Biomacromolecules 2005, 6, 168-173
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Molecular Weight Determination of Heparin and Dermatan Sulfate by Size Exclusion Chromatography with a Triple Detector Array Sabrina Bertini,*,† Antonella Bisio,† Giangiacomo Torri,† Donata Bensi,‡ and Maria Terbojevich§ Istituto di Ricerche Chimiche e Biochimiche “G. Ronzoni”, V. G. Colombo 81, 20133 Milano, Italy, Laboratori Derivati Organici, V. M. Barozzi 4, 20122 Milano, Italy, and Institute of Organic Chemistry, University of Padova, V. F. Marzolo 1, 35100 Padova, Italy Received May 24, 2004; Revised Manuscript Received September 6, 2004
The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/ TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [η]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters. Introduction Heparin (Hep) and dermatansulfate (DeS) are linear sulfated polysaccharides belonging to the family of glycosaminoglycans (GAGs) endowed with antithrombotic properties. They are widely distributed in a variety of animal tissues but are commercially prepared preferably from porcine or bovine intestinal mucosa. The structure of Hep is largely accounted for by alternating R1f4 linked residues of hexuronic acid, which can exist in two different epimeric forms (R-L-iduronic or β-D-glucuronic acid), and R-D-glucosamine, which can be either N-sulfated or N-acetylated. Moreover, some hydroxyl groups of both uronic acid and glucosamine can be differently O-sulfated: L-iduronic 2-O-sulfate R1f4 D-glucosamine N-sulfate 6-Osulfate is the most frequently occurring disaccharide unit in the Hep sequence.1 DeS is also an iduronic acid containing glycosaminoglycan; however, it significantly differs from Hep, since their iduronic acid residues are largely nonsulfated as well as for the position and configuration of glycosidic bonds. The structure of DeS is largely represented by monosulfated disaccharide repeating unit [IdoA-GalNac4SO3], where IdoA is R-L-idopyranosyluronic acid and GalNac4SO3 is N-acetyl-β-D-galactopyranose 4-O-sulfate, linked 1,3 and * To whom correspondence should be addressed. Tel: + 39 02 70641621. Fax: + 39 02 70641634. E-mail:
[email protected]. † Istituto di Ricerche Chimiche e Biochimiche “G. Ronzoni”. ‡ Laboratori Derivati Organici. § University of Padova.
1,4, respectively. Although iduronic acid is the major uronic acid found, glucuronic may be present as well. Moreover, a minor but significant proportion of DeS may be “oversulfated” either at position 2 of the uronic acid residue or at position 6 of the galactosamine residue, these oversulfated sequences being responsible for the antithrombotic properties of this glycosaminoglycan.2 The length of chains greatly contributes to the structural heterogeneity of Hep and DeS polymers: commercial GAG preparations are described as polydisperse mixture of molecules with molecular weight value up to 50 000 and 60 000 Dalton for Hep and DeS respectively.3 Such a structural variability makes these GAGs highly challenging molecules to characterize. Hep and DeS, well-known as anticoagulant and antithrombotic drugs, exert numerous important biological activities, associated with their ability to interact with different plasma and tissue proteins.4,5 Since one of the parameters affecting their biological activity is the molecular weight, an accurate determination of its value is particularly important.6 The high degree of polydispersity of chain length, together with the overall sulfation pattern and conformational differences, represents, besides important diversities between Hep and DeS, the main difficulty in the molecular weight evaluation.7 High-performance size-exclusion chromatography (HPSEC) with different columns and eluents represents the most commonly used method for the measurement of molecular weight and size distribution of Hep.8 This method rigorously
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MW Determination of Heparin and Dermatan Sulfate
needs a set of Hep reference standards with well defined M and narrow MD for column calibration, the accuracy of such a relative measurement being dependent mainly on the quality of standards themselves. During the past decades, a considerable effort has been directed toward the preparation of standards for the characterization of DeS, Heps, and low-molecular-weight Heps, but a universally recognized method for this purpose has not been found yet. At present, authentic Hep molecular weight standards mainly for unfractionated Heps (UFHs) and dermatan sulfates (DeSs) characterization are not commercially available. HP-SEC calibration is being commonly performed with homemade references, such as Hep and DeS fractions, and their low molecular weight derivatives,8,9 whose molecular weight were evaluated by different techniques, such as osmometry, intrinsic viscometry,10 or low angle light scattering.7,11 These techniques provide different types of average molecular weight varying from number-average mean molecular weight (Mn), as obtained from osmometry, to the weight average mean molecular weight (Mw), as for viscometry and light scattering. In literature examples of application of SEC associated with viscometry and light scattering for the determination of