Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides

Atsushi Suzuki, Hidenao Toyoda, Toshihiko Toida and Toshio Imanari1

Faculty of Pharmaceutical Sciences, Chiba University, 1–33, Yayoi, Inage, Chiba 263–8522, Japan

Received on June 14, 2000; revised on August 25, 2000; accepted on August 30, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Hyaluronan was partially depolymerized on a large-scale quantity using bacterial hyaluronidase (E.C. 4.2.2.1) for preparation of chemically fully O-sulfated oligosaccharides. The hyaluro-oligosaccharide (HAoligo) mixture obtained by partial digestion was repeatedly applied to low pressure gel permeation chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer. The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance (1H NMR) spectroscopy, capillary electrophoresis (CE) on normal polarity mode, and a newly established separation method by normal phase chromatography with Amide-80 column. The purified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulfation. Characterization of chemically fully O-sulfated HAoligos was performed by both chemical compositional analyses after hydrolysis and 1H NMR spectroscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAoligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase (bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ranged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfated hyaluronan (MW 100 kDa) through both competitive and noncompetitive effects.

Key words: HA oligosaccharides/hyaluronidase/NMR/CE/FIA/inhibition of hyaluronidase/chemically oversulfation/anti-coagulant activity


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Glycosaminoglycan (GAG) hyaluronan (HA) is a linear homogeneous polymer composed of N-acetyl-D-glucosamine and D-glucuronic acid and especially found as an unsulfated GAG. Generally, HA is found in the extracellular matrix and is well know that HA is important for the maintenance of tissue architecture (Laurent and Fraser, 1992; Fraser et al., 1997Go). At last decade, HA was supposed to be concerned with morphogenesis (Gakunga et al., 1997Go), regeneration (Wang et al., 1998Go), wound healing (Chen and Abatangelo, 1999Go) and cancer metastasis (Lokeshwar et al., 1997; Hayen et al., 1999Go; Itano et al., 1999Go); however, the biological and physiological functions of HA have not been elucidated. Interestingly, there have been several evidences that some of these functions mentioned above might be related to the molecular size of HA. Especially, high molecular weight HA was demonstrated enhancing cell growth in culture (Brecht et al., 1986Go; Yoneda et al., 1988Go) and anti-angiogenesis (Deed et al., 1997Go). On the other hand, the strong inhibitory activity of low molecular weight HA on angiogenesis has been reported (Deed et al., 1997Go). While the study was performed by using a mixture of depolymerized HA fraction, the effect of the sizes of HA on the regulation of angiogenesis was still unclear. Therefore, the preparation of size-uniformed HAoligos might be very important to investigate the physiological and biochemical functions of HA.

There are several methods for separation of HAoligos by HPLC. High performance liquid chromatography (HPLC) methods including gel-filtration, reverse-phase ion pair (Cramer and Bailey, 1991Go) and pellicular anion-exchange columns (Holmbeck and Lerner, 1993Go) have been used for the quantitative analysis of HA and for preparation of HAoligos. Capillary electrophoresis (CE) methods have also been established for determination of sizes of HA oligosaccharides (Stephen et al., 1991Go; Kakehi et al., 1999Go); however, it is impossible to prepare HAoligos in milli/microgram scale.

Chemical modification of HA such as O-sulfation has also been performed to create new medicines by many laboratories. Recently, anti-coagulant activity of fully O-sulfated GAGs including HA has been shown as a result of conformational change of glucuronic acid residues by O-sulfation (Maruyama et al., 1998Go; Toida et al., 1999aGo). Furthermore, fully O-sulfated HA has shown the strongest inhibitory activity against bovine testicular hyaluronidase among the fully O-sulfated GAGs (Toida et al., 1999bGo). In these studies, not only the sulfation degree but also the conformation of core structure and the sizes of the molecules might be important for the activities.

