urrutia et al.,
1992; Hon et al., 1995). Considering their large range of biological
functions, the precise role of TLPs as defence proteins is however
far from clear. To elucidate the physiological role of latex PTLP,
we tested several possible functions for latex PTLP.
Thermal hysteresis proteins, commonly termed antifreeze pro-
teins, lower the freezing temperature of body fluids by a noncolli-
gative mechanism without significantly affecting the melting
point. As a result, a gap is generated between the freezing and
melting temperatures, a phenomenon known as thermal hystere-
sis. Some plant TLPs interact with surfaces of ice crystals or ice nu-
clei and inhibit their growth (Urrutia et al., 1992).
The calorimetric measurements of a 2.7 mg/ml solution of latex
PTLP in pure water revealed that the protein depresses the freezing
point without significantly affecting the melting temperature.
Supercooled water gave a sharp freezing endotherm with an onset
of ?18.43 ?C, whereas the aqueous solution of the latex PTLP
showed an endotherm with an onset of ?20.76 ?C. These values
imply a freezing point depression of 2.33 ?C after substraction of
water supercooling, corresponding to 1250 mOsm (1.86 ?C equals
1 Osm). Although the melting exotherms of both samples are rela-
tively wide, the good overlap indicates that there is no significant
difference between the melting temperatures of the samples.
As shown here, latex PTLP is clearly able to interact with sur-
faces of ice crystals and may thus be qualified of an antifreeze pro-
tein. However, when compared to some other plant TLPs (Hon
et al., 1995), its effectiveness is weak.
Interestingly, some plant TLPs also exhibit a strong antifungal
activity (Ye et al., 1999; Wang and Ng, 2002; Krebitz et al., 2003;
Leone et al., 2006; Vitali et al., 2006; Gorjanovic ´ et al., 2007; Popo-
wich et al., 2007), while other TLPs have no or only weak antifungal
activities (Vigers et al., 1991; Min et al., 2004). This probably re-
sults from the existence of several forms of TLP, as found here for
C. papaya or elsewhere for H. vulgare (Reiss et al., 2006).
For the latex PTLP, no antifungal activity against Candida and
Saccharomyces could be observed, in contrast to some other TLPs
(Hejgaard et al., 1991; Gavrovic ´-Jankulovic ´ et al., 2002; Vitali
et al., 2006). However, one cannot conclude that latex PTLP do
not possess any antifungal activity as only two genera of yeast
have been tested. This point needs further investigations using a
large variety of yeast strains, especially plant pathogenic fungi.
Finally, we have observed an interaction between latex PTLP
and laminaritetraose (isolated from Poria cocs) leading us to exam-
[ [ Gu.HCl (M)] ]
2.50 2.75 3.00[ [ Gu.HCl (M) ] ]
Fig. 7. Gu?HCl induced unfolding of the latex PTLP at pH 6.5 and 25 ?C. The
transition was monitored by measuring the intensities of the fluorescence emission
spectra at 357.5 and 342 nm and plotting the intensities ratios as a function of
denaturant concentrations. Inset: dependence of the free energy of unfolding of the
latex PTLP upon Gu?HCl concentration.
Fig. 8. Experimental (squares) and reconstructed (triangles) circular dichroism
spectra of the latex PTLP in the far-UV region. The reconstructed spectrum was
obtained usingdichrowebtool (http://www.cryst.bbk.ac.uk/cdweb/html/
Y. Looze et al./Phytochemistry 70 (2009) 970–978
ine the possibility that this protein could actually act as an endo-b-
1,3-glucanase. However, colorimetric assays for b-1,3-glucanase
activity using laminarin azure as substrate, even up to 18 h as incu-
bation time, indicated that latex PTLP is completely devoid of b-
endoglucanase activity. In conclusion, the biological function of la-
tex PTLP remains unknown and further studies are needed to elu-
cidate the role of this protein in latex.
