60
CHAPTER 4
ISOLATION, PURIFICATION AND
CHARACTERIZATION OF THE PEPTIDOGLYCAN
FRACTION FROM CUTTLEFISH INK
61
4.1 INTRODUCTION
Cuttlefish ink consists of melanin granules in a viscous colourless medium (Russo
et al., 2003). Melanins are a group of natural black pigments. The structure of melanin
macromolecules is an irregular network arising from phenolic precursors in consequence
of enzymatic reaction and autooxidation (Barboi, 1999). Melanin isolated from the ink
sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural
eumelanin (Zeise and Chedekel, 1992). Melanogenesis in the ink sac of Sepia officinalis
in a simplified view seems to follow the general scheme of melanin formation in
vertebrates (Schraermeyer, 1994). The ink gland of cuttlefish, Sepia officinalis has
traditionally been regarded as a convenient model system for investigating
melanogenesis. The ink gland has been shown to contain a variety of melanogenic
enzymes including tyrosinase (Prota et al., 1981), a dopachrome- rearranging enzyme
(Palumbo et al., 1994) and peroxidase. Moreover, the peculiar and complex organization
of melanin in an invertebrate such as Sepia officinalis is surprising and could provide the
basis for understanding the process in more evolved systems such as that of mammals
(Palumbo et al., 1997b).
Crude melanin obtained from the ink bags of squid, Ommastrephes bartrami were
fractionated using gel filtration on Sephadex G100 column, which contained a
melanoprotein, composed of melanin pigment (90%), protein (5.8%), and carbohydrate
(0.8%). Chemical analysis revealed that amino acids such as glycine, aspartic acid and
glutamic acid were present in large amounts and there was a trace amount of sulphur
containing amino acids (Mimura et al., 1982a). High molecular weight sugars were
reported to exist in squid ink (Matsue et al., 1995). Squid ink was fractionated using ion
62
exchange and gel filtration chromatography and the peptidoglycan fraction obtained was
composed of 7.8% peptide, 57% polysacccharide and 30% pigment (Takaya et al.,
1994b). Three fucose rich glycosaminoglycans (GAGs) have been isolated from squid
ink of Illex argentinus. The unique repeating structure of the GAGs was determined to
be (-6GalNAc1-3glcA1-3Fuc1-) n (Takaya et al., 1994b). Squid ink is found to be
rich in taurine and hydroxy proline. Higher amounts of free aminoacids like aspartic acid,
glutamic acid, alanine, leucine and arginine have been detected in squid ink. A small
amount of trimethylamine oxide and large amounts of homarine and glycine betaine have
been detected in squid ink (Shirai et al., 1997).
The melanin free ink of the cephalopod, Sepia officinalis is shown to contain a
heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells.
Gel filtration chromatography indicated that the toxic component was concentrated in
those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the
highest tyrosinase activity (Russo et al., 2003). In the present chapter the isolation,
purification and characterization of a peptidoglycan fraction from the ink of cuttlefish,
Sepia pharaonis which is found to be bioactive in its crude form is described.
4.2 MATERIALS AND METHODS
4.2.1 REAGENTS AND CHEMICALS
Tris-HCl buffer (pH 6.8) (SRL, Mumbai)
DEAE Sephacel (Sigma, USA)
Sephacryl S-300 (Sigma, USA)
Dextran D 9260 Av. Mol. Mass. 10,000 (Sigma, USA)
Dextran D 1662 Av. Mol. Mass. 39,379 (Sigma, USA)
63
Dextran Av. Mol. Mass 75,000 (Himedia, Mumbai, India)
Coomassie Blue R-250 (SRL, Mumbai)
4.2.2 METHODOLOGY
4.2.2.1 COLLECTION AND EXTRACTION OF THE INK
Ink sacs from the cuttlefish, Sepia pharaonis were collected from the processing
plant, Bhatsons Aqatic Products, Aroor, Alleppy, Kerala. They were brought to the
laboratory in ice cold condition. A known weight (143 g) of the ink from the ink sac of
cuttlefish was delipidated with acetone at –200C for 96 h. The acetone was changed every
24 h after vacuum filtration. The defatted residue was vacuum dried to give a black
powder (73 g). This powder was extracted with 40 volumes of 0.1 M Tris- HCl buffer
(pH 6.8) at 40C for 48 h, followed by centrifugation at 13,000 x g at 4 0C for 30 min.
