End-Label Free-Solution Electrophoresis of the Low Molecular Weight

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Anal. Chem. 1997, 69, 3199-3204

End-Label Free-Solution Electrophoresis of the Low Molecular Weight Heparins Jan Sudor and Milos V. Novotny*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

The intact heparins are highly charged oligosaccharides. Their charge polydispersity and the possible occurrence of numerous isomers complicate the analysis of these biomedically important glycoconjugates. After unsuccessful attempts to resolve the low molecular weight heparins in entangled matrixes, or through the use of counterions (Stefansson, M.; Novotny, M. V. Anal. Chem. 1994, 66, 3466-3471), we have designed a unique end-label reagent to incorporate both a fluorescent moiety and a desirable frictional increment to the analyte molecules. The resolution of small oligomers was improved dramatically following this approach. We also propose a scheme, based on the end-label free-solution electrophoresis model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780), that could potentially predict the migration times of some oligomers of complex heparin mixtures. Heparins are an important part of the glycosaminoglycan (GAG) family.1 Their biochemical/physiological and pharmacological significance extends well beyond their widely recognized role in blood anticoagulant and antithrombin activities.2,3 In spite of their multilateral biological importance, heparins remain poorly characterized in terms of their chemical structure. The heparins isolated from biological materials may exhibit a considerable degree of polydispersity in both their size and charge. In a linear chain arrangement, heparins feature alternating disaccharide sequences of glucuronic acid and glucosamine residues that are further O-sulfated, N-sulfated, and N-acetylated (Figure 1) to a greater or lesser degree.4-7 Adding to the overall complexity of these GAGs are irregular sequences, such as occurrence of the hexasaccharide that is responsible for the highaffinity binding to antithrombin III.1,8 The structural analysis of heparins is further complicated by the fact that the polydispersity in size and charge appears interdependently. However, knowing the fine structural features of these molecules may be highly relevant to their binding to important biological entities.2 (1) Yalpani, M. Polysaccharides: Synthesis, Modifications and Structure/Property Relations; Elsevier: Amsterdam, 1988. (2) Lindahl, U.; Backstro¨m, G.; Ho¨o ¨m, M.; Thunberg, L.; Fransson, L.Å.; Linker, A. Proc. Natl. Acad. Sci. U.S.A., 1979, 76, 3198-3202. (3) Linhardt, R. J.; Loganathan, D. Biomimetic Polymers; Gebellein, G., Ed.; Plenum: New York, 1990; pp 135-175. (4) Lane, D., Lindahl, U., Eds. Heparin, Chemical and Biological Properties, Clinical Applications; CRC Press: Boca Raton, FL, 1989. (5) Linhardt, R. J. Chem. Ind. 1991, 2, 45-50. (6) Bae, J. H.; Desai, U. R.; Pervin, A.; Caldwell, E. O.; Wieler, J. M.; Linhardt, R. J. Biochem. J. 1994, 301, 121-129. (7) Rosenberg, R. D.; Lam, L. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 12181222. (8) Linhardt, R. J.; Rice, K. G.; Merchant, Z. M.; Kim, Y. S.; Lohse, D. L. J. Biol. Chem. 1986, 261, 14448-14454. S0003-2700(96)01297-8 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Structure of a heparin disaccharide: R ) SO3- (major) or H (minor); R’ ) SO3- (major) or Ac (minor).