radius of gyration and intrinsic viscosity are reported for a hyaluronan, a nonsulfated high molecular weight GAG,12 as well as some dynamic and static light scattering studies for the determination of dynamic properties of chitosan and chitosan derivatives.13 Also 13C NMR14 has been suggested for Mn determination of heparin, but the pure sensitivity of the technique associated with the heterogeneity of terminal residues represents a serious limitation to the application of a such method. Moreover, for HP-SEC calibration, an approximation is usually introduced: both Mn and Mw values are considered equal to Mp, which corresponds to the molecular weight value on the top of the chromatographic peak. Such a case is possible only for the theoretical situation of a polydispersity value (Mw/Mn) equal to one. For low-molecular weight Heps, a method was proposed that overcomes the problem of calibration with Hep standards by using a HP-SEC coupled with a multiangle laser light scattering detector which provides the molecular weight distribution of products.15 In the present work, we focused our attention on UFH and DeS, with the aim of obtaining the molecular weights and their distributions by HP-SEC technique combining three primary detectors connected in series: refractometer, viscometer, and right angle laser light scattering. One of the advantages of a triple detector array (TDA) assembly is that chromatographic calibrations are not necessary. The angular dissymmetry in the scattered intensity of light is accounted for by using the combined measurements of intrinsic viscosity and 90° light scattering intensity. The viscometer detector is also useful for determining the size of molecules in solution and particularly of small polymer molecules for which scattering dissymmetry is not observable. The molecular size can then be related to the secondary structure of a polymer, either in terms of chain stiffness or conformation.
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With the aim to generate standards for the evaluation of molecular weight of Hep samples, eight Hep fractions in the range 6000-60 000 Dalton were obtained by size exclusion chromatography starting from an unfractionated pig mucosal Hep. On consideration of their low polydispersity values, these fractions, characterized by HP-SEC/TDA, were regarded as narrow standards. Experimental Section Materials. Four Hep samples (UFH-A, UFH-B, UFH-C and UFH-D) were from pig mucosa. Eight fractions (HFah) were obtained by size-fractionation of UFH-D sample on a Sephadex G50 column (ø25 cm; h 100 cm), using 0.3 M NaCl with 0.2% NaN3 as eluent, with a flow rate of 2000 mL/h. NaCl and NaN3 were from Fluka. Five DeS samples (DS-A-E), from bovine, were kindly provided by Opocrin (Corlo di Formigine, Italy). For the instrument calibration, Viscotek GPC standards for aqueous applications were used: in particular, poly(ethylene oxide) (PEO) 23K (Mw 23 200, Mn 22 300, [η] 0.373 dL/g) and PEO 100K (Mw 97 900, Mn 31 500, [η] 0.809 dL/g) were used as narrow and broad standard, respectively. Apparatus. The HPLC equipment consisted of a Viscotek system equipped with a VE1121 Solvent Delivery pump, a metal free two channel on line degassing device GasTorr 150. The detector system used in this study was a Viscotek mod.300 Triple Detector Array. In this model, there is an oven compartment ranging between a temperature of 30 and 80 °C that contains space for up to three separation columns and the detectors. An optional manual sample injection valve was located inside of the oven module. In this way, the loop is maintained at the same temperature of the columns and the detectors. Right angle laser light scattering is the first detector after the columns, with the following technical specifications: a 90° angle geometry for maximum signalto-noise; cell volume of 10 µL; maximum backpressure on cell of 15 psi; maximum signal of 2.5 V; a 670 nm laser light source. Refractive index (RI) is the second detector with the following technical specifications: cell volume of 12 µL; maximum backpressure on cell of 5 psi; maximum signal of 10 V; light emitting diode (LED) at 660 nm wavelength. Viscometer is the last detector, characterized by four capillaries (0.01′′ id × 24′′ L) with a differential Wheatstone bridge configuration. Correction for any angular dissymmetry in the RALS data is carried out automatically in the TriSEC software using the viscometer signal. Of course, all of the molecules discussed in this paper are relatively small compared to the laser wavelength, and therefore, the angular dissymmetry correction, if any, is negligible. Two TSK gel columns in series (GMPWXL mixed bed column, 7.8 mm ID × 30 cm, Viscotek, V0 ) 6 mL, Vt ) 11 mL each one) were used. Columns, injector and detectors were maintained at 40 °C. An aqueous solution of 0.1 M NaNO3 pre-filtered on 0.22 µm filter (Millipore) was used as mobile phase at a flow rate of 0.6 mL/min. The system was calibrated with the PEO narrow and broad standards of known Mw, polydispersity, and intrinsic viscosity. The narrow standard is particularly useful for determining detector
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Figure 1. HP-SEC/RI response of Hep fractions HF-A-H.
