Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection

ANALYTICALBIOCHEMISTRY

184,200-206

(1990)

Analysis of Oligogalacturonic Acids with 50 or Fewer Residues by High-Performance Anion-Exchange Chromatography and Pulsed Amperometric Detection Arland

T. Hotchkiss,

Jr.,l and Kevin

B. Hicks

U.S. Department of Agriculture, ARS, Eastern Regional Research Center, 600 Easi Mermaidiaie, Philadelphia, Pennsylvania 19118

Received

August

21,1989

Underivatized oligogalacturonic acids with a degree of polymerization (DP) ranging from 2 to 60 have been separated for the first time on a high-performance CarboPac PA1 pellicular anion-exchange stationary phase column. Baseline separation of these pectic fragments was accomplished using a nonlinear gradient of pH 6 potassium oxalate buffer as the mobile phase. Acetate buffer linear gradients were also useful as mobile phases, but only for separations of oligogalacturonic acids that were soluble in this solvent (DP c 20). Additionally, oligogalacturonic acid separations were accomplished on a lower capacity AS4A stationary phase column. Triple pulse amperometric detection was selective, sensitive, and reproducible, nevertheless, oligogalacturonic acid response factors were affected by DP and compositional changes in the mobile phase. o 1990 Academic

Press,

Inc.

Pectin is a complex, extremely important and yet not fully characterized natural polysaccharide. The structure of the pectin gel matrix is important in determining plant cell wall strength and flexibility. Furthermore, calcium forms crosslinks between adjacent polygalacturanic acid chains (a component of pectic rhamnogalacturonan), conferring stiffness to the wall matrix. The disruption of these noncovalent forces may allow for cell expansion to occur during growth and development (1). The partial hydrolysis of polygalacturonic acid regions in pectin releases a series of oligogalacturonic acids. ’ To whom correspondence should be addressed. 2 Abbreviations used: DP, degree of polymerization; HPLC, performance liquid chromatography; HPAE, high-performance ion-exchange; PAD, pulsed amperometric detection.

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These acidic oligomers are now recognized as being capable of regulating a number of physiological responses in plants, which include the elicitation of increased phenylalanine ammonia lyase activity (2), proteinase inhibitor production (3), lignification (4), and phytoalexin production (5-7). In each of these responses, a specific degree of polymerization (DP)2 range of oligogalacturanic acids was associated with optimal biological activity. The ability to rapidly separate and detect oligogalacturonic acids with the widest possible range of DP values is therefore important in all these and related future investigations. Previous chromatographic separations of oligogalacturanic acids [reviewed in (8)] have included ambient pressure anion-exchange chromatography (6,7,9-12); thin-layer chromatography (13-15); gasiliquid chromatography (16); and high-performance liquid chromatography (HPLC) using gel-filtration (17), strong anionexchange (18), weak anion-exchange (18), ion-pair reversed-phase (l&19), and combined size- and ion-exclusion (20) modes. The largest oligogalacturonic acid separated with these techniques was DP 11. Recently, larger oligogalacturonic acids (over DP 25) were separated as their 2-aminopyridinyl derivatives by anion-exchange HPLC utilizing uv absorption (290 nm) for detection (21). We report the separation of oligogalacturonic acids by high-performance anion-exchange (HPAE) chromatography on pellicular anion-exchange resin stationary phases coupled with pulsed amperometric detection (PAD), which previously had provided excellent separation and detection of monosaccharides and oligosaccharides (22-26). Using this system it was possible to separate higher DP oligogalacturonic acids than had been possible previously. The HPAE-PAD method has the added advantage of not requiring sample derivatization. 0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. AI1 rights of reproduction in any form resented.