The structural studies of heparins represent a major methodological challenge in terms of sample complexity. The molecular weight distribution depends on a biological origin, isolation/ purification procedures and a method of degradation to yield the pharmaceutically desirable low molecular weight heparin preparations.9,10 Traditionally, slab gel electrophoresis11-14 and highperformance liquid chromatography (HPLC) in its size-exclusion15 and ion-exchange15 modes has been employed to characterize heparins, albeit with little chance to resolve the individual molecular entities. More recently, CE has been investigated16-18 as an alternative to slab gel electrophoresis and HPLC. The use of CE has been particularly successful16,17 in resolving the disaccharide mixtures formed enzymatically from heparin through the action of heparin lyases. However, there has been a little success with resolving additional low molecular weight heparins by CE.18 We propose here a different approach to the separation of low molecular weight heparins. This approach is based on the endlabeling idea in which an additional (constant) charge or friction is added to the size-polydisperse, uniformly charged solute, thus influencing the mobilities of smaller chains more profoundly than the mobilities of larger solutes. (We note that the end-labeling approach was earlier suggested as an improvement toward separations of large DNA molecules in gels under pulsed-field conditions a few years ago.19-21) Based on a similar idea, Mayer (9) Shively, J. E.; Conrad, H. E. Biochemistry 1976, 15, 3932-3942. (10) Linhardt, R. J. Carbohydrates, Synthetic Methods and Applications in Medicinal Chemistry; Ogura, H., Ed.; 1992; pp 387-403. (11) Rice, K. G.; Rottik, M. K.; Linhardt, R. J. Biochem. J. 1987, 244, 515-522. (12) Al-Hakim, A.; Linhardt, R. J. Electrophoresis 1990, 11, 23-28. (13) Al-Hakim, A.; Linhardt, R. J. Appl. Theor. Electrophor. 1991, 1, 305-312. (14) Edens, R. E.; Al-Hakim, A.; Weiler, J. M.; Rethwisch, D. G.; Fareed, J.; Linhardt, R. J. J. Pharm. Sci. 1992, 81, 823-827. (15) Pervin, A.; Gallo, C.; Jandik, K. A.; Han, X.-J.; Linhardt, R. J. Glycobiology 1995, 5, 83-95. (16) Desai, U. R.; Wang, H. M.; Ampofo, S. A.; Linhardt, R. J. Anal. Biochem. 1993, 213, 120-127. (17) Pervin, A.; Al-Hakim, A.; Linhardt, R. J. Anal. Biochem. 1994, 221, 182188. (18) Toida, T.; Linhardt, R. J. Electrophoresis 1996, 17, 341-346. (19) Ulanovsky, L.; Drouin, G.; Gilbert, W. Nature 1990, 343, 190-192.

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et al.22 have developed a model for DNA sequencing in CE with no sieving media involved. They called this approach an “endlabel free-solution electrophoresis” (ELFSE). The original ELFSE model22 was further revised by Long and Ajdari.23 These authors showed that flexible end-labeled solutes (e.g., DNA molecules) can undergo conformational changes as a consequence of the tension (due to “unbalanced” hydrodynamic forces) between the solute and the end label. Furthermore, the “local picture” of the electrophoretic mobility becomes violated under such circumstances.23,24 However, we have shown previously25 that the ELFSE model,22 based on the “local picture” of electrophoretic mobility, holds for uniformly charged (rodlike) oligosaccharides derived from a partially hydrolyzed κ-carrageenan. (Note that both the “electrophoretic” and “hydrodynamic” frictional coefficients of uniformly charged stiff rods scale linearly with their contour lengths.) Because of the high charge densities on heparin chains, the common “small” fluorescent tags [e.g., 8-aminonaphthalene-1,3,6trisulfonic acid (ANTS)26,27 and 6-aminoquinoline (6-AQ)28] have only negligible effects on their electrophoretic mobilities. Therefore, we have developed a unique end-label reagent (1-maltoheptaosyl-1,5-diaminonaphthalene) that is capable of modifying substantially the solutes’ frictional properties. This end label also incorporates a fluorophoric group with desirable properties for laser-induced fluorescence detection. The novel end label is shown here to improve the separation of low molecular weight heparins in free solutions, without the presence of either sieving media or “selective” counterions. Based on the presented ELFSE model, the migration velocities of certain components can also be predicted. In spite of the current unavailability of suitable heparin oligomeric standards (with the chain length L . κ-1, where κ-1 is the Debye screening length29), these possibilities are discussed. EXPERIMENTAL SECTION Apparatus. A home-built CE system was used in all experiments described in this work. The separation capillaries were purchased from Polymicro Technologies (Phoenix, AZ). Their inner surface was modified (for all experiments presented here) by the attachment of linear polyacrylamide.30 The separation capillary was enclosed in a Plexiglas box with an interlock safety system. On-column fluorescence measurements were carried out with a helium-cadmium laser (Model 56X, Omnichrome, Chino, CA) used at 325 nm as a light source. The incident laser beam was aligned to its optimum by adjusting the position of collecting optics between the optical cell and the detector. Fluorescence emission was collected through a 600-µm fiber optic placed at a right angle to the incident laser beam. Signals isolated by long-pass, lowfluorescence filters (λcutoff > 360 nm) (Oriel, Stradford, CT) were (20) Viovy, J. L.; De´fontaines, A. D. Pulsed-Field Gel Electrophoresis: Protocols, Methods, and Theories; Burmeister, M., Ulanovsky, L., Eds.; Humana Press: Totowa, NJ, 1992; pp 403-450. (21) Noolandi, J. Electrophoresis 1993, 14, 680-681. (22) Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780. (23) Long, D.; Ajdari, A. Electrophoresis 1996, 17, 1161-1166. (24) Long, D.; Viovy, J. L.; Ajdari, A. Phys. Rev. Lett. 1996, 76, 3858-3861. (25) Sudor, J.; Novotny, M. V. Anal. Chem. 1995, 67, 4205-4209. (26) Chiesa, C.; Horvath, C. J. Chromatogr. 1993, 645, 337-352. (27) Stefansson, M.; Novotny, M. V. Anal. Chem. 1994, 66, 1134-1140. (28) Nashabeh, W.; El Rassi, Z. J. Chromatogr. 1992, 600, 279-287. (29) Sherwood, J. D. J. Chem. Soc., Faraday Trans. 2 1982, 78, 1091-1100. (30) Hjerte´n, S. J. Chromatogr. 1985, 347, 191-198.