volume offsets and peak broadening parameters, whereas the broad standard is useful for checking the calculations for correct molecular weight distribution and Mark-Houwink parameters, etc. Analysis of data was performed with Viscotek TriSEC software, version 3.0.16 The HP-SEC analyses were performed with a Waters 515 pump, equipped with an auto-injector (Waters717plus), UV detector (Waters 2487), and RI detector (Waters 2410). Size exclusion cromatographies were carried out using TSK G2000 SW and TSK G3000 SW columns (Tosoh Corp. Japan) connected in series with a TSK guardcolumn, and eluting with 0.1 M NaNO3, at a flow of 0.6 mL/min. Processes were aided by Chromatography Manager Software Millenium -32 (Waters), with its GPC option. Sample Preparation. For HP-SEC/TDA analyses, samples were dissolved in a 0.1M NaNO3 aqueous solution at the following concentration: 20 mg/mL for HFa, HFb and HFc, and 8 mg/ml for HFd-HFh. 100 µL of each sample were injected. For HP-SEC all compounds were dissolved with the mobile phase, to a final concentration of 10 mg/mL and 20 µL were injected. The run time was of 55 min. Results and Discussion Part I: Heparin Fractions. A heparin sample (UFH) was fractionated by size exclusion chromatography under controlled experimental conditions to guarantee the reproducibility of results (see Experimental Section). Hep fractions were processed with HP-SEC/TDA at 40 °C by using 0.1 M NaNO3 as eluent. RI responses are shown in Figure 1. The dn/dc parameter is required to convert RI voltages to solute concentration at each data slice across a chromatographic peak. The area under the peaks was integrated and the sample recoveries were calculated. The dn/dc value was indirectly determined by comparing the RI detector response with the corresponding response of PEO, whose dn/dc value is 0.140 mL/g. A dn/dc of 0.120 mL/g was found for Hep samples in agreement with reported data.17,18 The TriSEC software calculates the actual concentration in each chromatographic slice by means of dn/dc combined with a detector constant for the RI. Full recovery was obtained for all of the Hep fractions indicating that no material remained
Figure 2. HP-SEC/TDA chromatograms of HF-H, the fraction with the highest molecular weight: RI detector vs Vr (A) and RALLS signal vs Vr (B).