3.1. Plant materials and chemicals
Sigma-Aldrich Chemie (Steinheim, Germany) provided methyl-
acid (ANS), succinimide, chitosan (low molecular weight), N-acet-
yltryptophanamide and dithiothreitol (DTT), SP-Sepharose Fast
Flow was from Amersham Biosciences GE Healthcare (Uppsala,
Sweden) while Fractogel EMD Propyl (S) was from Merck (Darms-
tadt, Germany). Tetra-N-acetylglucosamine was provided by Che-
mos Gmbh (Regenstauf,Germany).
paranitroanilide (BAPA), D-glucosamine, laminaritetraose, azocoll
and 2-mercaptoethanol were from Sigma Chemical (St. Louis,
USA) while Boc-Ala-Ala-Gly paranitroanilide (Boc-AAG-pNA) was
from Bachem AG (Bubendorf, Switzerland). N-acetyl-D-glucosa-
mine and guanidinium hydrochloride (Gu?HCl) were from Fluka
Papaya lattices were freshly collected in East Africa (Democratic
Republic of Congo, ex-Zaire). A papaya latex preparation (commer-
cially available and called liquid papain) was provided by BSC Bio-
chemicals (Hamme, Belgium).
In order to protect the papaya lattices from degradation by the
abundant proteinases, they were collected in an aqueous solution
containing an excess of MMTS which converts the active protein-
ases into their inactive S-thiomethyl derivatives as shown in Fig. 9.
This chemical modification is quite reversible since addition of
low molecular weight thiol compounds (2-mercaptoethanol or
DTT) allowed the quantitative regeneration of the essential thiol
functions of the proteinases when required (Wynn and Richards,
3.2. Fractionation of the papaya latex protein materials
Fractionation was performed using two chromatographic sup-
ports as described in details in the section results.
Buffers used for the ion-exchange chromatography on SP-Se-
pharose Fast Flow contained 50 mM sodium acetate at pH 5.0 to
which solid KCl was added to adjust the conductivity as indicated.
Aqueous solutions used for the hydrophobic interaction chroma-
tography on Fractogel EMD Propyl 650(S) contained ammonium
sulfate as indicated.
3.3. SDS–PAGE experiments
The SDS–PAGE experiments were carried out on precast gels
(ExcelGel, 245 ? 110 ? 0.5 mm, gradient 8–18%) using the Multi-
phore II kit from GE Healthcare. The running conditions were
600 V, 50 mA and 35 W at constant temperature (15 ± 0.1 ?C).
Molecular weight standards were hen egg white lysozyme
(14.4 kDa), soybean trypsin inhibitor (21.5 kDa), carbonic anhy-
drase (31.0 kDa), hen egg white ovalbumin (45.0 kDa), bovine ser-
um albumin (66.2 kDa) and rabbit muscle phosphorylase b
(97.4 kDa). Protein detection was performed using the silver stain-
ing procedure according to the instructions of the manufacturer.
3.4. Molecular mass determination
Latex PTLP was solubilized in 50% acetonitrile/1% formic acid (v/
v) and loaded into gold–palladium coated borosilicate nanoeclec-
trospray capillaries (Proxeon, Odense, Denmark). The mass spec-
trum was acquired on a Q-Tof Ultima mass spectrometer
(Waters/Micromass, Milford, USA), equipped with a Z-spray nano-
electrospray source and operating in the positive ion mode. The
time-of-flight analyser was operated in the W mode. Data acquisi-
tion was performed using a Mass Lynx 4.0 system. The molecular
mass of the proteins was determined after MaxEnt1 deconvolution
of the m/z raw data (Waters/Micromass, Milford, USA).
Latex PTLP was fully denatured, at pH 7.0, in 6 M Gu?HCl for
48 h at room temperature. Solid DTT was then added (25 mM)
and the reduction allowed to proceed for 4 h before addition of
MMTS (60 mM) (see Fig. 9). Unreduced fully denatured PTLP was
also reacted with MMTS. After dialysis, the molecular mass of these
derivatives was also determined.