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monitored using a R928 photomultiplier tube (Hamamatsu Photonics K. K., Shizuoka Prefecture, Japan) and amplified with a Model 128A lock-in amplifier (EG&G Princeton Applied Research, Princeton, NJ). In the case of UV/visible absorption detection, a variable-wavelength UV detector (UVIDEC-100-V; Jasco, Tokyo, Japan) was modified for the on-column measurements. A high-voltage power supply (Spellman High Voltage Electronics, Plainview, NY), capable of delivering 0-40 kV was employed for controlling the potential in CE. MALDI/TOF mass spectrometric measurements were performed on a Voyager Biospectrometry workstation (Perseptive Biosystems, Framingham, MA), and the fluorescence spectra of various fluorescent compounds were measured on a Perkin-Elmer 650 spectrofluorometer equipped with a xenon arc lamp and a Perkin-Elmer 150 power supply (Perkin Elmer, Norwalk, CT). Chemicals. Citric acid was purchased from EM Science (Cherry Hill, NJ), and sodium hydroxide and phosphoric acid (85%) were from Fisher Scientific (Fair Lawn, NJ). Acrylamide and ammonium persulfate were received from Bio-Rad Laboratories (Hercules, CA). Low molecular weight heparins (Mw ∼3000 and ∼6000), maltoheptaose, a mixture of maltooligosaccharide standards (G4-G10, where G is glucose), and heparin disaccharide standards (R-∆UA-2S-[1f4]-GlcNS-6S, R-∆UA-[1f4]-GlcNS-6S, R-∆UA-2S-[1f4]-GlcNAc, and R-∆UA-[1f4]-GlcNAc-6S, where ∆UA ) 4-deoxy-L-threo-hex-4-enopyranosyluronic acid; GlcN ) D-glucosamine; Ac ) acetyl; and NS, 2S, and 6S ) N-sulfo, 2-sulfate, and 6-sulfate, respectively) (Figure 2) were received from Sigma (St. Louis, MO). Heparin (Mw ∼13 500-15 000) was purchased from Calbiochem (La Jolla, CA). Sodium cyanoborohydride, 1,5-diaminonaphthalene, 2,5-dihydroxybenzoic acid, and sodium acetate were from Aldrich (Milwaukee, WI). Acetone was purchased from J. T. Baker (Philipsburg, NJ), while ethanol was the product of Aaper Alcohol and Chemical Co. (Shelbyville, KY). Synthesis of the End-Label Reagent. The end-label reagent (1-maltoheptaosyl-1,5-diaminonaphthalene; Figure 3) was synthesized through the Schiff base formation between the aromatic amine of 1,5-diaminonaphthalene and the aldehyde form of maltoheptaose, followed by reduction of the Schiff base to a stable product.31 A 500-mg sample of maltoheptaose and 362.5 mg of 1,5-diaminonaphthalene (∼1:5.3 molar ratio) were dissolved in a 10-mL volume of 1:1 mixtures of 20 mM phosphoric acid/ethanol (1,5-diaminonaphthalene dissolved completely at elevated temperature). The mixture was heated to 85 °C for 30 min prior to the addition of sodium cyanoborohydride to a final concentration of 0.3 M. The mixture was then maintained at 85 °C for 3 h. Subsequently, the solvent was removed under nitrogen (a major part of the unreacted 1,5-diaminonaphthalene precipitated out of the solution following the removal of ethanol) and the mixture was centrifuged. The supernatant was purified three times by precipitation from 90% acetone, and the product was dried under nitrogen. Finally, the solid material was dried under nitrogen and stored at 4 °C. Derivatization of Heparin Disaccharides and Intact Heparins. The heparin disaccharides (R-∆UA-2S-[1f4]-GlcNS-6S, R-∆UA-[1f4]-GlcNS-6S, R-∆UA-2S-[1f4]-GlcNAc, and R-∆UA[1f4]-GlcNAc-6S) and the intact heparins (Mw ∼3000 or ∼6000 preparations) were derivatized through Schiff base formation between the aromatic amine of the end label and the aldehyde form of a sugar, followed by reduction of the Schiff base to a stable (31) Jackson, P. Biochem. J. 1990, 270, 705-713.