adsorbed to the columns and that no low molecular weight components were eluted together with the salt peak. A minor peak, not visible with RI detector (Figure 2A), is observed in the RALLS signal for the highest molecular weight Hep fraction (Figure 2B). This peak is due to high molecular weight impurities, e.g., DeS as NMR analysis indicates (data not shown). The elaboration of the light scattering and concentration detector responses gives molecular weight values, whereas this latter together with the viscosity detector provides the [η] values. The hydrodynamic radius of the molecules (Rh), as a function of molecular size, can be calculated from the following equation: Rh ) [3/(4π)*([η]M/0.025]1/3
(1)
The coupling of a viscometer and a light scattering detector greatly improves the precision and dynamic range of SEC for polymer conformation studies. The results are insensitive to typical adverse variations of SEC experimental conditions, such as flow rate inconsistency, band broadening, column deterioration, non-SEC retention effect, moderate sample overloading, etc. The molecular weight values (Mn, Mw, Mp), [η], and Rh obtained with TriSEC software data analysis are reported in Table 1. The decreasing of the retention volume on increasing of the molecular weight, intrinsic viscosity, and hydrodynamic radius, as observed in HP-SEC/TDA experiments, indicates that Hep fractions were accurately fractionated according to size exclusion chromatography principles. To confirm Mw values obtained by coupling RALLS and Viscometer data, the RALLS signals were analyzed in batch mode since the
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MW Determination of Heparin and Dermatan Sulfate Table 1. Characterization of Eight Hep Fractions by HP-SEC/TDA in 0.1 M NaNO3, 40 °C
Table 2. Characterization by HP-SEC Analyses of Two Different UFH Samples in 0.1 M NaNO3, 40 °Ca
reference
Vra
Mn
Mw
Mp
[η]b
Rhc
sample
method
Mn
Mw
Mp
HF-A HF-B HF-C HF-D HF-E HF-F HF-G HF-H
16.89 16.61 16.33 15.99 15.74 15.58 15.37 15.03
5990 8520 12 200 17 200 25 000 30 600 41 100 52 500
6040 8610 12 400 18 700 26 200 32 700 44 300 60 700
5890 8340 12 100 16 900 24 300 29 600 41 700 56 200
0.062 0.093 0.131 0.187 0.257 0.321 0.383 0.469
1.76 2.28 2.93 3.82 4.64 5.43 6.32 7.50
UFH-A UFH-A UFH-B UFH-B
relative calibration TDA relative calibration TDA
7900 8200 14 500 15 000
9500 9240 15 800 16 300
6580 7150 14 200 14 500
a
b
c
Retention volume (mL). Intrinsic viscosity (dL/g). Hydrodynamic radius (nm).
a Evaluation of elution profile with LS-RI-DP detectors (TDA method) in comparison with RI detector alone (relative calibration).
Table 3. Characterization of Hep UFH-C by HP-SEC/TDA in 0.1 M NaNO3, 40 °C injection
Mn
Mw
Pda
[η]b
Rgc
Rhd
1 2 3 4 5
15 700 16 200 14 300 14 400 16 100
17 600 18 500 16 500 16 400 18 500
1.12 1.14 1.15 1.14 1.15
0.182 0.183 0.182 0.182 0.183
4.72 4.80 4.68 4.63 4.81
3.62 3.69 3.59 3.55 3.70
a Polydispersity (Mw/Mn). b Intrinsic viscosity (dl/g). c Radius of gyration (nm). d Hydrodynamic radius (nm).
Figure 3. Mark-Houwink plot of the eight Hep fractions in 0.1 M NaNO3, 40 °C.
small hep dimension does not induce any distortion symmetry: the same Mw values reported in Table 1 were obtained. The low polydispersity values of Hep fractions prevented the direct evaluation (from TriSEC data elaboration) of a and k parameters for the Mark-Houwink relationship [η ]) kMa
(2)
Such a parameters were then calculated by using data reported in Table 1, through the double logarithmic plot of intrinsic viscosity versus molecular weight, shown in Figure 3. The value of linear regression parameter (0.99) for this plot indicates that a scaling relationship, which is valid for linear homologous polymers, can be defined. Regression analyses provided scaling exponents a 0.88 and k value 3.16 × 10-5. These values are substantially comparable with literature data.19 Part II: Unfractionated Heparin and Dermatan Sulfate. On consideration of their low polydispersity values, the eight Hep fractions can be used as narrow standards for a relative calibration of a conventional HP-SEC/RI system.To evaluate the accuracy of the relative calibration, two unfractionated Heps (UFH-A and UFH-B) were analyzed by both HP-SEC/RI and HP-SEC/TDA. Results reported in Table 2 show that the molecular weight values obtained with the two different techniques are substantially in agreement with each
Figure 4. Mark-Houwink plot and molecular weight distribution of UFH-C in 0.1 M NaNO3, 40 °C.
other and confirm the accuracy of the relative calibration performed. A major advantage of using HP-SEC/TDA is that interference from aggregates or particles eluting early in the chromatogram may be excluded from the analysis and that the scaling relations for the structural parameters a and k may be obtained from polydispersed samples. To verify this statement, sample UFH-C was analyzed by HP-SEC/TDA. Table 3 shows the results obtained by five injections at different Hep concentration (about 15 mg/mL). The error of molecular weight values is 6% and that of intrinsic viscosity is 1%. Measurements repeated over 1 year of time span were in the same range, thus proving the reproducibility and the accuracy of the HP-SEC/TDA results. Polydispersity values are large enough to allow the calculation of Mark-Houwink parameters. Figure 4 reports the double logarithmic plot of intrinsic viscosity vs molecular weight of UFH-C, obtained using HP-SEC/TDA. In this case averages over the entire distributions were calculated. Regression analyses provided a scaling exponent of 0.88 ( 0.06 in good agreement with the value obtained from Hep fractions analysis. K value is equal to 3.20 × 10-5.
