CHAPTER II

MATERAILS AND METHODS

2.1 Chemicals

Bovine trachea cartilage chondroitin sulfate A was obtained from

Biochemika–Fluka Chemie AG CH-9471 (Buchs, Switzerland). Six unsaturated

chondroitin sulfate disaccharide standards were purchased from Seikagaku Corp., Ltd.

(Tokyo, Japan). Chondroitinase ABC (EC 4.2.2.4) from Proteus vulgaris and

Hyaluronidase (EC 3.2.1.35) from Sheep testes were purchased from Sigma–Aldrich

Chemical Corp., Ltd. (Missouri, USA). Recombinant human (rh) pleiotrophin (PTN),

rh-vascular endothelial growth factor (VEGF), and rh-hepatocyte growth factor

(HGF) were obtained from Sigma–Aldrich Chemical Corp., Ltd. (Missouri, USA).

Bio-Gel P-6 resin was obtained from Bio-Rad. The 2-aminobenzamide (2AB) and

sodium cyanoborohydride (NaCNBH3) were obtained from Sigma–Aldrich Chemical

Corp., Ltd. (Missouri, USA). Sephadex G-100 resin was obtained from Amersham

Bioscience Corp., Ltd. (UK). Other chemicals and materials used in this study were

shown in Appendix A. All other reagents and chemicals were of the highest quality

available.

2.2 Preparation of materials

2.2.1 Gel filtration chromatography

Chondroitin sulfate A 50 mg (from bovine trachea cartilage) was digested with

10 mg (approximately 15000 National formulary units; NFU) of sheep testicular

hyaluronidase (EC 3.2.1.35) in a total volume of 2.0 ml of 50 mM sodium phosphate

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buffer, pH 6.0, containing 150 mM NaCl (1 NFU corresponds to the amount of the

enzyme that hydrolyzes 74 µg of hyaluronate per min) at 37 ºC for 20 h, then an

additional 5 mg (approx. 7500 NFU) of the enzyme was added and the incubation

proceeded for 10 h to complete the digestion. Proteins were precipitated by heat at

100 ºC and removed by centrifugation at 3,000 rpm for 10 min. The sample was

fractionated on a Bio-Gel P-6 column (1.6×100 cm), using 0.2 M ammonium acetate

as an eluent, at a flow rate of 9 ml/h. The eluate was monitored by measuring the

absorbance at 210 nm, which is mainly due to the carbonyl groups. Major peaks were

pooled separately, concentrated, and desalted by evaporated. Each peak was

quantified by the carbazole procedure using GlcA as a standard (Bitter and Muir,

1962). The column was calibrated with blue dextran and phenol red for the void

volume (Vo) and the total bed volume (Vt), respectively.

2.2.2 High–Performance Liquid Chromatography (HPLC)

The reaction products were fractionated into fraction I-VII by gel filtration on

a Bio-Gel P-6 column (1.6×100 cm). The smallest fractions, fraction V-VII, were

subfractionated by anion-exchange HPLC on a short cartridge column (Mono-Q

column, 5 ml, Amersham Biosciences) of strong anion exchange (SAX), using a

linear gradient of LiClO4 (solvent A; deionized distilled water and solvent B; 0.25 M

LiClO4) from 0 to 100% solvent B over 60 min at a flow rate of 1.0 ml/min at room

temperature. The eluate was monitored by absorbance at 210 nm using a

SpectraSYSTEM™ UV1000 detector (Thermo Electron Crop.). Major peaks were

purified by rechromatography under the same conditions as above and desalted

through a column (1.6×5 cm) of Sephadex G-25 (medium) using distilled water as an

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eluent. Each peak was quantified by the carbazole method using GlcA as a standard

(Bitter and Muir, 1962).

2.2.3 Derivatization of reduced oligosaccharides with 2AB

The derivatization of oligosaccharides with 2AB was performed essentially as

described by Bigge et al. (1995). Briefly, 1.0 nmol of a given oligosaccharide was

lyophilized in a microcentrifuge tube. An aliquot (5 µl) of a derivatizing reagent

mixture (0.35 M 2AB/1.0 M NaCNBH3/30% (v/v) acetic acid in dimethyl sulfoxide)

was added to the oligosaccharide sample, and the mixture was incubated at 65 ºC for

2 h. An excessive 2AB will be removed by repeated chloroform extraction.

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Figure 2.1 Schematic representation of saccharide fluorotagged by reductive

amination with 2AB and sodium cyanoborohydride. In this reaction, a

Schiff’s base is formed between the aldol group of the reducing

saccharide and the amino group of a fluorotag, and this bond was then

reduced to the stable secondary amine linkage to complete the formation

of the fluorotagged products.