CHROMATOGRAPHY

MATERIALS

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UNDERIVATIZED

METHODS

Chromatogruphic apparatus. A Dionex (Sunnyvale, CA) Bio-LC system which included a quaternary gradient pump, eluant degas (He) module, postcolumn delivery system (DQP-1 pump), and pulsed amperometric detector II (with gold working electrode) was used. Chromatograms were recorded with a Hewlett-Packard (Palo Alto, CA) 3390A integrator. Oligogalacturonic acids were separated with two Dionex pellicular anionexchange columns (4 X 250 mm CarboPac PA1 and IonPat AS4A with AS4A precolumn). Gradient elution of different size oligomers was accomplished with either acetate- or oxalate-buffered mobile phases (0.8 ml/min). For optimal detector sensitivity, 500 mM hydroxide (either Na+ or K+ depending on the buffer salt used) was added postcolumn via a mixing tee. The postcolumn pump was adjusted such that the final flow rate from the PAD cell was 1.6 ml/min. The acetate buffer was prepared by diluting acetic acid in water, titrating with 50% NaOH to pH 5, and adjusting the volume with deionized water to provide a final concentration of 1.0 M acetate. Alternatively, a 500 mM oxalate (pH 5 or pH 6) buffer was prepared by a similar procedure except that oxalic acid was titrated with KOH. A pH 7.8 oxalate buffer (500 mM) was prepared by directly dissolving the appropriate amount of potassium oxalate in deionized water. Potassium rather than sodium oxalate was used because of its higher solubility. Mobile phase buffers (Eluant A) were mixed with deionized water (Eluant B) to produce chromatographic gradients. Acetate buffer linear gradients were produced by increasing the concentration of Eluant A from 10 to 60% (AS4A column) or from 27 to 82% (PA1 column) in 29 min. Both acetate buffer gradients followed a 0.9-min delay after the injection, during which the Eluant A concentration was held constant at its initial value. The oxalate buffer gradient was produced by the following Eluant A concentration changes: 5% at O-l min, 20% at 9 min, 40% at 40 min, 50% at 65 min, 56% at 95 min and 70% at 110 min. The triple-pulse sequence used for amperometric detection included the following potentials and durations: El = 0.1 V (ti = 300 ms), E2 = 0.6 V (t2 = 120 ms), and Es = -0.8 V (t3 = 300 ms). The integration time was set at 200 ms and the response time at 1 s.

Preparation of oligogulucturonic acids. Polygalacturanic acid (citrus, Sigma) was hydrolyzed according to a modification of the procedure reported by Robertsen (4). Hydrolysis involved autoclaving 1% polygalacturonit acid (in HPLC grade water, pH adjusted to 4.4 with NaOH or KOH) at 121°C for 40 min. Following hydrolysis, the appropriate amount of mobile phase buffer was added to hydrolysates such that the sample buffer concentration was similar to that of the initial mobile phase concentration. The addition of acetate buffer to hydroly-

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sates caused precipitation of higher DP oligogalacturonit acids which were then removed by filtration prior to injection. In order to analyze all oligomers present in hydrolysates, the pH was adjusted to 6 with KOH before adding the appropriate amount of oxalate buffer to produce a final oxalate concentration of 12.5 mM (no precipitate formed). Hydrolysates were fractionated by lowering the pH to 2 with HCl following autoclave hydrolysis. At pH 2 high DP oligogalacturonic acids precipitated, whereas lower DP oligogalacturonic acids remained in solution. The precipitate was collected by centrifugation (10,000 rpm, 20 min) and then both fractions were carefully neutralized (pH 7) with KOH, which solubilized the high DP oligogalacturonic acids. Identification of chromatographic peaks was aided by the injection of oligogalacturonic acid standards. DP 3 and 7 oligogalacturonic acid standards purified by preparative strong anion-exchange column chromatography with molecular weights confirmed by FAB-MS (12) were generously provided by Dr. L. Doner (USDA, ERRC). DP 15-19 oligogalacturonic acid standards were purified by anion-exchange column chromatography (21) and generously provided by Dr. N. Maness and Dr. A. Mort (Oklahoma State University). We purified DP 4-6 oligogalacturonic acid standards by preparative aminopropylsilica gel HPLC (27). Oligogalacturonic acid standards (desiccated with P205 under vacuum) also were used to determine molar response factors with PAD. Mean response factors were calculated from peak height per nanomole values expressed relative to galacturanic acid crystalline monohydrate (Sigma). RESULTS The use of alkaline mobile phases, typically employed for HPAE-PAD separations of neutral carbohydrates (23-26), was unsuccessful for oligogalacturonic acid separations due to the low pK, (3.4) of galacturonic acid, which resulted in long retention times. However, anionexchange separation of oligogalacturonic acids was achieved readily at pH 5 when linear acetate buffer gradients were utilized as mobile phases. Under these conditions, similar separations of DP l-6 oligogalacturonic acids were possible on both the AS4A and PA1 columns (Fig. 1). The AS4A column operated at lower back pressure and with a lower concentration of acetate buffer in the mobile phase than did the PA1 column. The maximum DP oligogalacturonic acid separated with a pH 5 acetate gradient was about 19 (Fig. 2). Oligogalacturonic acids with DP values up to 50 were separated with a pH 6 oxalate buffer gradient mobile phase using a PA1 column (Fig. 3). Nonlinear oxalate buffer gradients were used because they provided better separations of the later eluting peaks than did linear gradients. The pH 6 oxalate buffer was considered optimal