Figure 4. MALDI/TOF mass spectrometry of the end-label with (A) and without (B) the maltooligosaccharide standards (G4-G10).

Figure 2. Structures of selected heparin disaccharide standards: (A) R-∆UA-2S-[1f4]-GlcNS-6S; (B) R-∆UA-[1f4]-GlcNS-6S; (C) R-∆UA-2S-[1f4]-GlcNAc; (D) R-∆UA-[1f4]-GlcNAc-6S; where ∆UA ) 4-deoxy-L-threo-hex-4-enopyranosyluronic acid; GlcN ) D-glucosamine; Ac ) acetyl; NS, 2S, and 6S ) N-sulfo, 2-sulfate, and 6-sulfate.

Figure 3. Structure of a novel end label (1-maltoheptaosyl-1,5diaminonaphthalene).

product. Heparin disaccharides (0.5-1 mg) and 5-mg amounts of intact heparins were mixed with 5 (for disaccharides) and 10 mg (for intact heparins) of the end-label reagent and dissolved in 25 µL of 10 mM phosphoric acid prior to addition of 5 µL of 2 M sodium cyanoborohydride. The samples were heated to 65 °C for 3 h, diluted to the total volume of 0.5 mL with deionized water, and stored at -20 °C prior to analysis. Analysis of the End-Label Reagent and the Labeled Standards. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a linear time-of-flight (TOF)

instrument was employed for determination of the molecular weight of synthesized end-label reagent. Its purity was further assessed by CE in the presence of both the unreacted 1,5diaminonaphthalene and possible maltooligosaccharide impurities in the maltoheptaose reagent. Only unreacted 1,5-diaminonaphthalene and its single derivative with the maltooligosaccharides (other than maltoheptaose) can lead to a multiple peak formation in CE after a single heparin oligomer becomes derivatized with the end label. (However, the positional isomers of 1,5-diaminonaphthalene, if present, could also lead to additional peaks.) The part of 1,5-diaminonaphthalene that becomes doubly derivatized with maltoheptaose (or any other neutral or positively charged aldehyde) cannot react with the analytes of interest, and in addition, it can never migrate in the same direction as the negatively charged solutes (heparins derivatized with the end label) under the conditions of suppressed electroosmotic flow. For the MALDI/TOF measurements, the samples were prepared as follows: 1 µL of a 5 mg/mL solution of the maltooligosaccharide standards (G4-G10) was mixed with 1 µL of a 10 mg/mL solution of the end label. Subsequently, 1 µL of this mixture or 1 µL of the end-label solution was mixed separately on the metal plate with 1 µL of the matrix solution (10 g/L 2,5dihydroxybenzoic acid in 9:1 water/ethanol mixture). Further, 1-µL volumes of the ethanolic solution of sodium acetate (a few crystals of sodium acetate in 1 mL of ethanol) were added to the samples on the plate to maintain the oligosaccharides as their sodium adducts. The metal plate was inserted into the MALDI/ TOF instrument after the samples were completely dried. The output power of the nitrogen laser (337 nm) was ∼10 µJ/pulse (∼3 ns/pulse). The mixture of the end label and maltooligosaccharide standards (G4-G10) is shown in Figure 4A. The instrument was calibrated internally with maltohexaose (1013.89 Da; maltohexaose + Na+) and maltodecaose (1662.49 Da; maltodecaose + Na+). The measured mass of the (end label + Na+) adduct was 1318.64, which is in good agreement with the theoretical value of 1318.19. Figure 4B shows spectrum of the end label itself. The two smaller peaks that are close to the endlabel peak (1318.64 Da) were identified as the protonated form of the end label (1295.68 Da) and the adduct of the end label with Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

