Acta Biochim Biophys Sin (2009): 154 – 162 | ª The Author 2009.

Published by ABBS Editorial Office in association with Oxford University Press on behalf of
the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmn018.

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias

curassavica L.

Constanza Liggieri1 *, Walter Obregón1, Sebastián Trejo2, and Nora Priolo1

1
Laboratorio de Investigación de Proteı́nas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, C.C. 711, B1900AVW, La Plata, Argentina
2
Institut de Biotecnologia i de Biomedicina ‘Vincent Villar i Palasi’, Universidad Autònoma de Barcelona, 08193 Campus UAB, Bellaterra

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(Cerdanyola del Vallès), Barcelona, Spain
*Correspondence address. Tel: þ54-0221-423-0121; E-mail: cliggieri@biol.unlp.edu.ar

Most of the species belonging to Asclepiadaceae family Received: October 23, 2008 Accepted: November 19, 2008

usually secrete an endogenous milk-like fluid in a network

of laticifer cells in which sub-cellular organelles intensively
synthesize proteins and secondary metabolites. A new Introduction
papain-like endopeptidase (asclepain cII) has been isolated
and characterized from the latex extracted from petioles Proteases are enzymes that catalyze the degradation of
of Asclepias curassavica L. (Asclepiadaceae). Asclepain cII peptides and proteins, and occupy a significant role in
was the minor proteolytic component in the latex, but numerous physiologic processes in the living beings, as
showed higher specific activity than asclepain cI, the main well as by their use in different industrial processes. It
active fraction previously studied. Both enzymes displayed has been verified that proteases direct specific and selec-
quite distinct biochemical characteristics, confirming tive modifications of proteins, such as the activation of
that they are different enzymes. Crude extract was proenzymes, sanguineous coagulation, digestion of fibrin
purified by cation exchange chromatography (FPLC). clots, secretory protein processing and transport through
Two active fractions, homogeneous by sodium dodecyl membranes, germination, senescense, defense against
sulphate-polyacrylamide gel electrophoresis and mass plant pathogens (especially fungi and insects), and acqui-
spectrometry, were isolated. Asclepain cII displayed a sition of nutrients and apoptosis [1–7].
molecular mass of 23,590 Da, a pI higher than 9.3, In the world, the industries that apply enzymes
maximum proteolytic activity at pH 9.4–10.2, and showed for their products invest annually near a trillion of
poor thermostability. The activity of asclepain cII is inhib- dollars in their commercialization. Of them, 75% belong
ited by cysteine proteases inhibitors like E-64, but not by to hydrolytic enzymes and of this percentage, the pro-
any other protease inhibitors such as 1,10-phenantroline, teases represent 60% of total of the world-wide sales [2].
phenylmethanesulfonyl fluoride, and pepstatine. The N- The vast diversity of proteases, with its specification,
terminal sequence (LPSFVDWRQKGVVFPIRNQGQ has attracted the attention of the scientists worldwide
CGSCWTFSA) showed a high similarity with those that has taken them to explore its multiple physiologic
of other plant cysteine proteinases. When assayed on and industrial applications. In relation to this last aspect,
N-a-CBZ-amino acid-p-nitrophenyl esters, the enzyme the proteases develop to a protagonist role in the
exhibited higher preference for the glutamine derivative. biotechnological processes because they maintain their
Determinations of kinetic parameters were performed enzymatic activity within a wide range of pH and temp-
with N-a-CBZ-L-Gln-p-nitrophenyl ester as substrate: erature [8].
Km 5 0.1634 mM, kcat 5 121.48 s21, and kcat/Km 5 At the moment several examples of the use of enzymes
7.4 3 105 s21/mM. and of specifically proteases in different areas from the
industry can be mentioned: modified proteins for the food
Keywords Asclepias curassavica; Asclepiadaceae; industry, baking, beer elaboration, cheese production,
cysteine proteinase; latex; laticifers detergent dust preparation, treatment of industrial effluents,

Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 2 | Page 154

 Papain-like protease from latex Asclepias curassavica L.

