Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Abstract

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.

Full text

ORIGINAL ARTICLE
Balsamin, a novel ribosome-inactivating protein from the seeds
of Balsam apple Momordica balsamina
Inderdeep Kaur •Santosh K. Yadav •
Gururao Hariprasad •R. C. Gupta •Alagiri Srinivasan •
Janendra K. Batra •Munish Puri
Received: 10 July 2011 / Accepted: 14 November 2011
ÓSpringer-Verlag 2011
Abstract Plant seeds, a rich source of proteins, are con-
sidered important for their application as functional ingredi-
ents in a food system. A novel ribosome-inactivating protein
(RIP), balsamin was purified from the seeds of Balsam apple,
Momordica balsamina. Balsamin was purified by ion
exchange chromatography on CM Sepharose and gel filtra-
tion on superdex-75. It has a molecular weight of 28 kDa as
shown by SDS-PAGE analysis. Balsamin inhibits protein
synthesis in a rabbit reticulocyte lysate-based cell free
translation assay with an IC
50
of 90.6 ng ml
-1
. It has RNA
N-glycosidase activity and releases a 400-base long fragment
termed the Endo fragment from 28S rRNA in the same
manner as does saporin-6 from Saponaria officinalis. The
N-terminal sequence analysis of the first 12 amino acids of
balsamin revealed that it shares 83% similaritywith type I RIP
a-MMC from Momordica charantia and 50% similarity with
b-MMC (from Momordica charantia), bryodin I (from
Bryonia dioica) and luffina (from Luffa cylindrica). Balsamin
was further characterized by mass spectrometry. CD spec-
troscopic studiesindicate that secondary structure of balsamin
contains helix (23.5%), b-strand (24.6%), turn (20%) and
random coil (31.9%). Thus RIPs activity expressed in vege-
tables like Momordica sp. advocates its usage in diet.
Keywords Ribosome inactivating protein (RIP) 
Momordica balsamina RNA N-glycosidase Balsamin 
Cucurbitaceae
Introduction
Ribosome-inactivating proteins (RIPs) are a unique family
of proteins isolated from the seeds of many plants and are
abundant in angiosperms (Stripe and Barbieri 1986). RIPs
possess RNA N-glycosidase activity (EC 3.2.2.22) that is
involved in specific cleavage of a single N–C glycosidic
bond in universally conserved region of large ribosomal
ribonucleic acid (rRNA). This depurination of rRNA irre-
versibly inactivates the ribosomes by restricting their
binding to elongation factors, thereby blocking their par-
ticipation in protein synthesis (Endo et al. 1987). RIPs are
classified into two groups based on the difference in their
primary structure. Type I RIPs consist of a single poly-
peptide chain of approximately 26–35 kDa that possesses
RNA N-glycosidase activity. Type II RIPs consist of two
subunits, an A-chain similar to type I and a lectin subunit
B-chain that recognizes receptors on the membranes and
facilitates endocytosis. Though all RIPs share the identical
enzymatic activity, they differ in their biological activities.
Some RIPs display a variety of antimicrobial activities in
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-011-1162-1) contains supplementary
material, which is available to authorized users.
I. Kaur R. C. Gupta M. Puri
Fermentation and Protein Biotechnology Laboratory,
Department of Botany, Punjabi University,
Patiala 147 002, India
S. K. Yadav J. K. Batra
National Institute of Immunology, Aruna Asaf Ali Marg,
New Delhi 110067, India
G. Hariprasad A. Srinivasan
Department of Biophysics, All India Institute of Medical
Sciences, New Delhi, India
M. Puri (&)
Centre for Biotechnology, Chemistry and System Biology
(BioDeakin), Institute of Technology Research and Innovation
(ITRI), Geelong Technology Precinct, Deakin University,
Waurn Ponds, Geelong, VIC 3217, Australia
e-mail: munish.puri@deakin.edu.au
123
Amino Acids
DOI 10.1007/s00726-011-1162-1

vitro, such as antifungal, antibacterial and broad spectrum
antiviral activities against human and animal viruses
(Stripe et al. 1992). RIPs are of great interest because of
their potential application in medical biotechnology.
Majority of RIPs are found in plant families such as
Euphorbiaceae, Phytolaccaceae, Cucurbitaceae and Caryo-
phyllaceae. In the Cucurbitaceae family, several RIPs such
as bryodin from Bryonia dioica, luffin from Luffa cylind-
rica, trichosanthin from Trichosanthes kirilowii, momor-
charin and MAP30 from Momordica charantia have been
reported and investigated for their potential medicinal
usage. Plant RIPs have been shown to possess multiple
biological activities such as anti-tumor, anti-human
immunodeficiency virus and insecticidal properties (Ng
et al. 2010). RIPs are also used in crop biotechnology with
the aim of increasing resistance to insect, fungal and viral
pathogens (Hong et al. 1996). The A-chains of type-II and
type-I RIP have been linked to antibodies to construct
immunotoxins that are specifically toxic to the target cell.