Supernatant was dialysed against distilled water at 40C for 48 h and then lyophilised to
obtain an off white powder (279 mg) (Fig. 4.1). This powder was dissolved in normal
saline and used for primary culture studies. The antitumour activity against Dalton’s
lymphoma ascites (DLA) in BALB/c mice was also checked using the above powder
4.2.2.2
FRACTIONATION
BY
DEAE
SEPHACEL
ION
EXCHANGE
CHROMATOGRAPHY
The lyophilised powder (279 mg) obtained after Tris-HCl extraction was
dissolved in minimum volume of 0.1 M Tris- HCl buffer (pH 6.8) and transferred to a
column of DEAE Sephacel (1.6 x 38 cm) equilibrated with the same buffer. Elution was
carried out step- wise with 0, 0.1 and 0.2 M NaCl in the same Tris-HCl buffer. Fractions
(3 ml) were collected and absorbance at 280 nm was monitored .The eluant was separated
into 3 fractions A, B & C (87 mg). The fractions were desalted by dialysis and
Cuttlefish ink (143 g)
Defatting (acetone, -200C)
Lyophilization
Delipidated ink (73 g)
Extraction ( 0.1 M Tris HCl (pH 6.8), 40C, 48 h)
Centrifugation (13,000 x g, 40C, 30 min.)
Crude extract (yellow in colour)
precipitate
Dialysis & lyophilisation
Off white powder (279 mg)
DEAE Sephacel Ion exchange column (0.1 M Tris HCl, pH 6.8)
Fraction A
Fraction B
Fraction C (87 mg)
Sephacryl S-300 Gel filtration of 60 mg of
fraction C
Fraction C1 (4.9 mg)
Fraction C2 (29 mg)
Figure 4.1: Extraction and fractionation procedure of cuttlefish ink
64
lyophilised. The lyophilised samples were tested for antitumour activity against DLA in
BALB/c mice following intraperitoneal administration. Among the fractions the fraction
C which showed stronger antitumour activity (given in chapter 5) was further purified by
gel filtration.
4.2.2.3 GEL FILTRATION OF FRACTION C USING SEPHACRYL S-300
A known weight (60 mg) of the lyophilised fraction C was dissolved in minimum
volume of 0.1 M NaCl solution and transferred to a column of Sephacryl S-300 gel (1.6 x
36 cm) equilibrated with 0.1 M NaCl and eluted with 0.1 M NaCl at a flow rate of 36 ml
per h, monitoring the absorbance at 280 nm. 4.8 ml fractions were collected. Each
fraction was also examined using the carbazole sulphuric acid reaction for detecting
uronic acid. Two fractions C1 (4.9 mg) and C2 (29 mg) were obtained and they were
separately pooled, desalted and lyophilized and were tested for antitumour activity
against DLA in BALB/c mice. Of the two, fraction C2 showing higher antitumour activity
(data given in chapter 5) was subjected to further analysis such as SDS-PAGE, molecular
mass determination and chemical composition.
4.2.2.4 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) OF
PEPTIDOGLYCAN FROM CUTTLEFISH INK
The crude peptidoglycan obtained by Tris- HCl extraction of the cuttlefish ink and
the purified peptidoglycan fraction C2 were used for SDS-PAGE. The samples were
dissolved in sample buffer (Tris pH 6.8) containing 2% SDS, 5% mercaptoethanol and
10% glycerol and heated in a boiling water bath for 5 min. To the sample, 5 l of 0.025%
of Bromophenol blue solution was added and conducted SDS PAGE using the method of
Laemmli (1970), on a 10% polyacrylamide slab gel. The running buffer was Tris-glycine
65
(pH 8.3) containing 0.1% (w/v) SDS. Electrophoresis was carried out at 40 mA per slab
till the dye reached the bottom of the gel slab. The gel was stained for proteins with
0.02% w/v Coomassie Blue R-250 in methanol: water: acetic acid; 46:46:8 (v/v/v).