Based on these observations, this paper described an approach to (1) establishment of the preparation method for 4- to 20-mer HAoligos by low pressure gel permeation chromatography (LPGC) including optimization of the depolymerization condition for 4- to 20-mer; (2) establishment of the analytical method to demonstrate the purity of HAoligos using both CE and HPLC; (3) preparation of chemically fully O-sulfated 4- to 20-mer HAoligos to investigate the effects of size for the anti-coagulant activity and inhibitory action on hyaluronidase.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Preparation of HAoligos
Hyaluronan (HA) was depolymerized using bacterial hyaluronidase (lyase, E.C.4.2.2.1) to obtain HAoligos (4- to 20-mer) mixture. Although each oligosaccharide derived by this enzymatic treatment contains unusual double bond at the non-reducing end uronate, it might be advantageous to detect these oligosaccharides at UV 232 nm on purification procedures. The hyaluronidase digestion was achieved at 48 h by determined the separation pattern of HAoligos using HPLC with a gel filtrate Asahipak GF-510HQ column. The 40% digestion calculated by the absorbance at 232 nm thought to be the best source for HAoligo preparation, based on the HPLC chromatographic pattern (data not shown). The resulting powder (100 mg) for preparative scale was dissolved in 20 ml of water and was size-fractionated by low pressure gel permeation chromatography (LPGC) of a Sephadex G-50 column eluted with 0.2 M sodium chloride (Figure 1). Each size-uniformed fraction was collected concentrated, and desalted by a Hi-Trap Desalting column eluted with water. The fractions containing HAoligos were collected and freeze-dried. The purity of each HAoligo was analyzed using both capillary electrophoresis (CE) and HPLC with Amide-80 shown in Figures 2 and 3, respectively. The results obtained by both the CE and HPLC methods clearly show that each oligosaccharide ranged 4 to 20 mer was isolated more than 90% purity as shown in Table I.



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Fig. 1. Chromatographic profile of HAoligos mixture produced by partial bacterial hyaluronidase digestion. Each plot was depicted as absorbance at 232 nm.

 


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Fig. 2. Capillary electropherogram of hyaluro-oligosaccharide mixture produced by partial bacterial hyaluronidase digestion.

 


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Fig. 3. HPLC profile on an Amide-80 column of HAoligo mixture produced by partial bacterial hyaluronidase digestion.

 

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Table I. Purity of HAoligos from 4- to 20-mers analyzed by CE, HPLC, and proton NMR spectroscopy
 
High magnetic field (500 and 600 MHz) 1H NMR spectroscopic analysis was used to characterize each purified HAoligo (Toida et al., 1993Go). To confirm the sizes of HAoligos, one- (1D) and two- dimensional (2D) 1H NMR experiments were applied. Figure 4 shows the 1D 1H NMR spectra of purified HAoligo 20-mer. Based on 2D multiple-quantum filtered COSY and TOCSY experiments, all of the signals corresponding to internal GlcNAc and GlcNAc at the reducing end, internal GlcA and unsaturated uronate, which is located at the non-reducing end of each HAoligo, were assigned as shown in Figure 4. It might be difficult to measure molecular sizes of intact GAGs by 1H NMR because there is no landmark for suggesting their molecular weight (Pervin et al., 1995Go). On the contrary, there is a clear internal standard for molecular sizes in each HAoligo produced by bacterial hyaluronidase. Namely, the integration value of the H-4 signal of uGlcA at 5.88 p.p.m. can be used as an internal standard for the molecular size of each HAoligo.



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Fig. 4. One-dimensional 1H NMR spectrum of 20 mer hyaluro-oligosaccharide. GlcA, internal glucuronic acid residue; R-GlcA, glucuronic residue acid next to N-acetyl glucosamine residue at the reducing end; uGlcA, unsaturated glucuronic acid residue; GlcNAc, N-acetyl glucosamine residue.

 
Preparation of fully O-sulfated HAoligos
The optimum conditions for chemical O-sulfation reaction of GAGs were described previously (Maruyama et al., 1998Go; Toida et al., 1999aGo). The gradient PAGE analysis of O-sulfated HAoligos in Figure 5 clearly shows the purity of each oligosaccharide and indicates that none of HAoligos was depolymerized under the condition for O-sulfation of HAoligos. Interestingly, a band corresponding to the migration position of a dimmer of each O-sulfated HAoligo was detected on the PAGE gel (see Figure 5). While these bands were observed by PAGE, there was no peaks corresponding to dimmers by the HPLC. The isolation and characterization of these bands are under going and will be published elsewhere.