Immunotoxins are potentially useful in cancer and AIDS
therapy (Ramakrishan et al. 1992). The type-II RIP ricin has
been used as potential anti-cancer agent (Vitetta et al.
1987). Recently, interest in RIPs has been growing on
account of their anti-viral activities (Au et al. 2000).
Momordica balsamina (aka Balsam apple, bitter
cucumber or bitter melon) a high-climbing vine from family
Cucurbitaceae is native to the tropical regions of Africa,
Asia, Arabia and Caribbean. This plant is a monoecious
climber and found in India up to an altitude of 300 m. It has
been widely used traditionally in Africa to treat various
diseases such as diabetes and malaria (Ramalhete et al.
2011). A nucleic acid sequence submitted from this plant
indicating its homology to other proteins with Momordica
sp. remained inconclusive (Ortigao and Better 1992).
Leaves and fruit extracts of this plant have displayed
hypoglycemic, anti-inflammatory and analgesic effect in
rats (Karumi and Bobboi 1999; Karumi et al. 2003). The
solvent extract of M. balsamina has shown anti-malarial
activity in vitro and in vivo without any toxicity in healthy
mice (Benoit-Vical et al. 2006). Fruit pulp extract of this
plant has given valuable information on anti-HIV property
(Bot et al. 2007); however, such claims require validation
by sensitive techniques (Puri 2010). Here we report the
purification and characterization of balsamin, a novel type-I
RIP, from the seeds of M. balsamina.
Materials and methods
Materials
Momordica balsamina seeds were procured from National
seed stock and cultivated in Botanical garden, Punjabi
University, India. Plant specimens were identified and
confirmed by senior taxonomist. Gels for ion exchange and
gel filtration chromatography were obtained from Sigma
(St. Louis, US). All other chemicals were standard com-
mercial products of analytical grade.
Purification of balsamin
Seeds (20 g) of M. balsamina were decorticated and
ground by mortar and pestle to a powder form. The powder
(20 g) was homogenized in 50 ml 150 mM NaCl. The
mixture was stirred gently at 4°C. The slurry was filtered
through muslin cloth and centrifuged at 10,0009gfor
20 min at 4°C. The clear supernatant was collected and the
proteins were precipitated by slow addition of solid
ammonium sulfate (0–60%) with constant stirring using a
magnetic stirrer at 4°C. After 8 h, the crude extract was
centrifuged at 10,0009gfor 10 min at 4°C. The superna-
tant was removed and resulting precipitate was dissolved in
15 ml of 10 mM phosphate buffer, pH 6.5 (Buffer A) and
then dialyzed against same buffer. The protein solution was
loaded onto a CM-Sepharose fast flow column
(20 cm 91.5 cm), which was equilibrated with five col-
umn volumes of buffer A. The column was then washed
with the same buffer at a flow rate of 1 ml min
-1
until no
protein eluted. Bound proteins were eluted with a linear
gradient of 0–0.4 M NaCl in 10 mM phosphate buffer, pH
6.5 (Buffer B). The fractions that contained low-molecular
mass proteins (based on SDS-PAGE analysis) were pooled.
The fractions were concentrated by ultrafiltration using
Amicon Ultra-15 10 kDa membrane. Concentrated frac-
tions were loaded onto a superdex 75 column (10/300 GL,
Amersham Biosciences Co., Piscataway, USA), prior
equilibrated with (10 mM phosphate buffer, pH 6.5). The
protein was eluted with 10 mM phosphate buffer, pH 6.5 at
a flow rate of 0.5 ml min
-1
. The eluted fractions that
showed N-glycosidase activity were pooled, concentrated
and stored at -20°C.
Protein concentrations of crude extract and fractionated
sample at each step were calculated using the Bradford
method (Bradford 1976).
SDS-PAGE
The molecular weight of balsamin was determined by
SDS-PAGE performed according to the procedure of
Laemmli (Laemmli 1970), using a 12% resolving gel. After
electrophoresis, the gel was stained with Coomassie bril-
liant blue R-250 (CBB). The molecular mass of the bals-
amin was determined by comparison with the protein
markers; myosin (250 kDa), phosphorylase b (148 kDa),
bovine serum albumin (66 kDa), glutamate dehydrogenase
I. Kaur et al.
123

(64), alcohol dehydrogenase (50), carbonic anhydrase (36),
myoglobin red (22), lysozyme (16) and aprotinin (6).
rRNA N-Glycosidase activity assay
The assay was carried out as previously described (Bagga
et al. 2003; May et al. 1989). Rabbit reticulocyte lysate was
taken as a source of ribosome. Rabbit reticulocyte lysate
(50 ll) was treated with different concentration of bals-
amin (10, 20 and 50 lgml
-1
) and incubated at 30°C for
30 min. The reaction was terminated by the addition of
10% SDS (w/v). Total rRNA was extracted with trizol
reagent (Biorad). The RNA pellet was dissolved in 20 llof
water and divided into two parts. One part was treated with
aniline acetate, pH 4.5, whereas the other part was left
untreated. The aniline-treated and untreated samples were
electrophoresed on 2% agarose gel. The gel was stained
with ethidium bromide and the RNA was visualized on a
UV-transilluminator.