3.5. Protein sequencing
After denaturation, reduction of the disulfide bonds and conver-
sion of the cysteines into mixed disulfides using MMTS, latex PTLP
was dissolved in 80% acetonitrile/10 mM ammonium hydrogencar-
bonate at a concentration of 0.04 mg/ml. The protein was digested
during 1 h at 37 ?C with sequencing grade trypsin (Promega, Mad-
ison, USA) at an enzyme:protein mass ratio of 1:20. After evapora-
tion of the solvent, the tryptic peptides were dissolved in 0.5% TFA
(v/v), desalted on ZipTipC18(Millipore, Billerica, USA) and eluted in
50% acetonitrile/1% formic acid (v/v). The MS/MS spectra were ac-
quired. After processing of the MS/MS data using the maximum en-
tropy data enhancement software MaxEnt 3, the peptide amino
acid sequences were semi-automatically deduced using the Pep-
tide Sequencing program (Waters/Micromass, Milford, USA).
3.6. Fluorescence measurements
Fluorescence spectra were recorded using a Perkin Elmer LS 55
fluorimeter and measurements were made in the concentration
range where emission was linear with respect to fluorophore con-
centrations. In all cases, emission and excitation bandwidths were
5.0 nm each and the solution temperature in the cell was main-
tained at 25.0 ± 0.1 ?C. Emission spectra were the mean of five con-
For intrinsic fluorescence emission measurements, excitation
was at 295 nm and emission spectra were collected in the range
300–450 nm. Relative fluorescence quantum yields were deter-
mined using: Qx= Qs? As? Sx/Ax? Sswherein Q is the emission quan-
tum yield, A is the absorbance at the excitation wavelength and S is
the area under the emission curve. The subscripts s and x refer to
the standard and to the sample, respectively. The standard used
was N-acetyl-L-tryptophanamide with a published fluorescence
quantum yield of 0.14 (Eftink et al., 1995).
Fig. 9. Conversion of the papaya proteinases into their S-thiomethylderivatives.
Y. Looze et al./Phytochemistry 70 (2009) 970–978
3.7. Circular dichroism measurements
CD measurements were made at 25 ?C on a Jasco J-710 polarim-
eter using desalted and filtered protein solutions in water (pH
5.25). Far-UV spectra that were collected in the range 190–
240 nm in a 0.2 mm circular cell, were an accumulation of eight
scans recorded at 50 nm/min with a 1-nm slit width and a time
constant of 0.5 s for a nominal resolution of 1.7 nm. The far-UV
spectra were scaled to molar ellipticities [h] (deg ? cm2? dmol?1)
calculated from the formula: [h] = hobs? MRW/10 ? l ? c where
hobsis the measured ellipticity in degrees, MRW is the mean resi-
due weight, c is the protein concentration in g/ml and l is the path
length of the cell in centimeter.
3.8. Other spectroscopic and analytical methods
Absorbances were measured with a Perkin Elmer Lambda 45
spectrophotometer. Concentrations of latex PTLP were measured
spectrophotometrically using, for E0.1%at 280 nm, a value of 1.43.
This value was estimated with ProtParam (Gasteiger et al., 2005)
using the sequence EMBL:EX276621. ANS concentrations were also
determined spectrophotometrically using, for e, the value of
4950 M?1cm?1at 350 nm (Weber and Young, 1964).
3.9. Differential scanning calorimetry
Calorimetric analysis was performed using a TA-Instruments
DSC Q2000 device driven by a TA-Instruments Universal Analysis
2000 version 4-4a software. A sample containing 5 ll of 122 lM la-
tex PTLP in Milli-Q water was weighed and sealed in an aluminium
pan, and an empty aluminium pan was placed in the reference cell.