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Fig. 5. PAGE analysis of O-sulfated HAoligos. Lanes: a, 10-mer; b, 12-mer; c, 14-mer; d, 16-mer; e, 18-mer; f, 20-mer. Asterisk (*) indicates a faint stained band in each lane. Each band was stained by Alcian blue.

 
The data obtained by compositional analyses of inorganic sulfate and GlcN residues after acid hydrolysis of O-sulfated HAoligos suggest that most of hydroxy groups (96–100%) in O-sulfated HAoligos were sulfated as shown in Table II.


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Table II. Compositional analyses of O-sulfated HAoligos and their anti-IIa activity and IC50 on hyaluronidase
 
Double quantum filtered chemical shift correlated spectroscopy (DQF COSY) spectrum of O-sulfated HAoligo 18 mer is shown in Figure 6. Triple quantum filtered (TQF) COSY and TOCSY experiments were performed to confirm the assignments for H-5 and H-6 protons of GlcNAc and total connectivity of GlcA and GlcNAc residues, respectively (data not shown). All of the ring protons were downfield shifted by O-sulfation compared to that of intact unsulfated HAoligo 18 mer.



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Fig. 6. Two-dimensional DQF COSY spectrum of O-sulfated 18-mer. Cross peaks: a, GlcNAc(H-1/H-2) and rGlcNAc(H-1/H-2); b, GlcNAc(H-2/H-3), rGlcNAc(H-2/H-3), GlcNAc(H-3/H-4) and rGlcNAc(H-3/H-4); c, GlcNAc(H-4/H-5); d, GlcNAc(H-5/H-6); d',GlcNAc(H-5/H-6); e, rGlcNAc(H-4/H-5); f, rGlcNAc(H-5/H-6); g, uGlcA(H-2/H-3); h, uGlcA(H-3/H-4); i, GlcA(H-1/H-2); j, GlcA(H-2/H-3) and GlcA(H-3/H-4); k, GlcA(H-4/H-5).

 
Biological activity of O-sulfated HAoligos
Inhibitory actions of the prepared O-sulfated HAoligos on blood coagulation and hyaluronidase activity were investigated. The anti-coagulant activity of all O-sulfated HAoligos through anti-IIa activity, contrary to our expectation based on previous data (Toida et al., 1999aGo), was less than 3.1 units/mg as shown in Table II. The O-sulfated HAoligos ranged 4–8 have shown no significant anti-coagulant activity (data not shown). The activity of O-sulfated polymer HAs (M.W. 20,000 and 130,000) were 77.8 units/mg and 72.7 units/mg, respectively.

Because the inhibitory action of fully O-sulfated GAGs including HA on bovine testicular hyaluronidase has been described previously by using a new flow injection assay for the enzyme (Toida et al., 1999bGo), the activity of O-sulfated HAoligos was also investigated. As shown in Figure 7, the activity is increasing according to the size of O-sulfated HAoligos. Table II shows the IC50 on hyaluronidase of O-sulfated HAoligos and O-sulfated intact HA. Although the O-sulfated HAoligos ranged 10- to 20-mers did not show 100% inhibition activity at 50 µg/ml, the IC50 value of 20-mer O-sulfated HAoligos was 3.54 µg/ml, which is corresponding to nearly 80% of the activity of O-sulfated intact HA polymers. The small O-sulfated HAoligos 4~8 mer did not show any significant inhibitory activity on hyaluronidase (data not shown). Figure 8 depicts the Lineweaver-Burk plot of the inhibition of heparin and fully O-sulfated HAoligo 20 mer on hyaluronidase. Heparin was found to inhibit hyaluronidase activity non-competitively, while fully O-sulfated HAoligo 20-mer was found to inhibit hyaluronidase through both competitive and noncompetitive effects.



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Fig. 7. Inhibition of hyaluronidase activity by O-sulfated HAoligos.