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Table 1. Mobilities of Heparin Disaccharides, Intact Heparins, and the End Label µapp × 105 (cm2/V‚s)

solute

Figure 5. Capillary electrophoretic analysis of the end-label: capillary, 30-cm effective length, 40-cm total length, 50-µm inner diameter; buffer, 10 mM sodium citrate (pH 3.1); applied voltage 400 V/cm (16 kV). (A) 1,5-diaminonaphthalene (10 µM), (B) the end label (10 µM), and (C) heparin disaccharides derivatized with the end label. 1, R-∆UA-2S-[1f4]-GlcNS-6S (∼30 µM); 2, R-∆UA-[1f4]-GlcNS6S (∼35 µM); 3, R-∆UA-2S-[1f4]-GlcNAc (∼20 µM); 4, R-∆UA[1f4]-GlcNAc-6S (∼20 µM); * is the impurity from peak 3 and + is the impurity from peak 4.

potassium (1334.1 Da). The peak at mass 1177.56 is most probably the unreacted labeling sugar (maltoheptaose + Na+). There was no peak observed at 2455.29 Da, which would correspond to the adduct of sodium and 1,5-diaminonaphthalene doubly derivatized with maltoheptaose. The CE experiments shown in Figure 5 were performed with laser-induced fluorescence detection (325/>366 nm) in the positive mode 5(A,B), and in the negative mode (C). The excitation and emission maxima of the purified end label (324/ 400 nm) were determined from its fluorescence spectrum. Figure 5A shows the trace of 1,5-diaminonaphthalene (according to the information by the supplier, its purity was 97%). The trace of a purified end label is shown in Figure 5B. It is evident that the end label does not contain any free 1,5-diaminonaphthalene. Small satellite peaks close to the major peak (Figure 5B) are probably maltohexaose and maltooctaose derivatized with 1,5-diaminonaphthalene (purity of maltoheptaose was 92%, according to the supplier). The small peak emerging at ∼24 min could be 1,5diaminonaphthalene, doubly derivatized with maltoheptaose. Figure 5C shows the separation of the four heparin disaccharide standards (1, R-∆UA-2S-[1f4]-GlcNS-6S; 2, R-∆UA-[1f4]-GlcNS6S; 3, R-∆UA-2S-[1f4]-GlcNAc; 4, R-∆UA-[1f4]-GlcNAc-6S). Each singly sulfonated heparin disaccharide, derivatized with the end label, resulted in two peaks when analyzed under acidic conditions (pH 3.1). (The peak marked with the asterisk is an impurity from peak 3, and the peak marked with the plus sign is due to an impurity from peak 4). The smaller peak (marked with the asterisk) represented 22.4% of peak 3, and the peak marked with the plus sign represented 20.1% of peak 4 [calculated as (area/ mobility)impurity + (area/mobility)major peak ) 100%]. The secondary peaks from these heparin disaccharides have not been identified. RESULTS AND DISCUSSION Since intact heparins are multiply substituted, highly charged and mass-polydisperse glycoconjugates, the electrophoretic separation of the individual components in a mixture is extremely 3202 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