Cell-free protein synthesis inhibition assay
The assay was performed according to the procedure
described (Bagga et al. 2003; Sambrook et al. 1989). In the
cell-free translation assay, 6 ll of the protein solution was
incubated with rabbit reticulocyte lysate at 30°C for
60 min. The reaction was stopped by NaOH (250 ll, 1 N)
containing H
2
O
2
(0.2%). After further incubation at 37°C
for 10 min, the proteins were precipitated with trichloro-
acetic acid (15%) on ice for 30 min and harvesting was
done on 26 mm glass fiber filters (Whatman). The dried
filters were counted using a liquid scintillation counter.
Saporin (a type I RIP) from Saponaria officinalis was used
as a positive control (Bagga et al. 2003).
N-terminal sequencing
The sequence of first 20 amino acids from N-terminal end
was determined by Edman degradation method using an
automated protein sequencer. Protein was subjected to
SDS-PAGE using 12% separating gel. Blotting was per-
formed at 350–400 mA for 35 min using polyvinyl fluoride
membrane. After the transfer the membrane was stained
and de-stained and then washed extensively with Milli-Q
water. Protein blots were loaded on Blott
TM
cartridge
(reaction chamber) of Applied Biosystems PROCISE 491
cLC Protein Sequencer and amino acid sequencing was
conducted with protein sequencer.
Mass spectrometric analysis and protein identification
In gel trypsin digestion: The protein spots from coomassie
stained gel were cut into 1 mm
3
pieces and transferred into
a sterile micro centrifuge tube. In gel trypsin digestion was
done as per the described protocol (Promega). The digested
and extracted peptides were spotted onto MALDI sample
plate and mixed with equal volume of a-cyano-4-hydroxyl-
cinnamic acid matrix solution (10 mg/mL) in 0.1% TFA
and 50% ACN. Peptide mass spectra were obtained using a
MALDI-TOF/TOF 5800 mass spectrometer (ABSciex,
CA) operating in reflectron mode over a window of
m/z700 to m/z4,000. A combined MS peptide fingerprint
and MS/MS peptide sequencing search was performed
against the SwissProt 51.6 databases using the ProteinPilot
software v4.0 (Applied Biosystems) via MASCOT search
engine v2.2. Tryptic digestion with a maximum of one
missed cleavage was considered. The search parameters
allowed oxidation of methionine and carboxyamidome-
thylation of cysteine. The monoisotopic precursor ion tol-
erance was set to 100 ppm and the MS/MS ion tolerance to
0.4 Da. Protein identifications were accepted with a sta-
tistically significant probability based Mowse score
(pB0.01).
Sequence alignment
For multiple sequence alignment of balsamin RIP amino
acid sequences, a search for sequence similarities was
performed with BLAST program available on-line (http://
www.ncbi.nlm.nih.gov/BLAST). Sequence submitted was
then aligned using Clustal W software in the default set up
and alignment was analyzed.
CD spectroscopy
CD experiments were performed on Jasco J-815 spectropo-
larimeter. CD spectrum of the purified balsamin (1 mg ml
-1
)
prepared in a sodium phosphate buffer (10 mM, pH 6.5)
was measured in the far-UV range (200–240 nm) in 1 mm
path length cuvettes. Analysis of the protein CD spec-
trum was performed using Spectra manager
TM
software
(Jasco).
Results
Purification of balsamin
The proteins were precipitated by 60% ammonium sulfate
saturation from M. balsamina seed extract. The protein
precipitates were dialyzed against the buffer A and sub-
jected to CM-Sepharose fast-flow column which was pre-
viously equilibrated with 10 mM phosphate buffer, pH 6.5
(Buffer A). Most of the protein was retained on the
CM-Sepharose column; this indicates that proteins col-
lected are mainly basic in nature. One major peak (BI) and
Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina
123

two minor peaks (BII and BIII) were obtained using NaCl
linear gradient (0.1 and 0.2 M) to elute bound proteins. The
elution at 0.1 M NaCl resulted in one protein peak, peak
BI, whereas 0.2 M NaCl gave rise to two peaks (BII and
BIII) (Fig. 1). Negligible amount of protein was eluted at
higher salt concentrations. On SDS-PAGE peak BI con-
tained balsamin (data not shown). The fractions from peak
BI were pooled and after being desalted, further purifica-
tion of those fractions was achieved using superdex 75 gel-
filtration column. Four peaks (PI–PIV) were obtained
(Fig. 2). Table 1summarizes the results of purification of
balsamin indicating that 1 mg balsamin was purified. The
purified fraction PII was subsequently found to possess
rRNA N-glycosidase and cell-free protein synthesis inhi-
bition activity. The purified balsamin from M. balsamina
showed single band with a molecular weight corresponding
to *28 kDa by 12% SDS-PAGE under denaturing condi-
tions (Fig. 3).