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2.3 Analytical methods

2.3.1 Disaccharide composition analysis using 2AB labeling and HPLC

Major fractions (fraction from gel filtration chromatography and SAX

subfractions, 0.5 nmol each) were individually incubated with 10 mIU of

chondroitinase ABC (EC 4.2.2.4) in a total volume of 20 µl of 250 mM Tris-HCl

buffer, pH 7.3 at 37 ºC for 60 min. Each digest was labeled with a fluorophore 2AB

and analyzed by anion-exchange HPLC on an amine-bound silica PA-03 column

(4.6×250 mm; YMC Corp., Kyoto, Japan) eluted with a linear gradient of NaH2PO4 at

a flow rate of 1 ml/min at room temperature for the separation of CS disaccharides

(Sugahara et al., 1994; Sugahara et al., 1989; Yoshida et al., 1989). Samples prepared

as described above were diluted using 16 mM NaH2PO4 containing 5 pmol of each

oligosaccharide in 50 µl. Eluates were monitored using a SpectraSYSTEM™ FL3000

fluorescence detector (Thermo Electron Corp.) with excitation and emission

wavelengths of 330 and 420 nm, respectively. Unsaturated oligosaccharides unlabeled

with 2AB were monitored by absorbance at 232 nm using 300 pmol aliquots.

2.3.2 Molecular mass determination

The molecular mass of CS-A oligosaccharides were determined by gel

filtration using a column of Sephadex G-100 (1.6×40 cm) calibrated with molecular

mass markers including dextran preparations (average mass: 8, 18.1, and 37.5 kDa)

and Phenol red (average mass: 254.4 Da). Vo and Vt were determined using dextran

preparations (average mass: 200 kDa) and NaCl, respectively. CS-A oligosaccharides

(20 µg) were loaded onto the column and eluted with 0.2 ammonium acetate at a flow

rate of 0.5 ml/min, and the fractions collected at a 2-min interval, evaporated to

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dryness, and reconstituted in 100 µl of water. An aliquot was taken for estimating

GAG using 1,9-dimethylmethylene blue (DMMB) according to the method of

Chandrasekhar et al. (1987) except that the absorbance was read at 540 nm.

2.3.3 Protein assay

Protein concentration was determined using the bicinchoninic acid (BCA)

protein assay (Smith et al., 1985). BSA standards (0-1000 µg/ml) and samples were

added to the microtitre plates (20 µl/well) in duplicate. Copper (II) sulfate

pentahydrate 4% (w/v) and bicinchoninic acid solution were mixed together (1:50)

and added to each well (200 µl/well). The plates were incubated at 37 ºC for 30 min

and absorbance was measured at 570 nm. Protein concentrations were determined

from a standard curve.

2.3.4 Dye binding assay

The sulfated glycosaminoglycan (S-GAG) concentrations were determined

using a colorimetric dye binding assay modified from Ratcliffe et al. (1988). The

assay is based on a metachromatic shift in absorption maximum from 600 nm to 535

nm when a complex is formed between a mixture of 1,9-dimethylmethylene blue and

the S-GAG in the sample or standard. The dye solution was made by adding a 16 mg

of 1,9-dimethylmethylene blue dissolved in 5 ml ethanol to 2 g of sodium formate and

2 ml of formic acid in a total volume of 1 liter at pH 3.5. The maximum absorbance of

the dye solution was at 590 nm. This solution was stored at 4 ºC in a dark bottle.

Chondroitin 6-sulfate (CS-C) standards (0-30 μg/ml: 50 μl) or samples (50 μl) were

transferred to a microtitre plate. The dye solution (200 μl) was added immediately to

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each sample and standard, and the absorbance was measured at 525 nm. A standard

curve was plotted by using the standard data and the sample CS-C concentration was

then calculated from a standard curve.

2.3.5 Determination of uronic acid equivalents

Glucuronic acid (GlcA) concentrations were determined by the colorimetric

assay using m-hydroxydiphenyl sulfuric acid assay (Bitter and Muir, 1962). GlcA was

used as a standard. Unsaturated uronic acid was quantified by spectrophotometry

based on an average millimolar absorption coefficient of 5.5 at 232 nm caused by the

∆4,5 sites of the uronic acid at the non-reducing ends (Yamagata et al., 1968).