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Identical retention times (within experimental error) were observed for oligogalacturonic acid standards (DP 3-7,15-19) and the chromatographic peaks which were labeled accordingly (comparative retention times of DP 3 and 7 shown in Fig. 3). The DP values of the other peaks were assigned by extrapolation. Since the hydrolysate was completely soluble in oxalate buffer, the largest oligogalacturonic acid observed appeared to be the largest DP oligomer present in the polygalacturonic acid hydrolysate. Fractionation of the hydrolysate produced a DP 2-23, pH 2 soluble fraction and a DP 18-50 (with trace amounts of lower DP oligomers), pH 2 insoluble fraction (Fig. 4). The oxalate buffer gradient used in Fig. 4 was effective in separating oligogalacturonic acids up to DP 50 when the PA1 column was relatively new. After 6 months of use, column aging necessitated altering the oxalate buffer gradient from that used in Fig. 4 to that used in Fig. 3 in order to achieve optimal resolution of the later eluting peaks. PAD molar response factors varied according to oligogalacturonic acid DP and mobile phase gradient conditions (Fig. 5). Mean response factors ranged from 1.0 to 1.63 with the 0.27-0.82 M acetate buffer gradient, from 0.85 to 1.1 with the 0.45-1.0 M acetate buffer gradient, and from 0.97 to 1.53 with the oxalate buffer gradient. Preliminary studies suggest that higher DP (15-19) oligogalacturonic acids had molar response factors that were in the range of 40 to 60% of the galacturonic acid monohydrate molar response factor. Detector response was linear (R > 0.99) with both oxalate and acetate buffer gradient mobile phases for galacturonic acid monohydrate over a range of sample injections from 236 pmol to 236 nmol. The minimum amounts of sample detected (2X noise height) were 236 and 111 pmol for DP 1 and 5, respectively. DISCUSSION

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This report is the first to demonstrate separation and detection of underivatized oligogalacturonic acids up to

Time (min) FIG. 1. Separation of DP l-6 oligogalacturonic acids on two stationary phases (peaks are labeled according to DP value; detector sensitivity = 3 PA). (A) AS4A stationary phase with a pH 5 acetate buffer gradient consisting of 100-600 mM acetate in 30 min. (B) PA1 stationary phase with gradient conditions consisting of 270-820 mM acetate buffer (pH 5) in 30 min. (C) Separation of DP 3 and 7 oligogalacturonic acid standards (each peak = 20 ag) using the same conditions as in B.

because it provided shorter retention times than were possible with a pH 5 oxalate buffer. While little difference in retention time was observed between pH 6 and 7.8 oxalate buffers, baseline drift occurred during the first 25 min of the run at pH 7.8. Buffer salt concentration was lower in oxalate mobile phase gradients than that in acetate gradients.

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nttte(mln) FIG. 2. Oligogalacturonic acid separations up to DP 19 on a PA1 column with a pH 5 acetate buffer gradient (peaks are labeled according to DP value; detector sensitivity changed from 1 to 0.3 PA at the arrow; Eluant A concentration changes: 45% at O-l min, 100% at 60 min).

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of oligogalacturonic acids with a pH 6 oxalate buffer gradient on a PA1 column (peaks are labeled = 1 uA: other conditions are described under Materials and Methods). (A) Oligogalacturonic acids and’7 cligogalacturonic acid standards (each peak = 2.5 pg) under the same conditions as in A.