 


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Fig. 8. Lineweaver-Burk plot of inhibition of heparin and fully O-sulfated HAoligo 20-mer on hyaluronidase. Each point represents the mean value of triplicate. Bovine testicular hyaluronidase (50 TRU/ml) was used for the experiments. Open squares, untreated; solid circles, heparin; open circles, fully O-sulfated HA oligo 20-mer.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
There have been many reports on the anti-coagulant, anti-viral, and/or anti-inflammation activities of intact and chemically modified sulfated polysaccharides. Some of these compounds were applied for medicinal trial and low molecular weight heparin and sulfated xylane have already been introduced as anti-coagulant and anti-inflammation medicines in clinical field. We have also reported anti-coagulant and anti-hyaluronidase activities of chemically fully O-sulfated GAGs (Toida et al., 1999aGo,b). In those reports, we suggested that it might be very important to show these activities not only the sulfation degrees but also the sizes of core polysaccharides. Based on these observations, HAoligos ranged from 4- to 20-mers were purified and chemically O-sulfated to clarify the relationship between the biological activity and the size of HAoligos.

CE technique and a new HPLC separation method using Amide-80 column (Figure 3) performed analyses of the purified HAoligos. Especially, CE method was used to confirm the purity and sugar length of each purified HAoligo. CE represents an extremely high-resolution method establishing sample purity as described previously (Pervin et al., 1995Go). Although HAoligos were practically separated and prepared by LPGC in a large scale in this paper, the analytical separation of HAoligos on an Amide-80 column has suggested that the separation system might be suitable for the preparation of HAoligos in preparative scale. Additionally, 1H NMR spectroscopy was performed for the determination of the size of the purified HAoligos (Table I, Figure 4).

The reaction condition for chemical fully O-sulfation of GAGs was described previously (Maruyama et al., 1998Go; Toida et al., 1999aGo) and was improved for being suitable for fully O-sulfated HAoligos. The 1D 1H NMR spectra of fully O-sulfated 10- to 20-mer HAoligos were similar to that of fully O-sulfated intact HA polymer. Two-dimensional NMR experiments were also applied to confirm the structure of each modified HAoligo. Most of the signals of fully O-sulfated HAoligos were downfield shifted by O-sulfation, except the signals of H-1 and H-4 of uGlcA at the nonreducing end. The observation on shielding of H-1 and H-4 signals of uGlcA might be explained as the anisotropic effects of sulfate group and/or sugar moiety near to the non-reducing end.

While anti-coagulant activity of fully O-sulfated HAoligos was not significant, their inhibitory action on bovine testicular hyaluronidase was strongly depending on their sizes. Even though the size of fully O-sulfated HAoligo 20-mer is almost same as the molecular size of low molecular weight heparin (LMWHP), its anti-coagulant activity was much less weak than that of LMWHP. A possible reason for this observation might be explained that the anti-coagulant activity of LMWHP is shown through the binding with antithrombin III, which requires the specific sulfation pattern found in heparin/heparan sulfate. On the contrary, the activity of fully O-sulfated HAoligos must be through the interaction with heparin cofactor II, which may require non-specific large oversulfated sequence over 20-mer (Toida et al., 1999aGo).

Hyaluronidases are a family of ß-1,4-endoglucosaminidase that can afford to depolymerize HA into small oligosaccharides such as tetra- and hexasaccharides. A key role of this enzyme family has been supposed to be involved in a number of basic biological processes such as embryogenesis (Brown and Papaioannou, 1993Go; Meyer and Kreil, 1996Go), carcinogenesis (Knudson, 1996Go; Sherman et al., 1994Go), wound healing (Estes et al., 1993Go), angiogenesis (West and Kumer, 1989Go), and inflammation (Edelstam et al., 1992Go; Nobel et al., 1996Go). Pectin (Sawabe et al., 1992Go), glycyrrhizin (Fluruya et al., 1997Go), flabonoids (Li et al., 1997Go), and heparin (Wolf et al., 1984Go) are well known as the inhibitors of hyaluronidase. Inhibitor of hyaluronidase is expected to be a new medicine of anti-tumor and anti-inflammation. Inhibition activity of O-sulfated HA polymers on hyaluronidase is stronger than those of known inhibitors and other O-sulfated GAGs (Toida et al., 1999bGo). The IC50 values of O-sulfated HAoligos 18- and 20-mer were corresponding to nearly 80% of the IC50 of O-sulfated intact HA polymers. As a result of these experiments, the inhibitory action on hyaluronidase of fully O-sulfated HAoligos was appeared at the size of 16-mer. It may strongly suggest that the interaction between testicular hyaluronidase and O-sulfated HAoligos is caused not only by static electricity but also by the size of O-sulfated HAoligo. Additionally, it should be pointed out that the prepared HAoligos were damaged enzymatically on GlcA at the non-reducing end and N-acetyl-D-glucosamine at the reducing end, which exists both {alpha} and ß configurations in solution.