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Table 4. Characterization by HP-SEC/TDA of Dermatan Sulfate Samples in 0.1 M NaNO3, 40 °C
d
sample
Mn
Mw
Pda
[η]b
Rgc
Rhd
DS-A DS-B DS-C DS-D DS-E
46 600 38 000 33 000 29 200 24 400
53 100 42 500 37 600 33 200 29 900
1.14 1.12 1.14 1.14 1.22
0.713 0.607 0.552 0.476 0.418
10.89 9.60 8.91 8.13 7.47
8.36 7.37 6.84 6.24 5.73
a Polydispersity. b Intrinsic viscosity (dL/g). c Radius of gyration (nm). Hydrodynamic radius (nm).
Figure 6. Radius of gyration vs log (molecular weight) for dermatan sulfate and Hep in 0.1 M NaNO3, 40 °C.
Figure 5. Mark-Houwink plot of dermatan sulfate samples in 0.1 M NaNO3, 40 °C.
Since the a parameter for Hep is measured, it was possible to evaluate the radius of gyration (Rg) using the Flory-Fox and Ptitsy-Eisner equations Rg ) 1/60.5([η]M/F]1/3
(3)
where F ) 2.86 × 1021(1 - 2.63e + 2.86e2) and e ) (2a 1)/3. The values of Rg for UFH-C are given in Table 3. The error on the Rg values is about 2%. Since Hep fractions and DeSs are commonly used together as standards for HP-SEC to evaluate the molecular weight distribution of GAGs,9,20 similar studies were performed on DeS. As for Hep samples, the dn/dc value was indirectly obtained by comparing the RI signals with the PEO ones by five analysis of the same DeS sample. The injections average value was 0.125 ( 0.03 mL/g. Five unfractionated DeS samples were analyzed to evaluate the molecular weight distribution as well as the MarkHouwink parameters. Results are reported in Table 4. Figure 5 shows the dependence of intrinsic viscosity on molecular weight, in double logarithmic representation, for DeS samples. Regression analyses provide scaling exponents of 0.84 ( 0.07 in good agreement with the value obtained for Hep samples. On the contrary, Hep and DeS exhibit different k values of 3.16 × 10-5 and 4.7 × 10-5 respectively. According to this difference, intrinsic viscosity and retention volume values of DeS are respectively higher and lower with respect to Hep values, at comparable molecular weight. Moreover, the plot of the radius of gyration as a function of molecular weight, in double logarithmic scale, for typical DeS and Hep samples, has a similar slope but different interception with the y axis (Figure 6). This means that Hep
and DeS samples with the same molecular weight values show different intrinsic viscosity and different radius of gyration. Such a difference is also pointed out by comparing Rh values of Hep and DeS reported in Tables 1 and 4. In addition to the already mentioned structural differences between Hep and DeS (i.e., sulfation pattern and degree, glycosidic linkage, and uronic acid type and content) a further structural diversity contributes to their different dimensions in solution. This diversity is represented by the possible different conformations that uronic acid residues can assume, between the three possible equienergetic forms, namely 4C1, 1 C4, and 2S0: the 2-O-sulfated IdoA and IdoA residues, are in a dynamic equilibrium between the conformers 1C4 and 2 S0, whereas the glucuronic acid is in 4C1 conformation.2,21,22 The population of individual conformers depends on the structure and sulfation of neighboring units and on sulfation of the IdoA residue.23 Conclusions To generate standards for molecular weight evaluation of Hep samples, a pig mucosal Hep was fractionated by size exclusion chromatography into eight fractions ranging from 6000 to 60 000 Dalton. These fractions were characterized by HP-SEC/TDA and, on consideration of their low polydispersity values, were regarded as narrow standards. Their M values were then used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing UFH samples with both HP-SEC/TDA and HPSEC were in excellent agreement. Moreover, the a and k values calculated for these standards in our experimental conditions (a ) 0.88 and k ) 3.16 × 10-5), were in good agreement with the Mark-Houwink parameter values obtained by analyzing polydispersed hep samples. Since UFHs, Hep fractions, and DeSs are commonly used together as standards for HP-SEC calibration, Mark-Houwink parameters were evaluated also for dermatan sulfates. The finding that Heps and DeSs exhibit different k values in the same solvent suggests that the use of both polysaccharides as standards in the same calibration curve is not correct and can generate inaccurate results.