4.2.2.5 DETERMINATION OF THE MOLECULAR MASS OF THE PURIFIED
PEPTIDOGLYCAN FRACTION C2
The molecular mass of the purified peptidoglycan was determined by gel filtration
using Sephacryl S-300 gel column (1.6 x 36 cm). The column was calibrated using the
following authentic dextrans.
1. Dextran D 9260 Av. Mol. Mass. 10,000 (Sigma, USA)
2. Dextran D 1662 Av. Mol. Mass. 39,379 (Sigma, USA)
3. Dextran Av. Mol. Mass 75,000 (Himedia, Mumbai, India)
At a flow rate of 36 ml/h, 3 ml fractions were collected and each fraction was analysed
for carbohydrate content using Phenol- Sulphuric acid reaction.
4.2.2.6 COMPOSITION OF DIFFERENT FRACTIONS
Different fractions were subjected to protein analysis by Lowry et al. (1951)
method and uronic acid by Bitter and Muir (1962) method.
4.2.2.6.1 ESTIMATION OF PROTEIN BY LOWRY’S METHOD
REAGENTS
1. Sodium hydroxide
: 0.1 N
2. Sodium carbonate solution
: 2% in 0.1 N sodium hydroxide
3. Copper sulphate solution
: 0.5% in 1% Sodium potassium tartarate
4. Alkaline copper reagent
: prepared by mixing 50 ml of Reagent 2 with 1 ml
66
of Reagent 3
5. Folins phenol reagent
: diluted with distilled water to obtain a 1 N solution
6. Standard Bovine
Serum Albumin (BSA) solution
: 10-100 g/ml
PROCEDURE
Pippetted out 0.2 ml of sample to the test tube and added 0.8 ml of sodium
hydroxide solution and 5 ml of alkaline copper reagent. Shaken well and kept for 10 min.
After 10 min. 0.5 ml of Folins phenol reagent was added and mixed well. The mixture
was kept for another 30 min. The optical density was measured at 670 nm in
spectrophotometer. The system devoid of sample was used as the blank.
4.2.2.6.2 ESTIMATION OF URONIC ACID
REAGENTS
0.025 M sodium tetra borate in concentrated sulphuric acid
0.125% carbazol in methanol
Glucouronolactone standard
: (40 g/ml)
PROCEDURE
5 ml of sodium tetraborate was taken in a test tube and cooled to 40C by keeping it
in ice bath.1 ml of the sample was layered over this and stirred thoroughly with a glass
rod. It was allowed to attain room temperature and then heated in a boiling water bath for
10 min. After cooling to room temperature, 0.2 ml of carbazole was added and heated
again for 15 min. in a boiling water bath. It was then cooled and the optical density was
read at 530 nm.
67
4.2.2.7 CHEMICAL ANALYSIS OF FRACTION C2
The peptide content of fraction C2 was determined by Lowry et al. (1951) method.
The pigment content was measured by weighing the residue after hydrolysis with 6 N
HCl. Sugar content was measured by the difference. Amino acids were quantified by
HPLC as follows. A known weight of the peptidoglycan was hydrolysed with 6 N HCl at
1100C for 24 h in a sealed tube under nitrogen, after cooling, the solution was vacuum
filtered through Whatmann No. 42 filter paper and flash evaporated. The flash
evaporation was repeated after adding distilled water till HCl was removed. To the dry
residue 0.05 N HCl was added and made up to a known volume. An aliquot of the
solution was derivatised using Waters AccQ. Tag reagent kit and HPLC was run in a
Perkin Elmer System using RC183 Column, setting the absorbance at 250 nm.