R-∆UA-2S-[1f4]-GlcNS-6Sa R-∆UA-2S-[1f4]-GlcNS-6Sb R-∆UA-[1f4]-GlcNS-6Sa R-∆UA-[1f4]-GlcNS-6Sb R-∆UA-2S-[1f4]-GlcNAca R-∆UA-2S-[1f4]-GlcNAcb R-∆UA-[1f4]-GlcNAc-6Sa R-∆UA-[1f4]-GlcNAc-6Sb end label heparin Mw ∼3000a Mw ∼3000b Mw ∼6000a Mw ∼6000b Mw ∼13 500-15 000a

10 mM sodium phosphate (pH 7.6)

10 mM sodium citrate (pH 3.1)

57.43 ( 0.13 25.86 ( 0.31 48.25 ( 0.17 20.32 ( 0.36 32.28 ( 0.21 13.95 ( 0.28 32.28 ( 0.21 13.95 ( 0.28 0

45.92 ( 0.25 15.79 ( 0.15 34.86 ( 0.26 10.02 ( 0.20 22.67 ( 0.06 5.56 ( 0.16 22.29 ( 0.03 4.98 ( 0.14 -7.45 ( 0.26c

53.67 ( 4.19d 40.39 ( 0.88 50.76 ( 8.70d 40.82 ( 0.27 48.51 ( 8.11d

39.35 ( 3.18d 31.73 ( 0.63 36.03 ( 3.58d 34.31 ( 0.51 37.11 ( 4.56d

a Solute without the end label. b Solute with the end label. c Sign shows an opposite direction of the end-label migration compared to the heparin solutes. d Errors do not reflect the measurement reproducibility but rather the uncertainties attached to the choice of a single mobility for the broadly distributed heparin zones.

challenging. Typically, the intact heparins migrate as a broad “hump” in free-solutions, as seen from mobilities of three underivatized heparin fractions (Mw ∼3000, 6000, and 13 500-15 000) in Table 1. Generally, there are three ways of fractionating such polydisperse solutes through electrophoresis: (a) by size; (b) through charge density; or (c) by to the ratio of charge density to size. First, the size-dependent separations can be performed in a sieving medium such as a permanent gel or an entangled polymer solution. Since the heparin oligosaccharides of pharmaceutical interest are fairly small molecules,1,9,10 the pores in a sieving medium would also have to be small. This implies the use of highly concentrated polymer matrixes. Although highly concentrated polyacrylamide slab gels have been used for heparin separations,11-14 their translation into CE does not seem to be an easy task.32 Additionally, it is extremely difficult to fill a separation capillary with highly viscous and concentrated polymer solutions, which could potentially help the separation process. In a different approach, the separation of heparin oligosaccharides can be carried out through affecting the charged groups situated on the chains. This could be accomplished through employing the secondary equilibria between the negatively charged groups on heparins and certain cationic counterions, such as ethylenediamine, putrescine, spermine, spermidine, etc.33 This approach was tested for heparins33 but with only marginal success. Consequently, the end-label approach described here appears to be a promising solution for enhancing the differences between heparin oligomers. Through a modification of the solute’s molecular friction (without perturbing the rest of the oligomeric structure), the differences in electrophoretic mobility can be substantially increased. Importantly, the ELFSE strategy is free of any secondary equilibria of the heparin chains with “selective” counterions that should be very useful when there is a need to (32) Dolnik, V.; Cobb, K. A.; Novotny, M. V. J. Microcolumn Sep. 1991, 3, 155159. (33) Stefansson, M.; Novotny, M. V. Anal. Chem. 1994, 66, 3466-3471.