Samples were held at 25 ?C for 10 min, cooled to ?40 ?C at a scan
rate of 1 ?C/min, held 5 min at constant temperature, and warmed
to 5 ?C at a rate of 1 ?C/min. A sample of 5 ll Milli-Q water was
subjected to a similar procedure to establish the supercooling tem-
perature. The temperature at the onset of the freezing endotherm
was taken at the freezing point.
3.10. Antifungal activity
The activity of latex PTLP on the growth of Candida albicans and
Saccharomyces cerevisiae cultivated on L-Broth growth medium in
Petri dishes was studied. Inhibition was measured by placing ster-
ile filter paper disks containing latex PTLP (10, 20 or 200 lg), water
(negative control) and amphotericin B (positive control, 24 lg) on
the growth medium adjacent to the colony margin and allowing
the fungus to grow for several days at room temperature. The ab-
sence of growth over the disks and/or the occurrence of an inhibi-
tion zone surrounding the disks were used as an assessment of
3.11. b-1,3-Glucanase activity
A colorimetric assay for b-1,3-glucanase activity with laminarin
azure as substrate was performed as follows: 100 ll of latex PTLP
solution was incubated for 15 min with 1% (w/v) laminarin azure
at 37 ?C and pH 5.0, and the reaction was terminated by the addi-
tion of 250 ll of ethanol. The mixture was allowed to stand for
10 min at room temperature, then the precipitated substrate was
removed by centrifugation (10 min, 1500g), and the absorbance
of the supernatant was measured at 595 nm.
3.12. Sequence analysis
DNA sequences of TLP were retrieved from C. papaya EST data
deposited in EMBL databank by a tfastax query, which compared
protein sequence to a nucleic acid sequence database in all six
frame translations (Pearson and Lipman, 1988) using the sequence
of tobacco TLP (swissprot entry PRR1_TOBAC). Putative open
reading frames were estimated by sequence similarity with
tobacco TLP sequence. Sequences that were less than 150-amino
acid long were dropped and 33 PTLP sequences were kept in total.
Using Clustal (Tompson et al., 1994), the PTLP sequences were
aligned and a guide tree was constructed from the distance matrix.
This similarity tree was depicted with FigTree software (http://
tree.bio.ed.ac.uk/software/figtree/). A multiple alignment of 33
PTLP sequences can be obtained from authors by request or from
Supplementary information (Supplemental Fig. S1).
3.13. Homology modeling
A three-dimensional model of latex PTLP was built with the
automated comparative modelling program Modeller v9.5 (Šali
and Blundell, 1993) using as homologous protein templates the
X-ray structures of banana thaumatin-like protein (Protein Data
Bank entry: 1z3q, sequence similarity with latex PTLP: 85%, X-ray
resolution: 1.7 Å, Menu-Bouaouiche et al., 2003), tobacco osmotin
(1pcv, 84%, 2.30 Å, Min et al., 2004) and tomato PR-protein NP24
(2i0w, 84%, 2.50 Å, Ghosh and Chakrabarti, 2008). The stereochem-
ical quality of the model was evaluated with procheck-nmr pro-
gram (Laskowski et al., 1996), while the structural quality of the
model was assessed by three different web server programs, ProQ
(Wallner and Elofsson, 2003), Verify3D (Luthy et al., 1992) and Pro-
SA (Wiederstein and Sippl, 2007).
Mr. Bruno Geertsen is gratefully acknowledged for the generous
gift of the commercial papain sample. Ir. B. Nkoy is also acknowl-
edged for having collected and provided fresh papaya lattices.
We thank Nasiha Mrabet and Mehdi Kiass for their technical assis-
tance in antifungal experiments, and Dr. Eric Viscogliosi for helpful
discussions. J.H., R.W., P.B. and Y.L. gratefully acknowledge the
Communauté Française de Belgique (ARC) for its financial support.
R.W. and V.R. are Research Associate and Senior Research Associ-
ate, respectively, at the National Fund for Scientific Research
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