Heparin has a wide array of potential pharmacological uses as a medicine, however, the anti-coagulant activity of heparin limits its application for the clinical trials. The results obtained in this paper may suggest that fully O-sulfated HAoligos is one of the candidates for anti-hyaluronidase drug to prevent growth and metastasis of tumor cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Chemicals and instruments
Hyaluronan sodium salt (M.W. 20,000, 100,000 130,000) from Streptococcus zooepidemicus was purchased from Kibun Food Chemipha Co., Tokyo, Japan. Hyaluronidase from Streptomyces hyalurolyticus (lyase, E.C.4.2.2.1) for HAoligos preparation was purchased from Seikagaku Kogyo Co., Tokyo, Japan. Hyaluronidase (Hyaluroglucosaminidase from bovine testes, Type IV-S, E.C.3.2.1.35) was purchased from Sigma Chemical Co., USA. Chromogenic TH (tosyl-glycyl-prolyl-arginine-4-nitranilide acetate) and human thrombin were purchased from Boehringer Mannheim, Belgium. The USP heparin reference standard (172 units/mg) was supplied by the U.S. Pharmacopeial Convention, Rockville, MD. A column (4.4 cm I.D. x 1 m) for LPGC was purchased from Millipore Co., Germany. Sephadex G-50 (superfine) resin and a Hi-Trap desalting column were purchased from Pharmacia Biotech. Dialysis tubing (MWCO 500) was purchased from Wako, Japan. All other chemicals were analytical reagent grade. The gradient HPLC system to demonstrate the purity of HAoligos and the FIA was assembled with gradient pumps (Jasco 980-PU, intelligent HPLC pumps), an eluent mixer (Jasco HG-980-3, solvent mixing module) and a fluorescence-detector (Jasco FP-1520S intelligent fluorescence detector) from Nihon Bunko Co, Japan. A variable sample injector (VMD-350) was from Shimamura Instrument Co., Japan. A UV-detector (D-2500) was from Hitachi Seisakusho Co., Japan. A conductivity detector (CM-8) for Ion Chromatography and TSKgel Amide-80 resin was purchased from TOSOH Co., Japan. The CE system was assembled with Beckman capillary electrophoresis system (P/ACE 5010) equipped with a UV detector and an operation system using version 0.4P/ACE station on an IBM-compatible PC, from Beckman, USA. JEOL GSX500A and ECP600 NMR instruments, equipped with a 5 mm field gradient tunable probe with standard JEOL soft ware, were used for 1- and 2D 1H NMR experiments at 30°C on 500 µl each sample.

Optimization of the partial enzymatic depolymerization of HA
A solution containing 5 mg of HA in 500 µl of 0.04 M acetate buffer (pH 6.0) was prepared to optimize the partial enzymatic digestion condition. Hyaluronidase (Streptomyces hyalurolyticus) solution was prepared to be 0.2 TRU/µl with buffer. A 10 µl of enzyme solution was added to the HA solution, and the reaction was incubated in a microtube at 60°C. At various times intervals, a 5 µl aliquot was removed and inject directly to the column by monitoring at 232 nm. From these analysis, an optimal reaction time of 4.3 hr, corresponding to 40% reaction completion, was selected to obtain a partial HA digest providing a maximum concentration of 4- to 20-mer HAoligos.

HAoligos mixture solutions at various time intervals were injected directly to the following HPLC system. The condition are as follows; column, Asahipak GF-510HQ (4.6 mm I.D. x 300 mm); eluent, 50 mM ammonium bicarbonate; column temperature, ambient; flow rate, 0.50 ml/min; detector, UV monitored at 232 nm.

Large-scale depolymerization of HA
The large-scale, partial depolymerization of HA by bacterial hyaluronidase was carried out on 1.0 g of HA. To a solution containing 1.0 g of HA in 200 ml buffer, 2 ml of hyaluronidase solution (400 TRU/ml) was added, and the enzymatic digestion was performed in a glass-flask at 60°C. When the reaction reached to 40% digestion based on the absorbance at 232 nm, the reaction was stopped by dipping for 3 min in a boiling water bath. The sample was cooled in an ice bath and dialyzed for several days at 4°C in 500 MWCO dialysis tubing against deionized and distilled water, and then the sample was freeze-dried. The resulting white powder was dissolved in 20 ml water and was directly applied onto LPGC.