textile industry, manufacture of leather, pharmaceutical phosphate buffer ( pH 6.5) containing 5 mM ethylendia-
industry, cleaning of surgical supplies, and biomedicals minetetraacetic acid (EDTA) and cysteine, in order to
[9–12]. avoid phenoloxidase activity and oxidation, respectively.
Species belonging to the Asclepiadaceae family This suspension was first centrifuged at 16,000 g
usually contain proteolytic enzymes in latex. Latex is a for 30 min at 48C to discard gums and other insoluble
milky fluid with a complex mixture of constituents, like materials, and the supernatant was ultracentrifuged
proteins, vitamins, carbohydrates, lipids, terpenes, alka- at 100,000 g for 1 h at 48C. This new supernatant
loids, and free amino acids. The presence of certain containing soluble proteins (crude extract (CE), 12 ml),
enzymes like chitinases and proteases in latex vacuoles was fractionated and conserved at 2208C for further
suggest that they may help plants for defense against studies.
pathogens, parasites, and herbivores by attacking the
invader once the plant cell is lysed [13]. Purification of asclepain cII

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Our group has been largely devoted to screening new The CE was applied in a column packed with
plant sources from the local flora, which could provide SP-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden)
new proteases, as plant proteases used in Argentina are equilibrated with 0.05 M Tris –HCl buffer ( pH 8.25).
usually imported. Many members of the Asclepiadaceae Cation exchange chromatography (FPLC, Pharmacia)
family have shown to contain very active proteases in the was developed by adding the starting buffer (0.05 M
latex. The group has previously reported in the purification Tris–HCl, pH 8.25), followed by a sodium chloride
and characterization of proteases present in lattices of linear gradient (0–0.6 M) prepared in the same buffer.
several members of the Asclepiadaceae family [14–22]. Cation exchange chromatography was monitored by
Asclepias curassavica L. is native of South America measuring absorption at 280 nm. Caseinolytic activity
and grows from Mexico to Argentine but has become a was tested in on the eluted fractions, and those showing
naturalized weed in tropical and subtropical areas through- proteolytic activity were pooled and stored at 2208C for
out the world. The stems of this species exude large quan- further studies (cf next section).
tities of latex when leaves are cut off, which has been used
in folk medicine as emetic, vermifuge, and antigonorrheic Proteolytic activity assays
[23,24]. The main proteolytic component, named asclepain Proteolytic assays were made using casein as substrate.
cI, was isolated from the latex in a previous study [25]. In The reaction mixture was prepared by mixing 0.1 ml of
this article the biochemical characteristics of a second pro- the purified enzyme with 1.1 ml of 1% casein containing
tease is presented, which despite of being the minor pro- 12 mM cysteine in a 0.1 M Tris–HCl buffer ( pH 8.5).
teolytic component in the latex, it showed higher specific The reaction was carried out at 428C and stopped 2 min
activity than asclepain cI. Additionally, this new enzyme later by the addition of 1.8 ml of 5% trichloroacetic acid
exhibited different properties that could be profitable when (TCA). Each test tube was centrifuged at 3000 g for
used in industrial processes. 20 min and the absorbance of the supernatant measured
at 280 nm. An arbitrary enzyme unit (Ucas) was defined
Materials and Methods as the amount of enzyme (g) that produces an increase
of one absorbance unit per minute in the assay
Plant material conditions.
Asclepias curassavica L., ‘scarlet milkweed’,
(Asclepiadaceae) is an erect, evergreen perennial sub- Protein determination
shrub with woody base. Like most milkweeds, it has Proteins were measured according to Bradford’s method
opposite leaves and milky sap [26]. Latex was obtained [27] using bovine albumin (Sigma Chemical Co.,
from plants grown in Rosario, Province of Santa Fe, St. Louis, USA) as standard.
Argentina. A voucher specimen (UNR 1130) has been
deposited at the UNR herbarium (Faculty of Agricultural pH profile
Sciences, University of Rosario, Argentina). The effect of pH on enzyme activity was determined on
casein (pH range 6.0–10.5) at constant ionic strength using
Crude enzyme extract preparation 0.01 M sodium salts of the following buffers [28]:
Latex (2 ml), obtained by superficial incisions of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpho-
petioles, was collected on 13 ml of 0.1 M citric lino) propanesulfonic acid (MOPS), N-tris(hydroxymethyl)

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Papain-like protease from latex Asclepias curassavica L.

methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1-dime- Controls were prepared by preincubating the enzyme
thyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid with the appropriate solvent used to dissolve the inhibi-
(AMPSO), and 3-(ciclohexylamino)-1-propanesulfonic acid tors [30]. A control assay of the enzyme activity was
(CAPS). At each pH a control assay was done without done without inhibitors and the resulting activity was
enzyme and used as a blank. taken as 100%.