N-terminal sequencing
The first 12 amino acid residues from the N-terminal
sequence of balsamin are DVSFTLSGADPS which showed
homology with those of other type I RIPs in the Cucur-
bitaceae family recorded in GenBank (Table 2). The
sequence is similar to that of a-MMC (from Momordica
charantia; 83% amino acid identity) (Fong et al. 1996),
somewhat similar with TCS, TAP29 and trichokirin (from
Trichosanthes kirilowii; 66 and 58% amino acid identity,
respectively) (Shaw et al. 1991; Lee-Huang et al. 1991;
Casellas et al. 1988). Balsamin has 50% similarity to
several type-I RIPs purified from the Cucurbitaceae family
such as b-MMC (from Momordica charantia) (Fong et al.
1996); bryodin I (from Bryonia dioica) (Gawlak et al.
1997) and luffin a (from Luffa cylindrica) (Islam et al.
1990).
Protein identification
Balsamin, isolated from M. balsamina seed extract was
further characterized by determining its primary structure.
The protein was digested with trypsin and resulting
peptides analyzed by mass spectrometry. Five peptides
were identified out of which two peptides, REKVY
NIPLLLPSVSGAGRY and RKITLPYSGNYERL, were
unique to RIP from the seeds of Balsam apple (Momordica
balsamina) thereby confirming the identification of the
protein. The sequences of the peptide fragments were
obtained with 99% confidence. The calculated molecular
mass obtained from SDS-PAGE was 28 kDa. The theo-
retical molecular mass obtained by mass spectrometry
protein identification methods was 29.1 kDa. However,
both methods identified the protein to be ribosomal-inac-
tivating protein (Table 3). The confidence/accuracy levels
of peptide identification and sequences identified and the
molecular masses of the fragments are listed (see supple-
mentary information Table S1). Figure 4a shows the
alignment of balsamin amino acid sequences with other
type I RIPs. The amino acid sequences of balsamin were
used to find homology with other type I RIPs deposited in
GenBank. A BLAST search was performed to trace the
possible similarity between the amino acid sequences of
balsamin and other RIP sequences. Further analysis
showed that balsamin has the highest similarity with
a-MMC from M. charantia than to any other type I RIPs.
Sequence from mass spectrometric analysis has 78%
sequence identity with a-MMC, whereas, it has less iden-
tity with b-luffin, bryodin I, trichomislin, trichobakin and
trichosanthin. From multiple alignment (as shown in
Fig. 4a), a phylogenetic analysis of balsamin was carried
out by comparing its amino acid sequence with those of
known RIPs from other plants. Balsamin and a-MMC are
located on the same branch and other RIPs originated from
this branch. The phylogenetic tree shows the close rela-
tionship of balsamin with a-MMC and its distances
from other RIPs. Trichomislin from T. kirilowii is more
closely related to bryodin from Bryonia dioica, trichosan-
thin and trichobakin from T. kirilowii than balsamin
(Fig. 4b).
RNA N-glycosidase activity
It has been demonstrated that RIPs are RNA N-glycosidases
and they catalytically cleave the N-glycosidic bond specific
to adenine residue at position 4324 in rat 28S rRNA. After
further treatment with acidic aniline, the phosphodiester
bond splits at the depurination site, and a specific RNA
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 5 10 15 20 25 30 35
Absorbance at 280 nm
Fraction Number
B I
B II
B III
Fig. 1 Ion-exchange chromatography of extract. Profile of CM
Sepharose Ion exchange chromatography. Fractions corresponding
the peaks were pooled separately and designated as BI, BII and BIII
I. Kaur et al.
123

fragment of about 400 nucleotides, named Endo-fragment,
is released (Karumi et al. 2003). The N-glycosidase activity
of balsamin from M. balsamina was examined by incubat-
ing ribosomes with different concentration of the protein.
As shown in Fig. 5, reticulocyte lysate treated with 10 ng
balsamin and depurinate 28S rRNA released a characteristic
RNA fragment, the Endo fragment. Saporin-6 from Sap-
onaria officinalis was taken as positive control and bals-
amin released Endo fragment same as saporin-6 (data not
shown). The release of Endo fragment suggested that
balsamin possesses rRNA N-glycosidase activity.
Cell free protein synthesis
Balsamin was tested in a rabbit reticulocyte lysate trans-
lation system to investigate whether it inhibits protein
synthesis in a cell-free environment. The lysate was treated
with different concentrations of sap-6 and balsamin.
Balsamin from M. balsamina and saporin-6 (sap-6, a
positive control) from Saponaria officinalis both inhibited
protein synthesis in the rabbit reticulocyte lysate system.