LPGC condition for HAoligos preparation
The 4~20 mer HAoligos were fractionated on a Sephadex G-50 (superfine) column (4.4 cm x 1 m) eluted with 0.2 M sodium chloride at an optimum flow rate defined by the Dracy’s low. Freeze-dried HAoligos mixture sample (~100 mg) was dissolved in 20 ml water and applied to the column, 300 fractions were collected (5 ml/tube each) and absorbance at 232 nm of each fraction was measured. Each unified HAoligo fraction was collected and concentrated by evaporation. If necessary, this chromatographic separation was performed repeatedly.

Each size-uniformed HAoligo fraction obtained from LPGC repeatedly twice was desalted by a Hi-Trap Desalting column eluted with water at 1.0 ml/min, and then the fractions were freeze-dried.

Analysis of HAoligos by normal-phase HPLC using Amide-80 column
Purified HAoligos were analyzed by a new separation method of normal-phase HPLC using Amide-80 column. The gradient elution condition was as follows. The stepwise gradient elution was started at 78% eluent A (a mixture of acetonitrile/distilled water/0.2 M phosphate buffer (pH 7.0)/3.0 M ammonium chloride = 32/11/1/1, by vol.) and 22% eluent B (a mixture of acetonitrile/distilled water/0.2 M phosphate buffer (pH 7.0)/3.0 M ammonium chloride = 16/21/1/1, by vol.), and was subsequently changed to 65% eluent A and 35% eluent B for 15 min, 50% eluent A and 50% eluent B for 35 min, 25% eluent A and 75% eluent B for 75 min, and finally maintained 100% eluent B for 10 min, and then returned to the initial condition; flow rate, 1.0 ml/min; column temperature, 50°C; detection, UV at 232 nm.

CE analysis
The purity of HAoligos was confirmed by CE on the normal polarity mode using a mixture of 40 mM disodium phosphate/40 mM sodium dodecylsulfate/10 mM tetraborate adjusted to pH 9.0 with 1.0 M hydrochloride as described previously (Stephen et al., 1991Go). The fused silica capillary (75 µm I.D. x 375 µm O.D., 67 cm long) was automatically washed before use with 0.1 M sodium hydroxide, followed nitrogen gas pressure injection (5 s) at a constant current 15 kV. The samples (0.1 mg/ml) were dissolved in water and loaded (7 nl) with nitrogen gas pressure injection.

NMR spectroscopy
One and 2D 1H NMR spectroscopy was performed under the condition described previously (Toida et al., 1999aGo). Briefly, each sample (~2.0 mg) was dissolved in 0.5 ml of D2O (99.9%) and freeze-dried repeatedly to remove exchangeable protons. The sample was kept in a desiccator over phosphate pentoxide in vacuo overnight at room temperature. The thoroughly dried sample was the dissolved in 500 µl of D2O (99.96%) and passed through 0.45 µm syringe filter and transferred to an NMR tube (5.0 mm O.D. x 25 cm). The HOD signal was suppressed by presaturation during 3 and 1.5 s for 1D- and 2D-NMR experiments, respectively. To obtain 2D spectra, 1024 x 512 data matrix for a spectra width of 2000 Hz were measured, and the time domain data were multiplied after zero-filling (data matrix size 1K x 1K) with a shifted sine-bell window functions.