pH stability Electrophoresis
Two milliliter of the purified enzymatic preparation was Purified samples of asclepain cII were analyzed by
precipitated with three volumes of acetone, centrifuged sodium dodecyl sulphate-polyacrylamide gel electrophor-
at 3000 g during 20 min, and then the precipitated was esis (SDS-PAGE) with Tris–glycine cathodic buffer in
redissolved in the corresponding ‘Good’ buffer adjusted 10% polyacrylamide gels [31]. Voltage was kept constant
to pH 10.0. Samples (0.1 ml) were incubated at 208C for at 40 mV for the stacking gel and at 150 mV for the resol-

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1–3 h and the residual activity was assayed according to ution gel. The gels were stained with coomassie brilliant
the method described above. blue R-250 and the molecular weight of asclepain cII was
estimated by using the Scion Image software. Protein mol-
Thermal stability ecular weight markers (SDS-PAGE Molecular Weight
After incubating the enzyme solution (0.1 ml) for 30, Standards, Low Range; Bio Rad, Hercules, USA) were
60, 90, and 120 min at 408C, 508C, 608C, and 708C, the used as standards to generate the calibration curve.
residual proteolytic activity was determined as indicated
previously. Isoelectric focusing and zymogram
Isoelectric focusing (IEF) was performed in a mini IEF
Effect of inhibitors on proteolytic activity cell (Model III, Bio-Rad). The sample was concentrated
The effect of specific inhibitors on proteolytic activity and deionized with five volumes of cold acetone and
was determined by preincubating the protease prep- further centrifugation at 3000 g for 15 min. The precipi-
aration on casein or azocasein [29] with inhibitors and tate obtained was redissolved with deionized water and
then estimating the residual activity. The enzyme the treatment repeated twice. Polyacrylamide gels con-
preparation (0.99 ml) was incubated with 0.01 ml of taining broad pH range ampholytes (3.0–10.0) were
the one following inhibitors: 10 mM (2S,3S)-3-(N- used. Focusing was carried out under constant voltage
f(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-methylbutylg conditions in a stepped procedure: 100 V for 15 min,
carbamoyl)oxirane-2-carboxylic acid (E-64), 100 mM 200 V for 15 min, and 450 V for 60 min. Gel was fixed
phenylmethanesulfonyl fluoride (PMSF), and 0.1– and then stained with Coomassie brilliant blue R-250.
0.5 mM pepstatin A, during 30 and 60 min at 308C. The In order to ascertain if the protein fraction had proteo-
residual proteolytic activity was determined on casein as lytic activity, the unstained gel was contacted for 10 min
indicated above. at 508C with an agarose gel imbibed for 20 min in 1%
Due to 1,10-phenantroline exhibits high absorbance at casein solution (0.1 M Tris –HCl buffer, pH 8.3) with
280 nm, for this assay casein was changed by azocasein. 12 mM cysteine and washed twice with distilled water.
The enzyme preparation (0.99 ml) was incubated with Then, the agarose gel was stained by Coomassie brilliant
0.01 ml of 100 mM 1,10-phenanthroline and then the blue R-250. Clear bands on the stained agarose gels evi-
residual activity was determined as follows: 0.25 ml of dence the presence of proteolytic activity [32].
2% azocasein in 0.1 M glycine–NaOH buffer ( pH 9.5)
containing 20 mM cysteine was added to 0.15 ml of Mass spectrometry
enzyme sample and incubated at 458C for 30 min. The The molecular weight and purity of asclepain cII were
reaction was stopped by adding 1 ml of 10% TCA. After determined by Matrix assisted laser desorption ionisation
centrifugation at 4000 g for 15 min, 0.9 ml of the mass spectrometry/time-of-flight (MALDI-MS/TOF).
supernatant obtained was added to 1 ml of 1 M NaOH, Mass spectrum was acquired on a Bruker Daltonicsw
and the absorbance was measured at 440 nm. In this model Ultraflex spectrometer equipped with a pulsed
case, one unit of proteolytic activity (Uazocas) was nitrogen laser (337 nm), in a linear positive ion mode,
defined as the amount of enzyme (g) that produced an using a 20 kV acceleration voltage. Samples were pre-
absorbance increase of one unit per minute under the pared by mixing equal volumes of a saturated solution of
assay conditions. the matrix (sinapic acid) in 0.1% Trifluoroacetic acid

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 Papain-like protease from latex Asclepias curassavica L.