2.3.6 Determination of neutral sugars

Since dextran is not sulfated, the elution of it on Sephadex G-100 is monitored

by estimating neutral sugars by the colorimetric assay using orcinol method

(Bruckner, 1955). To 80 µl of each sample, 200 µl of a reagent A and 1.2 ml of a

reagent B were added. After heating at 80 ºC for 20 min, the samples were cooled at

room temperature and the absorbance was measured by spectrophotometer at 470 nm.

2.3.7 Polyacrylamide gel electrophoresis (PAGE)

The electrophoresis method was adapted from Cowman et al. (Cowman et al.,

1984) for standard mini-gels. Concentrated (2x) Tris-Borate-EDTA buffer stock (0.18

M Tris/0.18 M boric acid/0.005 M EDTA buffer, pH 8.3) was prepared and stored at

4 ºC. The acrylamide stock solution contained 30% (w/v) acrylamide and 1% (w/v)

N,N'-methylenebisacrylmide. Equal volumes of the buffer and the acrylamide stock

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solution were mixed together. Ammonium persulfate [10% (w/v); 500 µl] and

TEMED (50 µl) were added to the 100 ml of the acrylamide mixture. A gel

(16×20×0.15 cm) was cast immediately, and allows standing to polymerize for up to 3

h at room temperature.

Chondroitin sulfate chains (3-5 µg) or digested samples (containing 20 µg by

DBA of initial chondroitin sulfate chains) were mixed with 20 µl of sample loading

buffer [0.01% (w/v) bromophenol blue in 60% (w/v) sucrose] before loading on the

gel. The running buffer was (1X) Tris-Borate-EDTA, pH 8.3. The gels were

electrophoresed at 150 volt for 3 h. The gel were then stained using a combined alcian

blue-staining method.

2.3.8 Alcian blue staining for polyacrylamide gels

Alcian blue dye is a copper phthalocyanine derivative containing multiple

cationic substituent groups. The dye precipitates and immobilizes GAGs within the

polyacrylamide matrix, there by serving both as a fixative and staining (Cowman et

al., 1984). The sensitivity of dye is in microgram range. Gels were stained according

to the methods of Moller et al. (1993). Gel were stained according to the methods

which shown in Table 2.1.

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Table 2.1 Method for successive staining with alcian blue and neutral silver of

polyacrylamide gel after gel electrophoresis (Moller et al., 1993).

Step Solution Preparation Time

(min)

Temp

(ºC)

1 Fix/Wash

I 25%(v/v) Ethanol, 10%(v/v) Acetic acid in water 5 50

2 ” ” 10 50

3 ” ” 15 50

4 Stain I 0.25%(v/v) Alcian blue in fix/wash I 75 50

5 Fix/Wash

I 25%(v/v) Ethanol, 10%(v/v) Acetic acid in water 30 50

6 ” ” 30 50

7 ” ” 30 50

8 ” ” O/N 50

9 Wash II 10%(v/v) Ethanol, 5% (v/v) Acetic acid in water 10 50

10 ” ” 10 50

11 Sensitizer 5%(v/v) Glutaraldehyde 50 50

12 Wash II 10%(v/v) Ethanol, 5% (v/v) Acetic acid in water 5 50

13 ” ” 10 50

14 Wash III Water 5 50

15 ” ” 5 50

16 Stain II 0.4%(w/v) Silver nitrate 65 50

17 Wash III Water 5 50

18 ” ” 5 50

19 Developer 2.5%(w/v) Sodium carbonate, 0.013%(v/v)

Formaldehyde 2 50

20 Stop 10%(v/v) Acetic acid, 10%(v/v) Glycerol 10 50

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2.4 Examination of specific interaction of CS-A oligosaccharide preparations

2.4.1 Determination of CS-E binding to pleiotrophin by BIAcore™ 2000 system

To study whether isolated oligosaccharides could bind to pleiotrophin which is

an important cytokine or not, this experiment will be designed by using BIAcore™

2000 system. From previous study, it is known that CS-E could bind to various

heparin binding growth factors including pleiotrophin (Deepa et al., 2002). Therefore,

to determine the binding to pleiotrophin of CS-A oligosaccharide preparations, firstly,

CS-E will be injected onto the pleiotrophin sensor chip, which was used as positive

control, at flow rate of 10 µl/min and given a period of 3 minutes for the association

phase and 2 minutes for the dissociation phase. The sensorgrams will be shown as a

resonance unit (RU).