The lysate was treated with different concentrations of sap-
6 and balsamin. Balsamin effectively inhibited protein
synthesis with an IC
50
90 ng ml
-1
(Fig. 6). In this exper-
iment balsamin appeared to be less toxic than the saporin-6
which has an IC
50
3ngml
-1
.
Secondary structure determination of balsamin
The far-UV CD spectrum suggests the presence of both
a-helical and b-sheet conformations. The CD spectrum
used to calculate secondary structures of balsamin by using
spectra manager software (Figure 7). Balsamin contains
23.5% helix, 24.6% b-strand, 20% turn and 31.9% random
coil (data not shown).
Fig. 2 Gel Filtration
chromatography. Superdex 75
column profile of BI from ion
exchange chromatography
Table 1 Purification summary for balsamin
Purification step Total protein (mg) Protein yield (%)
Extract 6.6 100
Ammonium sulfate 6.0 90
CM-Sepharose 4.0 60
Superdex 75 1.0 15
M 1 2
250
148
98
64
50
36
22
16
6
kDa
Fig. 3 SDS-PAGE of the purified balsamin fractions. Lane M
contains molecular weight standards (molecular weight in kDa given
on the left). Lane 1: crude extract; Lane 2: Superdex-75 purified
fraction
Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina
123

Discussion
A novel type I RIP named as balsamin has been isolated and
characterized from the seeds of Balsam apple (Momordica
balsamina), a member of the Cucurbitaceae family.Bals-
amin has a molecular mass of 28 kDa, as determined by
SDS-PAGE and gel filtration chromatography on Superdex
75. Multiple forms of RIP from M. charantia, namely
a-momorcharin, b-momorcharin (Fong et al. 1996), c-mo-
morcharin (Pu et al. 1996), d-momorcharin and e-momor-
charin (Tse et al. 1999) and MAP30 (Lee-Huang et al. 1990)
have been reported. Most of the RIPs possess molecular
masses in the range of 24–30 kDa and exhibit similarity in
amino acid sequences (Puri et al. 2009). RIPs from Cu-
curbitaceae family have a notable heritage in traditional
Chinese medicine. Proteins from T. kirilowii have been
reported for centuries in abortion and in therapy for certain
types of carcinoma (Casellas et al. 1988).
Balsamin cleaves a single N-glycosidic bond between
base and ribose at position A4324 in 28S rRNA, similar to
the other type-I RIPs. Balsamin displayed N-glycosidase
activity by producing the Endo fragment and also cell-free
protein synthesis inhibitory activity which are similar to
other RIPs (Bagga et al. 2003). The action of balsamin on
rRNA in the presence and absence of aniline is similar to
that of other RIPs (Barbieri et al. 1993). Balsamin pos-
sesses a potent cell-free translation inhibitory activity
(IC
50
=90 ng ml
-1
), which is a property for type I RIPs.
It adsorbed to CM-Sepharose column, similar to the chro-
matographic behavior of other RIPs (like a-MMC, b-MMC
and MAP30) from M. charantia (Lifson et al. 1989; Lee-
Huang et al. 1990). N-terminal sequence of balsamin
shares 83% similarity with a-MMC from Momordica sp.
Homology comparison of N-terminal amino acid sequence
suggested a high similarity of balsamin to already known
RIPs.
Table 2 Comparison of N-terminal amino acid sequence of balsamin with those of other type 1 RIPs
Plant RIP Sequence Homology with
balsamin (%)
Reference
Momordica balsamina Balsamin DVSFTLSGADPS 100 This paper
Momordica charantia a-MMC DVSFRLSGADPRSYGMFI 83 21
M. charantia b-MMC DVNFDLSTATAKTYTKFI 50 21
M. charantia MAP30 DVNFDLSTATAKTTTKFIFD 50 29
Trichosanthes kirilowii TCS DVSFRLSGATSSSYGVFI 66 22
T. kirilowii TAP29 DVSFRLSGATSKKKVYFISNL 66 23
T. kirilowii Trichokirin DVSFSLSGGGTASYEK 58 24
Bryonia dioica Bryodin I DVSFRLSGATTTSYGVFI 66 25
Luffa cylindrica Luffin a DVRFSLSGSSSTSYSKFIGDL 50 26
Residues identical to corresponding residues with balsmin are underlined. Sequence comparison of first 20 amino acids from the amino terminal
of balsamin showing sequence identity with other type 1RIPs; while only 50–65% sequence identity with the RIPs from other plant families
(luffin, Trichokirin and TCS)
Table 3 Identification profile of the purified protein
Protein name Species Protein
accession
number
pI/mass (kDa) Sequence
coverage
(%)
No. of
peptides
detected
(Position) peptide sequence
(peak mass)
Threshold
confidence for
accuracy (%)
Observed Theoretical
Ribosome-
inactivating
protein
momordin
Momordica
charantia
RIP1_MOMCH –/28.6 8.67/29.1 17.0 5 46–53: RNALPFREKV
(974.5054)
99
52–69:
REKVYNIPLLLPSVSGAGRY
(1913.1525)
125–136: RKITLPYSGNYERL
(1440.7858)
54–69: KVYNIPLLLPSVSGAGRY
(1656.0001)
221–233: KQIQLAQGNNGIFRT
(1458.7930)