DP 50. This was possible with HPAE-PAD using nonlinear oxalate buffer gradients, which allowed for the greater solubility of higher DP oligogalacturonic acids compared to the use of an acetate buffer. Maness and Mort (21) were able to separate 2-aminopyridinyl-oligogalacturonic acid derivatives with DP values exceeding 25 using a linear acetate gradient (77-406 mM) mobile phase with a Spherogel TSK DEAE-2SW anion-exchange column. However, with underivatized oligogalacturonic acids, we were only able to separate up to DP 19 with an acetate buffer gradient due to the limited solubility of larger size oligogalacturonic acids. Confirmation of the molecular weight of oligogalacturonic acids as large as DP 50 by comparing retention times with standards was not possible since the largest oligogalacturanic acid standard available with FAB-MS evidence supporting its molecular weight was DP 7 (12,21). However, retention time agreement was observed between hydrolysate chromatographic peaks and all those oligogalacturonic acid standards used (maximum DP = 19).

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Based on the assumption that a homologous series of oligogalacturonic acids was present [GC-MS analysis of per-0-methylated alditol acetates demonstrated that rhamnose was the only other monosaccharide present (trace amounts) in addition to galacturonic acid, data not shown] in the hydrolysates produced in this investigation, the DP value of the latest eluting peaks was determined to be about 50. Debate exists concerning the DP of commercially prepared polygalacturonic acid. Various DP average values (25-70) for polygalacturonic acid have been proposed based on end-group, osmometry, size-exclusion, and EPR analytical methods (2% 31). Unlike previous methods that gave average molecular weight values, the method presented here provides unique information concerning both the amount and the distribution of individually resolved oligogalacturonic acids. The observed PAD oligogalacturonic acid molar response factors are unusual because smaller DP oligomers exhibited enhanced response, whereas larger DP

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Time (mln) FIG. 4. Oxalate buffer (pH 7.8) gradient separation of fractionated oligogalacturonic acids (peaks are labeled according to DP value; detector sensitivity = 1 PA; Eluant A concentration changes: 5% at O-l min, 20% at 10 min, 45% at 50 min, 57% at 100 min, and 75% at 120 min). (A) Oligogalacturonic acids soluble at pH 2. (B) Oligogalacturonic acids insoluble at pH 2.

oligomers had reduced response relative to galacturonic acid. The reduced PAD response of acidic oligosaccharides has been reported previously (26) for an oligomeric series of mannosyl g-phosphate glycosides, whereas structurally diverse neutral oligosaccharides generally had increased molar response with increasing DP (DP 9 largest observed). The increase in molar response in homologous series of glucooligosaccharides (DP 2-7) was observed to be proportional to the increased number of HCOH groups (32). It was proposed that the bulky hydrophilic phosphate groups might impair the adsorption of the analyte to the positively charged hydrophobic gold electrode surface (26). The detection mechanism for oligogalacturonic acids by PAD must involve additional factors besides the oxidation of the reducing end Ci mechanism (22) due to the reduction of PAD response for larger oligogalacturonic acids. It should be emphasized that changing the mobile phase gradient will affect the value of the oligogalacturonic acid molar response factors reported here. We have demonstrated that different oligogalacturonic acid response factors were produced when the mobile phase buffer was changed from acetate to oxalate and when the acetate buffer concentration gradient was changed. Therefore, in order to obtain reproducible and accurate quantitation of oligogalacturonic acids, gradient uniformity must be main-

tained from run to run. Previously reported PAD minimum detection limits for carbohydrates fall in the single to 100 pmol range (23,24). Minimum detector response limits reported here (111-236 pmol) were influenced by excessive baseline noise at lower detector sensitivity range values (~300 nA). To our knowledge, this report is the first to utilize an oxalate buffer gradient with HPAE-PAD. Rocklin and Pohl (23) compared the use of acetate, carbonate, nitrate, and sulfate as eluant additives to alkaline mobile phases and stated that acetate was better for neutral oligosaccharide separations since the other anions possessed too much affinity for the anion-exchange resin stationary phase. This report also represents the first HPAE-PAD separation of completely acidic oligosaccharides (those containing 100% acidic carbohydrate residues). With partially acidic oligosaccharides (those containing both neutral and acidic carbohydrate residues), Townsend et al. (26) used a pH 4.65 sodium acetate gradient to separate sialylated oligosaccharides and a pH 6 sodium acetate gradient to separate phosphorylated oligosaccharides. However, alkaline (pH 13) mobile phases ultimately were used to provide optimal separations of those partially acidic oligosaccharides by shifting the mechanism of retention from that based on the charge (ionized carboxyl groups only) to mass ratio