Figure 6. Capillary electrophoretic separation of low molecular weight heparin (Mw ∼3000) end-labeled with 1-maltoheptaosyl1,5-diaminonaphthalene: capillary, 50-cm effective length, 60-cm total length, 50-µm inner diameter; buffer, 10 mM sodium citrate (pH 3.1), applied voltage, 350 V/cm (21 kV); laser-induced fluorescence detection, 325/>360 nm; heparin concentration, 2mg/mL. The peak numbers are as in Figure 5C.

study the affinity of heparin components with certain peptides (structure vs activity studies). Although the ELFSE approach using the novel end label (1-maltoheptaosyl-1,5-diaminonaphthalene) resulted in an excellent separation of small or less charged oligomers from the low molecular weight heparin sample (Figure 6; the peak numbers are as in Figure 5C), identification of the individual components will present a considerable challenge in future work. For instance, peaks migrating later than 10 min (Figure 6) are evidently grouped in the clusters. These clusters represent oligomers of similar charge-to-friction ratios (e.g., diand tetrasaccharides). However, the ELFSE model can be utilized to interpret experimental data (and predict migration of unknowns) when the sample is either size- or charge-polydisperse. When both polydispersities are present at the same time, the availability of standards to “spike” the mixtures is the only reasonable option. Except for the heparin disaccharides used in this work, there are no commercial standards. We spiked the intact heparin with selected heparin disaccharide standards, but little correspondence between the peaks was observed (Figure 6). Nonetheless, we feel justified in proposing a strategy for the analysis of complex heparin mixtures based on end-label freesolution electrophoresis. We can measure the electrophoretic mobility of a monomer, with and without the end label (the unlabeled heparin oligosaccharides can easily be detected by UV absorption at 232 nm16), obtaining µN)1 (mobility of the end-labeled monomer) and µo (mobility of the unlabeled solute) values. Note that the mobility of the unresolved “hump” (i.e., very large oligosaccharides on which the end label has negligible effect) does not provide a reliable µo, because the presence of any homooligomer chain in a heparin mixture cannot be automatically assumed. Subsequently, the following equations can be utilized to calculate mobility of any component which is a multiple of the selected monomer unit (for µN>1)22,23

µT/µo ) (N + µELζEL/µoζo*)/(N + ζEL/ζo*)

(1)

where µT is the total mobility of labeled heparin oligosaccharides,

Figure 7. Calculated mobility hyperbolic curves for heparin homooligomers “derivatized” with the end labels of frictional coefficients equal to 1 (solid line), 2 (dashed line), and 5 (dotted line), relative to the frictional coefficient of 1-maltoheptaosyl-1,5-diaminonaphthalene. The upper curves correspond to the oligomers built from R-∆UA-2S-[1f4]GlcNS-6S heparin disaccharide and the lower hyperboles show mobility of oligomers built from R-∆UA-[1f4]-GlcNS-6S heparin disaccharide. We assume that the mobility of unlabeled heparin disaccharide is the same as the mobility of any subsequent unlabeled heparin oligomers. Obviously, this is an oversimplification. The relative trends of hyperbolic curves should be viewed as more important than the actual mobility values.

N is the number of monomers in an oligomeric chain, µEL and ζEL are the mobility and the friction of the end label, respectively, and ζo is the friction of an unlabeled heparin “monomer”. This equation assumes the “local picture” of electrophoretic mobility, which should be valid as long as both the solute and the end label are approximated as stiff rods. The ratios of the electrophoretic friction of the end label relative to that of the monomer (ζEL/ζo*) can be calculated by rearranging eq 1 into

ζEL/ζo* ) N(µT - µo)/(µEL - µT)

(2)

However, to be justified in using eq 1, the monomer unit will have to satisfy the free-draining condition (L . κ-1). As an example, the length of a heparin disaccharide is ∼1.02 nm.34 When working at buffer concentrations of ∼0.1 M (κ-1 ∼0.96 nm for a 0.1 M 1:1 electrolyte solution), the monomer unit should thus be at least a hexasaccharide or even larger oligomer. In other words, if one succeeds in isolation and characterization of a heparin oligosaccharide (e.g., a hexasaccharide), it should be feasible to predict mobilities for its multiples from the ELFSE model. Nevertheless, eqs 1 and 2 and the data listed in the Table 1 can be used to demonstrate the effect of an increased friction of the end label on the heparin oligomers’ mobility. Figure 7 shows the mobility hyperboles calculated from eq 1 for the heparin homooligomeric ladders built from two different disaccharides and derivatized with the novel end label and for any end labels that may have 2 and 5 times greater frictional coefficients. It is evident from Figure 7 that, for smaller end labels, the mobility reaches a plateau value (i.e., a size-independent regime) at smaller N (number of monomers). In other words, the resolution between adjacent oligomers will diminish, at smaller N, for smaller end labels. (34) Tivant, P.; Perera, A.; Turq, P. Biopolymers 1989, 28, 1179-1186.