4.2.2.8 PAPAIN DIGESTION OF FRACTION C2 AND ISOLATION OF TOTAL
POLYSACCHARIDE
A known weight of the peptidoglycan was subjected to digestion with papain in
0.1 M phosphate buffer (pH 6.5) containing 0.005 M EDTA and 0.005 M cysteine HCl
for 48 h at 650C. Fresh papain was added every 16 h. The papain digest was deproteinised
with trichloroaceticacid (final concentration of TCA 10%) and the supernatant dialysed
till free of TCA. Total polysaccharide (especially glycosaminoglycans) was precipitated
from the solution by the addition of 4-5 volumes of 95% ethanol containing 1-2%
potassium acetate. The precipitate collected by centrifugation was dissolved in known
volume of water and lyophilized. The antitumour activity of the polysaccharide thus
obtained was studied against DLA in BALB/c mice.
68
4.2.2.9 MEASUREMENT OF ABSORPTION SPECTRUM OF PIGMENT
PRESENT IN THE PURIFIED PEPTIDOGLYCAN
The pigment isolated from the purified peptidoglycan was further subjected to
carotenoid extraction. For this a known weight of the pigment was dissolved in 13.5 ml
of acetone and mixed with 5 ml of petroleum ether (Boiling point 40-600C) and 3.7 ml of
NaCl solution (0.73%) in a separating funnel. Phase separation was allowed, epiphase
was recovered into a round flask and an equal volume of water was added to the
remaining solution. After mixing and phase separation, the epiphase was again recovered
into the same flask and dried under vacuum in a rotary evaporator at 400C. The extracts
were then redissolved in acetone and absorption spectrum was measured at 380-600 nm
by UV visible spectrophotometer (Jasco, Japan).
4.3 RESULTS
4.3.1 PREPARATION OF PEPTIDOGLYCAN FRACTIONS
The cuttlefish ink extract with Tris-HCl buffer (pH 6.8) was fractionated using a
DEAE Sephacel ion exchange column. Equilibrated with Tris-HCl buffer (pH 6.8) and
then step wise elution with increasing concentration of NaCl was carried out. The elution
pattern is given in figure 4.2. Three fractions A, B and C were obtained, of these the
largest amount of material was found in fraction C (87 mg) eluted with 0.2 M NaCl. The
three fractions were dialysed and lyophilized and they were tested for antitumour activity
against DLA in BALB/c mice as described in chapter 5. Of the three fractions, fraction C
showed antitumour activity and this fraction was further fractionated using gel filtration
chromatography using Sephacryl S-300 column. The figure 4.3 shows the elution patterns
which give two peaks C1 and C2. These two fractions were dialysed and lyophilized and
Ion exchange chromatography
0.8
C
0.6
0.4
B
absorbance
215
199
182
125
102
40
29
0.2
0
20
absorbance
A
Tube no.
Fig.4.2: DEAE Sephacel fractionation of Cuttlefish ink Extract.
Table 4.1: Composition of different fractions
Fraction
Protein %
Uronic acid %
Crude preparation
34.68
24.66
(Tris-HCl extract)
C
29.69
21.03
C1
5.19
12.94
C2
5.64
37.74
Table: 4.2: Chemical analysis of fraction C2
Fraction
Sugar
Peptide
Pigment
C2
84.36%
5.64%
10%
69
tested for antitumour activity against DLA in BALB/c mice. Of the two fractions fraction
C2 showed strong antitumour activity.
4.3.2 SDS PAGE
The electrophoretic pattern obtained by SDS PAGE of Tris-HCl extract of the
cuttlefish ink and the purified peptidoglycan fraction (fraction C2) is shown in figure 4.4.
The results showed that a number of bands were visible in the crude extract of cuttlefish
ink, where as no band was observed in purified fraction C2.