I. Kaur et al.
123

Balsamin matched with a-MMC according to mass
spectrometry results. Alignment of the balsamin sequence
shows that some residues are conserved between other
RIPs like luffin b, bryodin, trichosanthin, trichomislin and
trichobakin. Balsamin secondary structure contains helix
(23.5%), b-strand (24.6%), turn (20%) and random coil
(31.9%). Phylogenetic relationship showed that balsamin
and alpha MMC can be grouped together and other RIPs
originated from this branch. The tree showed close rela-
tionship of balsamin with a-MMC than other RIPs. Alpha
MMC has abortifacient (Law et al. 1984), anti-tumor,
immune response suppressor (Leung et al. 1987) and anti-
HIV-1 (Lifson et al. 1989), deoxyribonuclease (Go et al.
1992) and ribonuclease (Mock et al. 1996) activities.
Thus, balsamin also appears to be active as alpha MMC;
it remains to be elucidated whether the balsamin is
equipotent in other biological activities. These results
may provide significant insights into the relationship
between the various biological and enzymatic activities
observed.
Fig. 4 a Alignment of amino
acid sequences of balsamin
from M. balsamina with other
type I RIPs; alpha momorcharin
from M. charantia
(gi: 60459323), b-luffin from
Luffa aegyptica (gi: 19150),
bryodin from Bryonia dioica
(gi: 2981957),trichosanthin
from T. kirilowii (gi: 547149),
trichomisilin from T. kirilowii
(gi: 46403107),trichobakin
from Trichosanthes sp. Bac kan
8-98 (gi: 7242890). (star)
conserved, (dot) conserved
substitutions, (colon)
semi-conserved residues.
bPhylogenetic tree is built from
the sequences of Momordica
balsamina (balsamin),
Momordica charantia
(a-MMC), Luffa aegyptica
(b-luffin), Bryonia dioica
(bryodin), Trichosanthes
kirilowii (trichosanthin),
T. kirilowii (trichomislin),
Trichosanthes sp. Bac Kan 8-98
(trichobakin)
Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina
123

It is noteworthy that some of the plants containing RIPs
are eaten raw by humans (e.g., spinach, pumpkin) which
mean that materials containing those levels of RIPs are not
harmful. Pharmaceutical-grade trichosanthin has been used
for many years in China as an abortifacient and as an
anticancer agent for choriocarcinoma (Lau et al. 1980).
Phase I trial has been done with subcutaneous application of
aviscumin in patients with progressive malignant tumors.
Aviscumin induced the secretion of IL-1band IFN-cin
plasma and stimulation of T-cells that mediated an in vivo
anti-tumor T cell response (Bergman et al. 2008). Several
health organizations around the world have recommended
an increase in the intake of plant-derived food in order to
improve human health status (Espin et al. 2007). Particu-
larly the proteins from bitter melon are suitable for specific
products (where the native form is needed) since they can
resist higher temperature during processing. All the essen-
tial amino acids of bitter melon proteins with the exception
of Threonine met the minimum requirements for preschool
children by FAO/WHO/UNU (Horax et al. 2010). Thus,
RIPs expressed in vegetables like Momordica sp. may be
promoted as ‘‘functional ingredient’’ in a food system since
it does not possess any toxic effect for humans and are
useful as they possess anti-viral activity. Thus efforts are
being made in many laboratories to purify RIPs from plants
and check its activity against HIV and various tumors,
which may allow their usage as potential nutraceutical.
In conclusion, balsamin, the basic protein isolated from
Momordica balsamina has the properties of type I RIP as it
(i) is a single-chain protein with a molecular mass of
approximately 28 kDa; (ii) possesses enzymatic N-glyco-
sidase activity on rabbit reticulocyte rRNA and (iii)
releases Endo fragment after aniline treatment (Fig. 2).
Furthermore, balsamin inhibits protein synthesis in cell-
free system with IC
50
value of 90 ng ml
-1
. Based on its
above-mentioned activities, balsamin may be promoted as
a nutraceutical.
Acknowledgments The authors (MP, JKB and AS) acknowledge
collaborative research supported by participating Institutions from
Australia and India. They greatly acknowledge the help of Trayambak
Basak with Mass Spec (MS) and the permission for the use of MS
from Dr. Shantanu Sengupta of Institute of Genomic and Integrative
Biology (IGIB), Delhi.
Conflict of interest The authors declare no conflict of interest.