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Due to the highly charged nature of heparin oligosaccharides, their electrophoretic mobility is strongly influenced by counterion condensation.35 According to the Manning counterion condensation theory,35 the electrophoretic mobility of a polyelectrolyte is controlled through a dimensionless charge density parameter ξ:

ξ ) e2/(4πkBTd) ) lb/d

(3)

where e is the electron charge,  is the fluid permittivity, d is the axial spacing between neighboring (bound) charged groups and, lb is the Bjerrum length. The Bjerrum length is the separation required between two unscreened electron charges to make their electrostatic interaction energy equal to the thermal energy kBT. For ξ < ξcrit (ξcrit ) |z-1|, with z ) 1 for a univalent counterion), the electrophoretic mobility of a rodlike polyelectrolyte is proportional to the actual polyelectrolyte charge density, ξ. For ξ g ξcrit, the Manning counterion condensation theory predicts that the mobility is independent of ξ, but it becomes rather proportional to the critical charge density ξcrit, which is a constant for a given condition. On average, the dimensionless charge density of the intact heparins (with the anticoagulant activity equal to ∼160 IU/mg) is greater34 than 2 (for a univalent counterion). Thus, the apparent electrophoretic mobility should be independent of the actual charge density (i.e., the actual number of substitutions) within the heparin chains, so long as ξ g ξcrit. This suggests that the ELFSE model could also be used to calculate the mobilities of the heterooligomers with average charge densities greater than ξcrit. However, the counterion condensation theory assumes that the polyelectrolyte is uniformly charged and does not consider the effect of the local charge density (at the length scales greater than κ-1) on the overall mobility of heterooligomers. Therefore, an additional study on the influence of the local charge density on heparin heterooligomers’ mobility is needed. Furthermore, the electrophoretic mobility of a heparin oligomer also depends on its conformation, which should not be overlooked when the mobility of a heterooligomer is being calculated. CONCLUSIONS It has been shown here that highly charged, stiff oligosaccharides can be separated in free-buffer solutions. However, the size (35) Manning, G. S. Q. Rev. Biophys. 1978, 11, 179-246.

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selectivity in the electrophoretic mobility must be induced by breaking the symmetry between the solute’s charge and friction through an end-labeling procedure. In fact, the end-labeling strategy is very common to carbohydrate analysis by capillary electrophoresis. Before this study, the only rationale for labeling was to enhance spectroscopic detection (either UV/visible absorbance or fluorescence) of the analyzed sugars. However, it is clear that the ELFSE strategy also introduces a new possibility of tuning the size range of electrophoretically separated carbohydrate entities through the chemical design of end labels. We synthesized a hydrophilic end label (1-maltoheptaosyl-1,5diaminonaphthalene) that combines enhanced frictional properties and fluorescent capabilities. This novel end label was applied here to the fractionation of low molecular weight heparins. A significant effect on the mobilities of small heparin oligomers was noticeable, making them well-separated from each other. However, it is obvious that the high charge densities on the heparin chains will demand even larger end labels (The current end label had only a small effect on the larger, highly charged oligomers, e.g., N > 3, with N being the number of disaccharide, monomer units in a chain). Based on the ELFSE strategy for stiff solutes, the mobilities of heparin multimers (built from a given monomer) can be calculated. This should have some importance for the analysis of complex heparin mixtures. However, the free-draining condition for the monomer must be satisfied in such a case; i.e., a larger oligomer should be considered as a monomer. In addition, the mobilities of heparin heteromultimers can also be calculated using the ELFSE model, but the effect of the local charge densities (at length scales greater than κ-1) on the chain mobility and a possible change of oligomer conformation with change of its primary structure must be evaluated first. ACKNOWLEDGMENT This work was supported by Grants CHE-9321431 from the National Science Foundation and GM 24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services. Received for review December 30, 1996. Accepted June 3, 1997.X AC961297F X

Abstract published in Advance ACS Abstracts, July 1, 1997.