The molecular mass of the purified peptidoglycan fraction C2 was estimated to be
10,000 Daltons by sephacryl S-300 gel chromatography calibrated with authentic
dextrans as standards.
4.3.3. CHEMICAL COMPOSITION OF DIFFERENT FRACTIONS
The crude preparation obtained by Tris-HCl extraction of cuttlefish ink,
fraction C obtained by ion exchange chromatography and fractions C1 and C2 obtained
by gel filtration of fraction C were analysed for protein and uronic acid contents. The
results are shown in table 4.1.
The results showed that the protein content of the crude preparation was highest
and it decreased as the purification of the peptidoglycan was carried out. The fraction C2
had the least amount of protein and this fraction had the highest amount of uronic acid.
The results thus indicated that the fraction C2 was probably a peptidoglycan rich in uronic
acid. The purified peptidoglycan fraction C2 was then analysed for its polysaccharide,
peptide and pigment content. The results are shown in table 4.2.The results showed that
the fraction C2 is a peptidoglycan containing small amount of pigment.
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
C2
absorbance
22
20
18
16
14
12
10
8
6
C1
0
absorbance
Gel filtration chromatography
tube no.
Figure 4.3: Fractionation of fraction C using Sephacryl S- 300 Gel filtration column.
FIGURE 4.4. SDS-PAGE
SDS PAGE of Tris HCl extract and C2 fraction from the ink of Cuttlefish,
Sepia pharaonis.
T-Tris HCl extract
C2 - Fraction C2
70
HPLC analysis of the peptide fraction showed that it is made up of five major
amino acids namely aspartic acid, serine, threonine, glutamic acid and alanine.
The pigment obtained from the peptidoglycan fraction C2 was subjected to
carotenoid extraction and the absorption spectrum of the extract was measured. The
results showed that the carotenoids present in the extract had 3 absorption peaks at 400,
474 and 512 nm.
71
4.4 DISCUSSION
Crude melanin obtained from the ink bags of squid, Ommastrephes bartrami were
fractionated using gel filtration on Sephadex G100 column, which contained a
melanoprotein, composed of melanin pigment (90%), protein (5.8%), and carbohydrate
(0.8%). Chemical analysis revealed that amino acids such as glycine, aspartic acid and
glutamic acid were present in large amounts and there was a trace amount of sulphur
containing amino acids (Mimura et al., 1982a). In the present study the cuttlefish ink was
extracted with Tris-HCl buffer and fractionated using DEAE Sephacel ion exchange
column. Three fractions (A, B and C) obtained were dialysed and lyophilized and they
were tested for antitumour activity against DLA in BALB/c mice. The fraction C which
showed antitumour was further fractionated into two fractions C1 and C2 by gel filtration.
Molecular mass of the fraction C2 was determined which was found to be 10 kDa. Further
analysis of this fraction revealed that the protein content of the crude preparation was
highest and it decreased as the purification of the peptidoglycan was carried out. The
fraction C2 contained a uronic acid rich polysaccharide which formed 84.4% of the total
mass. The peptide part was 5.6% and the rest was pigment. On SDS–PAGE of fraction
C2, no bands were observed, possibly due to higher sugar content. The results thus
indicated that the fraction C2 is probably a peptidoglycan rich in uronic acid. HPLC
analysis of the peptide part showed that it is made up of five amino acids aspartic acid,
serine, threonine, glutamic acid and alanine in the cuttlefish. Takaya et al. (1994b) had
showed that peptide part of a peptidoglycan from squid ink also consists of same
aminoacids. The pigment obtained from the peptidoglycan fraction C2 was subjected to
carotenoid extraction and the absorption spectrum of the extract has shown three peaks.
72
Thus a peptidoglycan containing 84.4% uronic acid rich polysaccharide, 5.6% peptide
part and 10% pigments (carotenoids) was isolated and purified from the ink of the
cuttlefish, Sepia pharaonis. The anticancer property of the purified peptidoglycan was
studied and the results are discussed in the subsequent chapters.