2.4.2 Immobilization of various growth factors on the CM5 sensor chip

The binding of CS-A oligosaccharide preparations to various recombinant

human growth factors were evaluated in the BIAcore™ 2000 system (BIAcore AB),

which had been equilibrated with the running buffer (10 mM HEPES-NaOH, pH 7.4,

containing 0.15 M NaCl, 3 mM EDTA and 0.005% Tween 20) at a flow rate of 10

µl/min. Firstly, all growth factors were immobilized on the surface of a CM5 sensor

chip by amine coupling, where the primary amino groups on the protein were coupled

to the carboxymethylated dextran on a sensor surface. The carboxymethylated dextran

surface on the sensor chip was activated by injection of 40 µl of a mixture [1:1; (v/v)]

of 200 mM N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and 100

mM N-hydroxysuccinimide at a flow rate of 5 µl/min. An 80 µl aliquot of the various

recombinant human growth factors (50 µg/ml in 10 mM acetate/NaOH buffer, pH

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5.08) was then injected at a flow rate of 10 µl/min onto the activated sensor surface.

The remaining activated N-hydroxysuccinimide ester groups were blocked by

injecting 40 µl of 1 M ethanolamine hydrochloride/NaOH, pH 8.5, and washed with

10 µl of 1 M NaCl.

2.4.3 Interaction analysis of CS-A oligosaccharides by BIAcore™ 2000 system

Binding assays were performed at 25 ºC with a constant flow rate of 10 µl/min

in both association and dissociation phases. For the kinetic analysis, the CS-A

oligosaccharide preparations in a series of concentrations ranging from 15.0 to 30.0

µg/ml in the running buffer, pH 7.4 (HBS-EP; BIAcore AB), containing 10 mM

HEPES-NaOH, 0.15 M NaCl, 3 mM EDTA, and 0.005% Tween 20, were injected

onto the flow cell, and the changes in resonance units were recorded. After each run,

regeneration of the sensor chip surface was accomplished by an injection of 10 µl of 1

M NaCl. The kinetic parameters were evaluated with BIAevaluation software 3.1

(BIAcore AB) using a 1:1 binding model. Association rate constants (ka) were

calculated from the linear portion of sensorgrams during the early association phase.

Dissociation rate constants (kd) were calculated from the early portion of the

dissociation phase after the completion of the sample injection during the washout

period. The apparent equilibrium dissociation constant (Kd) was calculated as the ratio

kd/ka. The kinetic constants were determined by three or more independent

experiments.

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Figure 2.2 An example of a sensor surface designed for direct, covalent

immobilization of interacting partners. The carboxymethyl groups are

open to covalent amine, thiol or aldehyde coupling. Although BIAcore

assays are ostensibly solid phase, flexible dextran linkers provide a

scaffold on which interactions proceed under conditions that closely

mimic a fluid environment, allowing the immobilized partner

considerable freedom of movement.

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2.5 Molecular docking

2.5.1 Coordinates

The X-ray crystallographic structure of midkine (PDB code 1MKN) was

obtained from the Protein Data Bank. The hetero atoms (cofactors, water molecules,

and ligands) were removed and polar hydrogens were added. The Kollman unit-atom

charges were assigned to protein atoms using Auto Dock Tool 1.4.6 program.

2.5.2 Ligand preparations

For docking experiments with AutoDock 3.0, ligand molecules were drawn,

optimized, and saved as in pdb format with the aid of HyperChem 6.0 program. Full

hydrogens were added to the ligands and Gasteiger partial atomic charges were

computed using the AutoDock Tool 1.4.6 program and saved in the required format.

All possible flexible torsions of the resultant ligand molecules were defined by using

AUTOTORS. The prepared ligands were used as input files for AutoDock 3.0.5 in the

next step.

2.5.3 Docking simulations

The automated molecular docking simulations were performed with AutoDock

3.0.5 using a Lamarckian genetic algorithm (LGA). The standard docking procedure

was used for a rigid protein and a flexible ligand whose torsion angles were identified

(for 100 independent runs per ligand). A grid size was ser to 60×65×70 points in x, y,

and z directions were built, with a grid spacing of 0.375 Å centered at the center of

mass of the ligand. Each LGA job consisted of 100 runs, and the number of

generation in each run was 27,000 with an initial population of 50 individuals. The

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default settings were used for all other parameters. At the end of docking, ligands with

the most favorable free energy of binding were selected as the resultant complex

structures. All calculations were carried out on PC based machines running Linux as

operating systems. The resultant structure files were analyzed using WebLab

ViewerPro 4.0 program.

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Figure 2.3 The X-ray structure of midkine (MK62–104) (PDB code 1MKC). The

ribbon structure is protein backbone. The balls and sticks is amino acid

side chains.