References
Au TK, Collins RA, Lam TL, Ng TB, Fong WB, Wan DCC (2000)
The plant ribosome inactivating protein luffin and saporin are
potent inhibitors of HIV-1 integrase. FEBS Lett 47:169–172
00 1
-An
10
nili
2
ne
20 550 0
+
0
+An
1
nili
10
ine
220 550 (ng/ml)
Endo fragmen
t
Fig. 5 RNA N-glycosidase activity of balsamin when incubated with
rabbit reticulocyte lysate. The rRNA was extracted and treated (?)or
not treated (-) with aniline-analyzed hydrolysis and analyzed by 2%
agarose gel electrophoresis. The arrow denotes the position of rRNA
fragments (Endo’s fragment) resulting from aniline-catalyzed
hydrolysis
Fig. 6 Effect of balsamin on the protein synthesis of rabbit reticu-
locyte lysate on a cell-free system and Sap-6 as a positive control [
conc (ng/ml) vs. % activity]
Fig. 7 CD spectra of balsamin measured in protein solution of
1 mg/mL (10 mM sodium phosphate buffer, pH 6.5). CD spectra
measured in far-UV range in 1-mm path length quartz cuvettes
I. Kaur et al.
123

Bagga SB, Seth D, Batra JK (2003) The cytotoxic activity of
ribosome inactivating protein saporin-6 is attributed to its rRNA
N-glycosidase and internucleosomal DNA fragmentation activ-
ities. J Biol Chem 278:4813–4820
Barbieri L, Battelli MG, Stripe F (1993) Ribosome-inactivating
proteins from plants. Biochim Biophys Acta 1154:237–282
Benoit-Vical F, Grellier P, Abdoulaye A, Moussa I, Ousmane A,
Berry A, Ikhiri K, Poupat C (2006) In vitro and in vivo
antiplasmodial activity of Momordica balsamina alone or in a
traditional mixture. Chemotherapy 52:288–292
Bergman L, Aamdal S, Marreaud S, Lacembe D, Herold M,
Yamaguchi T, Wilhelm-Ogunbiyi K, Lentzen H, Zwierzina H
(2008) Phase I trial of r viscumin (INN: aviscumin) given
subcutaneously in patients with advanced cancer: a study of the
European Organization for Research and Treatment of cancer
(EORTC protocol number 13001). Eur J Cancer 44:1657–1662
Bot YS, Mgbojikwe LO, Nwosu C, Ambiku A, Dadik J, Damshak D
(2007) Screening of the fruit pulp extract of Momordica
balsamina for anti HIV property. Afr J Biotechnol 6:47–52
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein dye binding. Anal Biochem 72:248–254
Casellas P, Dussossoy D, Falasca AI, Barbieri L, Guillemot JC,
Ferrara P, Bolognesi A, Cenini P, Stripe F (1988) Trichokirin, a
ribosome inactivating protein from the seeds of Trichosanthes
kirilowii Maximowicz. Purification, partial characterization and
use for preparation of immunotoxins. Eur J Biochem
176:581–588
Endo Y, Mitsui K, Motizuki M, Tsurugi K (1987) The mechanism of
action of ricin and related toxic lectins on eukaryotic ribosomes.
J Biol Chem 262:5908–5912
Espin JC, Garcia-Conesa MT, Toma
´s-Barbera
´n FA (2007) Nutraceu-
ticals; facts and fiction. Phytochemistry 68:2986–3008
Fong WP, Poon YT, Wong TM, Mock JWY, Ng TB, Wong RNS,
Yao QZ, Yeung HW (1996) A highly efficient procedure for
purifying the ribosome-inactivating proteins a- and b-momor-
charins from Momordica charantia seeds, N-terminal sequence
comparison and establishment of their N-glycosidase activity.
Life Sci 59:901–909
Gawlak SL, Neubauer M, Klei HE, Chang CY, Einspahr HM, Siegall
CB (1997) Molecular, biological and preliminary structural
analysis of recombinant bryodin 1, a ribosome-inactivating
protein from the plant Bryonia dioica. Biochem 36:3095–3103
Go TTM, Yeung HW, Fong WP (1992) Deoxyribonucleolytic activity
of a- and b-momorcharins. Life Sci 51:1347–1353
Hong Y, Saunders K, Hartley MR, Stanley J (1996) Resistance to
geminivirus infection by virus induced expression of dianthin in
transgenic plants. Virology 220:119–127
Horax R, Hettiarachchy N, Over K, Chen P, Gbur E (2010)
Extraction, fractionation and characterization of bitter melon
seed proteins. J Agric Food Chem 58:1892–1897
Islam MR, Nishida H, Funatsu G (1990) Complete amino acid
sequence of luffin-a, a ribosome inactivating protein from the
seeds of Sponge gourd (Luffa cylindrica). Agri Biol Chem
54:2967–2978
Karumi Y, Bobboi A (1999) Hypoglycemic effects of Balsam apple
(Momordica balsamina Linn) in alloxan diabetic male rabbits.