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FIG. 5. Oligogalacturonic acid molar response factors relative to galacturonic acid monohydrate as a function of DP (error bars represent +standard deviation, n = 3). Oligogalacturonic acid separation with a 0.27-0.82 M acetate buffer gradient (A) compared with a 0.45-1.0 M acetate buffer gradient (B), and an oxalate buffer (pH 6) gradient (C, conditions are described under Materials and Methods).

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umn will replace the PA1 column, which has a wider range of previously demonstrated applications for carbohydrate separations. It has been demonstrated that the HPAE-PAD technique possesses tremendous potential for making significant contributions to future investigations of plant cell wall carbohydrate structure. Positional isomers of neutral and partially acidic oligosaccharides derived from glycoproteins were previously separated by HPAE-PAD (24,26). Initial attempts were made (24,26) to develop rules which explain the relationship between oligosaccharide substitution position and retention time with HPAE-PAD based on the different pK, values of ring hydroxyl groups (36). Structural investigation of plant cell wall carbohydrates has proved difficult in many instances due to the large size and complex branching pattern of repeating units. We have suggested in this report that oligogalacturonic acids were separated up to a molecular weight of 10,000 with HPAEPAD. It is conceivable that larger oligosaccharides could be separated by using a steeper gradient and/or higher final oxalate concentration. The evidence presented here, combined with the earlier work cited for neutral oligosaccharides, demonstrates that this method has great potential for separating a broad spectrum of linear and branched plant cell wall carbohydrates ranging from neutral to completely acidic. ACKNOWLEDGMENTS

to that based on oxyanion formation by sugar hydroxyl groups (26). Oligogalacturonic acids have a much higher affinity for the PA1 stationary phase than do neutral or partially acidic oligosaccharides. Therefore, a pH 6 mobile phase consisting of a stronger buffer (oxalate) than acetate was ultimately required for optimal separations with this anion-exchange chromatography system. We also have demonstrated the novel HPAE-PAD separation of oligogalacturonic acids on a Dionex AS4A pellicular anion-exchange column. Previously Smith and MacQuarrie (33) and Smith et al. (34) separated sugar phosphates on an AS4A column; however, chemically suppressed conductivity detection was used rather than PAD. The lower degree of oligogalacturonic acid retention on the AS4A column compared to that on the PA1 column reflected the lower ion-exchange capacity of the former (35). Differences in column construction, which included smaller packing material particle size (10 pm vs 15 pm), larger size pellicular latex microbeads (350 nm vs 200 nm), and higher degree of latex crosslinking (5% vs 0.5%) in the PA1 column vs the AS4A column, were responsible for the column capacity and back pressure differences observed. Therefore, the AS4A column should be considered as a good alternative to the PA1 column for acidic carbohydrate separations by HPAEPAD. However, it is not anticipated that the AS4A col-

We thank Rebecca Haines for technical assistance and Julia Goplerud for editorial assistance. We thank Mark Mercer of Dionex for loaning us the IonPac AS4A column. An ARS administratorfunded research associateship, which supported A.T.H., is gratefully acknowledged.

REFERENCES 1. Carpita, Growth American

N. C. (1987) in Physiology of Cell Expansion during Plant (Cosgrove, D. J., and Knievel, D. P., Eds.), p. 28, The Society of Plant Physiologists, Rockville, MD.

2. De Lorenzo, G., Ranucci, A., Bellincampi, vone, F. (1987) Plant Sci. 51.147-150. 3. Bishop,

P. D., Pearce,

G., Bryant,

D., Salvi,

J. E., and Ryan,

G., and CerC. A. (1984)

J.

Biol. Chem. 259,13,172-13,176. 4. Robertsen, B. (1986) Physiol. Mol. Plant Path&. 28,137-148. 5. Nothnagel,

E. A., McNeil,

M., Albersheim,

P., and Dell,

A. (1983)

Plant Physiol. 7 1,916-926. 6. Jin,

D. F., and West,

C. A. (1984)

Plant Physiol. 74,989-992.