(TFA) (aq.): acetonitrile (2:1), and protein solution. Kinetic parameters

From this mixture, 1 ml was spotted on the sample slide N-a-CBZ-L-Gln-p-nitrophenyl ester was used to deter-
and allowed to evaporate to dryness. Bovine trypsinogen mine Vm, Km, kcat, and kcat/Km of asclepain cII.
was used for internal calibration. Estimation of kinetic parameters was performed follow-
ing the method described previously for this substrate
N-terminal sequence [34]. Substrate final concentration ranged from 125 
The N-terminal sequence was determined by Edman’s 1026 to 2  1023. Km, kcat, Vm, and kcat/Km values were
automated degradation using a Beckman LF3000 protein calculated by hyperbolic regression analysis using the
sequencer equipped with a System Gold (Beckman, nonlinearized form of the Michaelis–Menten equation.
Fullerton, USA) PTH-amino acid analyzer. The Basic
Local Alignment Search Tool (BLAST) network service Results
[33] was used to perform protein homology studies but

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considering only those specific residues that are identical Purification
(‘identities’). Cation exchange chromatography allowed the separation
of three fractions (Fig. 1). No proteolytic activity was
Measurement of endoesterolytic activity found in the former fraction eluted, but the two other
These assays were carried out by the Silverstein’s [34] fractions eluted with the linear salt gradient used were
method modified to reach optimal conditions of active. The active fractions were named asclepain cI and
the enzyme. The activity was studied using cII, according to nomenclature recommendations for pro-
N-a-Cbz-L-Gln-p-nitrophenyl esters of some L-amino teases obtained from latex of species of the
acids (Ala, Asn, Gln, Gly, Ile, Leu, Trp, Pro, and Val) Asclepiadaceae family [36,37].
as substrates. Assays were made at 378C in 0.1 M Tris – Asclepain cI was the main fraction [25]. Asclepain cII
HCl buffer ( pH 8.0) containing 2 mM EDTA, 25 mM was purified with 12.1-fold, the percent recovery in
cysteine and 1 mM of each substrate in the reaction terms of total activity was 9.8% and the specific activity
mixture. Liberation of p-nitrophenol was followed spec- was 12.8 Ucas/mg (Table 1).
trophotometrically at 405 nm in an Agilent 8453 E
UV-visible spectroscopy system (Santa Clara, CA, USA) Homogeneity of the enzyme
equipped with a chamber termostatized at 378C. An arbi- Purity of asclepain cII was checked both by SDS-PAGE
trary enzyme activity unit (Ucbz) was defined as the and mass spectrometry: 23,500 and 23,590 Da, respect-
amount of protease (g) that released one micromol of ively (Figs. 2 and 3). The presence of a single band both
p-nitrophenolate per min in the assay conditions. To in SDS-PAGE and IEF (Fig. 4) show that the enzyme is
determine the micromoles of p-nitrophenolate produced homogenous, like asclepain cI.
during the reaction, a standard curve with p-nitrophenol As it has already been mentioned before, IEF and
(5 –50 mM) in 0.1 M Tris –HCl buffer pH 8.0 containing zymogram confirmed the presence of a unique band
5% acetonitrile was carried out. with proteolytic activity, corresponding to a basic

Measurement of amidolytic activity

Amidolytic activity was determined by hydrolysis of
L-pyroglutamil-L-phenylalanyl-L-leucine-p-nitroanilide
(PFLNA) according to Filippova et al. [35]. This assay
was performed using a solution of 1 mM PFLNA in
dimethyl sulfoxide (DMSO). The reaction mixture con-
tained 1.5 ml of 0.1 M phosphate buffer, pH 6.5, 0.3 M
KCl, 1024 M EDTA, 0.003 M dithiothreitol (DTT),
0.18 ml substrate, and 0.12 ml enzyme. The
p-nitroaniline released at 378C was detected spectropho-
tometrically at 410 nm. An arbitrary enzyme activity unit Fig. 1 Cation exchange chromatography Elution buffer: 0.05 M
(UPFLNA) was defined as the amount of protease (g) that Tris – HCl ( pH 8.25). Gradient: sodium chloride 0 – 0.6 M. Flow rate:
released one micromol of p-nitroaniline per min in the 0.75 ml/min. Fraction volume: 2.0 ml. SP-Sepharose Fast Flow,
assay conditions. column Pharmacia Biotech XK 16.

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Papain-like protease from latex Asclepias curassavica L.