Biochem 9:795–808
Karumi Y, Onyeyili P, Ogugbuaja OV (2003) Anti-inflammatory and
antinociceptive (Analgesic) properties of Momordica balsamina
Linn (Balsam Apple) leaves in rats. Pak J Biol Sci 6:1515–1518
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680–685
Lau LF, Saksena SK, Chang MC (1980) Further studies on the
trichosanthin-induced termination of pregnancy. Contraception
21:77–86
Law LK, Tam PPL, Yeung HW (1984) Effects of a-momorcharin on
preimplantation development in the mouse. J Reprod Fert
69:597–604
Lee-Huang S, Huang PL, Nara PL, Chen HC, Kung HF, Huang P,
Hunag HI, Huang PL (1990) MAP30: a new inhibitor of HIV-1
infection and replication. FEBS Lett 272:12–18
Lee-Huang S, Huang PL, Kung H, Li BQ, Huang PL, Huang P,
Huang HI, Chen HC (1991) TAP29: an antihuman immunode-
ficiency virus protein from Trichosanthes kirilowii that is
nontoxic to intact cells. Proc Nat Acad Sci USA 88:6570–6574
Leung SO, Yeung HW, Leung KN (1987) The immunosuppressive
activities of two abortifacient proteins isolated from the seeds of
bitter melon (Momordica charantia). Immunopharmacol
13:159–171
Lifson JD, McGrath MS, Yeung H-W, Hwang KM (1989) Method of
inhibiting HIV. US Patent 4,795,739
May MJ, Hartley MR, Roberts LM, Kreig PA, Osborn RW, Lord JM
(1989) Ribosome inactivation by ricin A chain: a sensitive
method to assess the activity of wild-type and mutant polypep-
tides. EMBO J 8:301–308
Mock JWY, Ng TB, Wong RNS, Yao QZ, Yeung HW, Fong WP
(1996) Demonstration of ribonuclease activity in the plant
ribosome-inactivating proteins alpha- and beta-momorcharins.
Life Sci 59:1853–1859
Ng TB, Wong JH, Wang H (2010) Recent progress in research on
ribosome inactivating proteins. Curr Protein Pept Sci 11:37–53
Ortigao M, Better M (1992) Momordin II, a ribosome inactivating
protein from Momordica balsamina, is homologous to other
plant proteins. Nucleic Acids Res 20:4662
Pu Z, Lu BY, Liu WY, Jin SW (1996) Characterization of the
enzymatic mechanism of gamma-momorcharin, a novel ribo-
some-inactivating protein with lower molecular weight of 11,500
purified from the seeds of bitter gourd (Momordica charantia).
Biochem Biophys Res Commun 229:287–294
Puri M (2010) ‘‘Momordica balsamina: a medicinal and nutraceutical
plant for health care management’’: biotechnological potential of
M. balsamina revealed. Curr Pharm Biotchnol 11:229
Puri M, Kaur I, Kanwar RK, Gupta RC, Chauhan A, Kanwar JR
(2009) Ribosome-inactivating proteins (RIPs) from Momordica
charantia for antiviral therapy. Curr Mol Med 9:1080–1094
Ramakrishan S, Fryxell D, Mohanraj D, Olson M, Li BY (1992)
Cytotoxic conjugates containing translational inhibitory proteins.
Annu Rev Pharmacol Toxicol 32:579–621
Ramalhete C, Spengler G, Martins A, Martins M, Viveiras M,
Mulhovo S, Ferreira M-JU, Amaral L (2011) Inhibition of efflux
pumps in methicillin resistance Staphylococcus aureus and
Enterococcus faecalis resistant strains by triterpenoids from
Momordica balsamina. Int J Antimicrob Agents 37:70–74
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a
laboratory manual, 2nd edn, vol 3. Cold spring Harbor Labo-
ratory Press, Cold spring Harbor, NY, pp 18.76–18.80
Shaw PC, Yung MH, Zhu RH, Ho WK, Ng TB, Yeung HW (1991)
Cloning of trichosanthin cDNA and its expression in Escherichia
coli. Gene 97:267–272
Stripe F, Barbieri L (1986) Ribosome-inactivating proteins up to date.
FEBS Lett 195:1–8
Stripe F, Barbieri L, Battelli MG, Soria M, Lappi D (1992) Ribosome
inactivating proteins from plants: present status and future
prospects. Nat Biotechnol 10:405–411
Tse PMF, Ng TB, Fong WP, Wong RNS, Wan CC, Mak NK, Yeung
HW (1999) New ribosome-inactivating proteins from the seeds
and fruits of the bitter gourd Momordica charantia. Int J
Biochem Cell Biol 31:895–901
Vitetta ES, Fulton RJ, May RD, Till M, Uhr JW (1987) Redesigning
nature’s poisons to create anti-tumor reagents. Science
238:1098–1104
Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina
123