7. Davis, K. R., Darvill, A. G., Albersheim, Plant Physiol. 80,568-577. 8. Hicks,

K. B. (1988)

P., and Dell,

A. (1986)

Adv. Carbohydr. Chem. Biochem. 46,17-72.

9. Derungs, R., and Deuel, H. (1954) Helu. Chim. Actu 37,657-659. 10. Nagel, C. W., and Wilson, T. M. (1969) J. Chromutogr. 41,410416. 11. Rexovi-Benkovi, L. (1970) Chem. Zvesti 24,59-62. 12. Doner,

L. W., Irwin,

togr. 449,229-239.

P. L., and Kurantz,

M. J. (1988)

J.

Chroma-

206

HOTCHKISS

13. Koller,

A., and Neukom,

H. (1964)

Biochim.

Biophys.

AND

Acta 83,

366-367. 14. Lui, Y. K., and Luh, B. S. (1978) J. Chromatogr. 161,39-49. 15. Doner, L. W., Irwin, P. L., and Kurantz, M. J. (1988) Carbohydr. Res. 172,292-296. 16. Raymond, W. R., and Nagel, C. W. (1969) Anal. Chem. 41,17001703. 17. Thibault, J.-F. (1980) J. Chromatogr. 194,315-322. 18. Voragen, (1982)

A. G. J., Schols,

H. A., De Vries,

J. Chromatogr. 244,327-336. 19. Heyraud, A., and Rochas, C. (1982) 412. 20. Hicks, K. B., and Hotchkiss, A. T. 382-386. 21. Maness, N. O., and Mort, A. J. (1989) 254. 22. Hughes, S., and Johnson, D. C. (1981) 22. 23. Rocklin, R. D., and Pohl, C. A. (1983)

J. A., and Pilnik,

W.

J. Lz$. Chromatogr. 6, 403(1988) J. Chromatogr. 441, Anal. Biochem. 178,248-

HICKS

26. Townsend, R. R., Hardy, M. R., Hindsgaul, O., and Lee, (1988) And. B&hem. 174,459-470. 27. Hotchkiss, A. T., Hicks, K. B., Doner, L. W., and Irwin, (1988) Third Chemical Congress of North America, Toronto, ada. [Abstract 951 28. Powell,

D. A., Morris,

E. R., Gidley,

Y. C. P. L. Can-

M. J., and Rees, D. A. (1982)

J. Mol. Biol. 155,517-531. 29. Fishman, M. L., Pfeffer, P. E., Barford, R. A., and Doner, L. W. (1984) J. Agrie. Food Chem. 32,372-378. 30. Konno, H., Yamasaki, Y., and Katoh, K. (1986) Phytochemistry

25,623-627. 31. Irwin, P. L., Sevilla, J. 54,337-344. 32. Koizumi,

K., Kubota,

M. D., and Chamulitrat, Y., Tanimoto,

W. (1988)

T., and Okada,

Biophys.

Y. (1989)

J.

Chromatogr. 464,365-373. 33. Smith,

R. E., and MacQuarrie,

R. A. (1988)

Anal. Biochem. 170,

308-315. And. Chim. Acta 132, llJ. Liq. Chromutogr. 6,X77-

1590.

24. Hardy, M. R., and Townsend, R. R. (1988) Proc. Natl. Acad. Sci. USA 86,3289-3293. 25. Hardy, M. R., Townsend, R. R., and Lee, Y. C. (1988) Anal. Bio&em. 170,54-62.

34. Smith,

R. E., Howell, S., Yourtee, D., Premkumar, N., Pond, T., Sun, G. Y., and MacQuarrie, R. A. (1988) J. Chromatogr. 439,83-

92. 35. Weiss, J. (1986) in Ion Chromatography (Johnson, E. L., Ed.), Dionex Corp., Sunnyvale, CA. 36. Rendleman, J. A. (1971) in Carbohydrates in Solution, Advances in Chemistry Series (Gould, R. F., Ed.), Vol. 117, pp. 51-69, American Chemical Society, Washington, DC.