Table 1 Purification of the proteolytic components present in the latex of Asclepias curassavica L

Sample Volume Protein Total Arbitrary enzyme unit Total Specific activity Purification Yield
(ml) (mg/ml) proteins Ucas (ml21) Ucas (Ucas/mg) (fold) (%)

CE 1.5 0.9333 1.399 0.9880 1.480 1.0586 1 100

Asclepain cI 7.5 0.0243 0.182 0.2649 1.980 10.877 10.27 26.80
Asclepain cII 10.5 0.0075 0.078 0.0968 1.016 12.836 12.125 9.80

An arbitrary enzyme unit (Ucas) was defined as the amount of enzyme (g) that produces an increase of one absorbance unit per minute in the
assay conditions.

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Fig. 2 SDS-PAGE M, Bio Rad molecular weight standards:
phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa),
ovoalbumin (45.0 kDa), carbonicanhydrase bovine (31.0 kDa), soybean
trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa); 1, crude
extract; 2, asclepain cI; 3, asclepain cII.
Fig. 4 isoelectric focusing and corresponding zymogram M, pI
markers, amyloglucosidase ( pI 3.50), trypsin inhibitor ( pI 4.55),
b-lactoglobulin a ( pI 5.20), carbonic anhydrase II ( pI 5.85), carbonic
anhydrase I ( pI 6.55), myoglobin ( pI 6.85, 7.15, and 7.35), lectins from
Lens culinaris ( pI 8.15, 8.45, and 8.65), and trypsinogen ( pI 9.30); 1,
crude extract; 2, asclepain cI; 3, asclepain cII; 4, zymogram.

Dependence of enzyme activity on pH

Asclepain cII showed maximum activity within the
range of pH 9.4 and 10.2 (casein), which is higher than
that exhibited by the CE of Asclepias curassavica and
asclepain cI [25], and similar to that of funastrain cII
[22]. On the other hand, asclepain cII showed a high
stability at pH 10 retaining about 95% of the residual
activity after 1 h (Fig. 5).

Fig. 3 Mass spectroscopy of asclepain cII MALDI-MS/TOF was Dependence of enzyme activity on temperature
used for the determination of purity degree as well as of molecular Asclepain cII exhibited poor thermostability, as only
weight of asclepain cII. Sample was mixed with sinapinic acid (matrix)
30% of its activity was retained after 2 h of incubation at
dissolved in 0.1% trifluoroacetic acid. Bovine trypsinogen was used for
internal calibration. 508C (Fig. 6). At 60–708C the lost of activity is rapid.

protein, which focused at pI higher than 9.3 (Fig. 4). Effect of inhibitors on proteolytic activity
The pI value of asclepain cII is comparable with those Different inhibitors specific for distinctive classes of pro-
of other plant proteases from the Asclepiadaceae family teases were used to determine the class to which the pur-
(Table 2). ified protease belongs (Fig. 7). The enzyme was

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 Papain-like protease from latex Asclepias curassavica L.

Table 2 Comparison of biochemical characteristics of

phytoproteases belonging to Asclepiadaceae family

Phytoproteases Molecular pI pH References

mass (Da) optima

Asclepain cII 23,590 9.3 8.5 This study

Asclepain cI 23,200 9.3 8.5 [25]
Araujiain aI 23,464 9.3 8.00 Our previous
work
Araujiain aII 23,528 9.3 8.25 Our previous
work
Araujiain aIII 23,488 9.3 9.25 Our previous

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work Fig. 6 Thermal stability on the proteolytic activity of the asclepain cII
Araujiain hI 24,031 9.3 8.75 [19] at 408C, 508C, 608C, and 708C following 30, 60, 90, and 120 min
Araujiain hII 23,718 8.9 8.5 [19] incubation.
Araujiain hIII 23,446 10.5 8.5 [19]
Asclepain f 23,652 9.3 9.5 [21]
Funastrain cII 23,636 9.3 9.5 [22]
Morrenain bI 23,205 9.3 8.70 [15]
Morrenain bII 26,000 9.3 8.25 [17]
Morrenain oI 27,000 9.3 8.5 [17]
Morrenain oII 25,800 9.3 8.5 [17]

Fig. 7 Effect of inhibitors on the proteolytic activity of asclepain cII

incubated on each inhibitor solution during 30 and 60 min at 308C.

immediately and irreversibly inhibited by 10 mM E-64

(cysteine proteases inhibitor). Incubation with 100 mM
PMSF (serine proteases inhibitor) for 30 min decreased
activity to 63% of the initial value, but inhibition
was partially reverted when 12 mM cysteine was
added to the reaction mixture (data not shown). As
1,10-phenantroline (metaloproteases inhibitor) and
pepstatine (aspartic proteases inhibitor) do not affect the
activity of asclepain cII.