MW Determination of Heparin and Dermatan Sulfate
Acknowledgment. The authors thank Dr. M. Gianelli of Labservice for the technical support and Dr. P. Clarke of Viscotek for useful discussion. References and Notes (1) Casu, B. AdV. Carbohydr. Chem. Biochem. 1985, 43, 51-134. (2) Casu, B.; Guerrini, M.; Torri, G. Curr. Pharm. Des. 2004, 10, 939949. (3) Comper, W. D.; Laurent, T. C. Physiol. ReV. 1978, 58, 255-315. (4) Capila, I.; Linhardt, R. J. Angew. Chem. 2002, 41, 390-412. (5) Casu, B.; Lindahl, U. AdV. Carbohydr. Chem. Biochem. 2001, 57, 159-208. (6) Nieduszynski, I. In Heparin; Lane, D. A., Lindahl, U., Eds.; Edward Arnold: Great Britain, 1989; Chapter 3, pp 51-63. (7) Ehrlich, J.; Stivala, S. S. J. Pharmacol. Sci. 1973, 62, 527. (8) Harenberg, J.; De Vries, J. X. J. Chromatogr. 1983, 261, 287-292. (9) Malsch, R.; Harenberg, J.; Piazolo, L.; Huhle, G.; Heene, L. J. Chromatogr. 1996, 685, 223-231. (10) Johnson, E.; Mulloy, B. Carbohydr. Res. 1976, 51, 119-27. (11) Miklautz, H.; Riemann, J.; Vidic, H. J. J. Liq. Chromatogr. 1986, 9, 2073-2093. (12) Mendichi, R.; Solte´s, L.; Schieroni, A. G. Biomacromolecules 2003, 4, 1805-1810.
Biomacromolecules, Vol. 6, No. 1, 2005 173 (13) Buhler, E.; Rinaudo, M. Macromolecules 2000, 33, 2098-2106. (14) Desai, U. R.; Linhardt, R. J. J. Pharmacol. Sci. 1995, 84 (2), 212215. (15) Knobloch, J. E.; Shaklee, P. N. Anal. Biochem. 1997, 245, 231241. (16) Beer, U.; Wood, P. J.; Weisz, J. Carbohydr. Polym. 1999, 39, 377380. (17) Barlow, K.; Sanderson, P. N.; McNeill, J. Arch. Biochem. Biophys. 1961, 94, 518-521. (18) Terbojevich, M.; Cosani, A.; Liverani, L. EuroCarb VIII, SeVilla (Spain), 2-7 July 1995, B-56. (19) Lasker, S. E.; Stivala, S. S. Arch. Biochem. Biophys. 1966, 115, 360372. (20) Linhardt, R. J.; Al-Hakim, A.; Yu Liu, S.; Shik Kim, Y.; Fareed, J. Semin. Thromb. Hemost. 1991, 17, supplement 1, 15-22. (21) Ferro, D. R.; Provasoli, A.; Ragazzi, M.; Torri, G.; Casu, B.; Gatti, G.; Jacquinet, C.; Sinay, P.; Petitou, M.; Choay, J. J. Am. Chem. Soc. 1986, 108 (21), 6773-6678. (22) Bossennec, V.; Petitou, M.; Perly, B. Biochem. J. 1990, 267 (3), 625630. (23) Ferro, D. R.; Provasoli, A.; Ragazzi, M.; Casu, B.; Torri, G.; Bossennec, V.; Perly, B.; Sinay, P.; Petitou, M.; Choay, J. Carbohydr. Res. 1990, 195, 157-167.
BM049693S