Preparation of chemically fully O-sulfated 4- to 20-mer HAoligos
Preparation of fully O-sulfated HAoligos were based on the method described previously (Nadkarni et al., 1996Go; Maruyama et al., 1998Go). This method was slightly improved for HAoligos in this paper. To obtain the tributylamine (TBA) salt of HAoligos, 50 µl of TBA was added to each size-uniformed HAoligo sample (1.0 mg) in 1.0 ml of distilled water adjusted to pH 2.8 with 0.1 M hydrochloride. The mixture was mixed vigorously and freeze-dried. The resulting salt was dissolved 0.2 ml of N,N-dimethylformamide (DMF) to which a required excess (15 mol/equivalent of available hydroxy group in HAoligos) of pyridine–sulfur trioxide complex was added. After 3 h at 40°C, the reaction was interrupted by addition of 0.5 ml of water and the raw product was precipitated with 3 vol. of cold ethanol saturated with anhydrous sodium acetate, and then collected by centrifugation at 3000 x g for 15 min. The resulting fully O-sulfated HAoligo was dissolved water. Five hundred microliters of solution was centrifuged, the supernatant was concentrated, and reagents were removed by using a M.W. 3000 cut-off filter device (Microcon YM-3) repeatedly until reagents were not detectable for O-sulfated HAoligos 10- to 20-mer. The concentrated supernatant was removed and freeze-dried. The small O-sulfated HAoligos 4- to 8-mer were prepared by ethanol precipitation procedure, in which final ethanol concentration was 80–90%; however, the recovery of the products was significantly low (35–50%).

Gradient PAGE analysis of O-sulfated 10- to 20-mer HAoligos
Gradient PAGE was used to monitor the preparation and purification of O-sulfated HAoligos 10~20 mer, as well as to check depolymerization of HAoligos by the O-sulfation reaction. Polyacrylamide linear gradient resolving gels (14 x 28 cm, 10–20% acrylamide gel) were purchased and run as previously described (Edens et al., 1992Go). O-Sulfated HAoligos were visualized by Alcian blue staining.

Compositional analysis
The determination of sulfate groups was performed by the anion exchange HPLC, named Ion Chromatography, using a conductivity detector after acid hydrolysis of the sample in 6 M hydrochloride at 100°C for 2.5 h. Hexosamine was also analyzed by the post-column HPLC derivatization method (Toyoda et al., 1991Go) after acid hydrolysis under the same conditions as described for inorganic sulfate analysis.

Assay for anti–factor IIa activity
Normal human plasma (NHP) was collected from healthy volunteers for the assay. Anti–factor IIa activity was determined by incubating 50 µl of O-sulfated HAoligos, 30 µl of NHP and 20 µl of human thrombin (1.2 NIH units/ml) in 850 µl of Tris-buffer (50 mM Tris, pH 8.3, 227 mM sodium chloride) at 25°C for 3 min. Then 50 µl (1.9 µmol/ml) of chromogenic TH (tosyl-glycyl-prolyl-arginine-4-nitranilide acetate) was added, and the amidolytic thrombin activity was measured at 405 nm under 25°C.

Assay for hyaluronidase inhibition by FIA
Hyaluronidase (hyaluroglucosaminidase from bovine testes) inhibition by fully O-sulfated HAoligos was determined by the FIA method described previously (Toida et al., 1999bGo). Briefly, 20 µl of O-sulfated HAoligos solution, 10 µl of HA (as a substrate, 400 mg/ml) and 60 µl of 0.2 M sodium acetate buffer (pH 5.0) containing 2.5 mM calcium chloride were mixed. Ten microliters of enzyme solution (0.25 units/µl) was added to this mixture. The mixture solution was incubating at 37°C for 2 h. The enzyme action was terminated after a fixed time by incubating the mixtures at boiling bath for 5 min. And then, 2 µl of sample solution was applied to the FIA system. The concentrations of O-sulfated HAoligos and O-sulfated intact HA were adopted in triplicate.


    Acknowledgment
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
This work was supported in part by Grants-in-Aid from the Ministry of Culture, Sports and Education of Japan (11672136 (T.T.) and 09672185 (T.I.)).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
CE, capillary electrophoresis; 1D, one dimensional; 2D, two dimensional; DQF COSY, double quantum filtered correlation spectroscopy; E.C., enzyme code; FIA, flow injection assay; GAG, glycosaminoglycan; GlcA, D-glucuronic acid; GlcNAc, N-acetyl-D-glucosamine; HA, hyaluronan; HAoligo, hyalurooligosaccharide; HPLC, high performance liquid chromatography; IC50, concentration of 50 % inhibition; LPGC, low pressure gel permeation chromatography; M.W., molecular weight; MWCO, molecular weight cut-off; NHP, normal human plasma; NMR, nuclear magnetic resonance.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
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