N-terminal protein sequence

The N-terminus sequence of asclepain cII (30 amino
Fig. 5 Effect of pH (A) Proteolytic activity of asclepain cII
acids) was analyzed with the BLAST (http://www.ncbi.
measured on casein (range pH 6.0– 11.0) using 0.01 M sodium salts of
the following buffers: MES, MOPS, TAPS, AMPSO, and CAPS. (B) nlm.nih.gov/BLAST/). This sequence showed higher
Stability at pH optimum on the proteolytic activity of asclepain cII, identity (86% and 80%, respectively) with proteases iso-
using buffers adjusted to pH 10.0 following 1 –3 h incubation at 208C. lated from species of Asclepias and Funastrum genera

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Papain-like protease from latex Asclepias curassavica L.

(Table 3). Both, asclepain cI and cII, shared the motifs ester. The obtained results were: Km ¼ 0.1634 mM; Vm ¼
surrounding the catalytic cysteine (CGS and WTFS), that 0.000974 mM/s; kcat ¼ 121.48 s21; and kcat/Km ¼ 7.4 
also occur in most of the sequences compared. The 105 s21/mM. The kcat/Km values within a range from
DWR and QG motifs are notably conserved and are 105 –106 s21/mM revealed a high affinity of the enzyme for
present in all the cases, as well as the proline residue this substrate.
located in position 2, to which is attributed a protective
anti-aminopeptidase role [38].
Discussion
Measurement of endoesterolytic activity
When the enzyme was assayed using N-a-Cbz-L-amino Although asclepain cII is the minor fraction, it showed
acid-p-nitrophenyl esters, the highest relative esterolytic higher specific activity (12.8 Ucas/mg) than asclepain cI
activity was obtained for the glutamine derivative (10.8 Ucas/mg). The molecular mass of this new enzyme

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(Table 4), as happened with asclepain cI [25]. is very close to that of asclepain cI [25] and similar to
those obtained for other species of the Asclepias genus,
Measurement of amidolytic activity like Asclepias syriaca, that displays proteases of molecu-
Asclepain cII exhibited very low activity on PFLNA, a lar weight 21,000 and 23,000 Da [39,40], Asclepias
typical substrate for cysteine endopeptidases (data not glaucescens, with a single protease of molecular weight
shown), unlike asclepain cI, which showed an important 23,000 Da [36,37] and Asclepias fruticosa, also contain-
activity on this substrate [25]. ing a single protease of molecular weight 23,652 Da
[21], as well as those from other genera of the same
Kinetic parameters family, within the interval from 20 to 35 kDa [16,18,22].
Km, kcat, and kcat/Km ratio of asclepain cII were determined The important loss of proteolytic activity at 60–708C
with the substrate for which the CE showed the greatest of asclepain cII denotes a different behavior from other
preference, that is N-a-Cbz-L-amino acid-p-nitrophenyl proteases studied in our laboratory [15,16,18,19].
Table 3 Comparison of N-terminal amino acid sequences of asclepain cII and other cysteine plant endopeptidases

Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 2 | Page 160

 Papain-like protease from latex Asclepias curassavica L.

Table 4 Asclepain cII’s endoesterolytic activity using As most cysteine proteases obtained from other plant
N-a-Cbz-L-Gln p-nitrophenyl esters of some L-amino acids as latex sources, asclepain cII, the new protease isolated and
substrates purified from the latex of A. curasavica, could be useful in
N-a-Cbz-L-amino acid p-nitrophenyl ester Ucbz pharmaceutical and biotechnology industries due to their
wide ranges of activity over temperature and pH [43,44].
Gln 75.35
Asn 130.00
Ala 181.28
Funding
Gly 226.06
This work was supported by the grants from ANPCyT
Tyr 361.88
(PICT 9-9916), CONICET (PIP 2813) and the
Asp 904.26
University of La Plata, Argentina, as well as for CYTED
Val 0

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IV.22. C.L. is member of the CIC Support Professional
Leu 0
Career Program. W.D.O. is CONICET fellowship holder.
Ile 0
Lys 0

An arbitrary enzyme activity unit (Ucbz) was defined as the amount

References
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5 Boller T. Roles of proteolytic in interactions of plant with other organ-
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