Anal. Chem. 1993, 65, 563-567
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Separation of Amino Acid Homopoiymers by Capillary Gel Electrophoresis Vladislav Dolnik7 and Milos V. Novotny*
Anal. Chem. 1993.65:563-567. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/29/19. For personal use only.
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
we have chosen several mixtures of poly (amino acids) (of both anionic and cationic nature) as model solutes. We report
Gel-filled capillaries utilizing highly concentrated and moderately cross-linked acrylamide-type gels In capillary electrophoresis were successfully applied to the separation of the Individual oligomers of various poly(amino acids). Mixtures of both anionic and cationic nature were adequately resolved. While UV detection at 220 nm was mostly utmzed, the polyanions with N-termlnal groups can also be tagged with 3-(4-carboxybenzoyl)-2-qulnollnecarboxaldehyde (CBQCA) for a more sensitive detection by a laser-induced fluorescence detector.
here certain analytical aspects of these investigations. While the poly(amino acids) and their derivatives have traditionally been popular as model compounds for physicochemical studies of biopolymeric behavior,16-18 their practical use has also been increasing during the last several years. Poly (L-glutamic acid) increases the therapeutic efficacy of cis-platinum complexes against murine carcinomas,19 while polyaspartate preparations have been shown to inhibit toxicity of amino glycosides to liposomes,20 to protect against gentamycin nephrotoxicity in rats,21 and to exhibit catalase-like activity.22 In addition, preparations of thermally-produced poly-D,L-aspartate have been evaluated as corrosion inhibitors.23 Within the group of cationic poly (amino acids), polyL-lysine and poly-L-arginine were demonstrated to induce cytotoxicity to human vascular endothelial cells24 and promote autophosphorylation and aggregation at the insulin receptor site.25 Polylysine also interacts with the cellular receptor for Herpes simplex type I virus;26 interestingly, its potency is greatly dependent on the molecular-weight range used. Basic poly(amino acids) have shown considerable potential in
INTRODUCTION In 1983, Hjertén was the first to demonstrate the use of capillaries filled with gels while separating the components of bovine serum albumin.* 11Later, Cohen et al.2 showed the value of gel-filled capillaries in high-resolution analysis of charged biological macromolecules by capillary electrophoresis (CE). Since then, various procedures have been reported for the preparation of immobilized gel3-6 inside fused-silica capillaries. Most recently, entangled polymer matrices7-9 have received additional attention as alternative media for CE of certain biopolymers. Polyacrylamide gels with widely varying concentrations of a monomer and a cross-linking agent have been successfully explored for separating oligonucleotides2·4·610-12 and oligosaccharides.13-15 However, in contrast to slab gel techniques, attempts at separating proteins in gel-filled capillaries have thus far met with only a limited success. This could be due to a variety of reasons, including pore-size limitations, denaturation due to local overheating, matrix incompatibility, etc. In order to elucidate the roles of some of these factors, 1
promoting the cellular uptake of modern protein drugs.27 In spite of an increasing multilateral value of poly(amino acids) in science and technology, analytical methodology for their characterization remains relatively undeveloped. Viscosity measurements and low-angle light scattering have been used to assess the average molecular weight and degree of polymerization.28·29 Polydispersity profiles can be generated by means of size exclusion chromatography combined with low-angle laser light scattering (LALLS) detection.29 Obviously, common separation techniques do not possess the separating power to resolve the oligomeric constituents of various poly(amino acid) preparations.
*
To whom all correspondence should be addressed. On leave from the Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, Bmo, Czechoslovakia. (1) Hjertén, S.; J. Chromatogr. 1983, 270, 1-6. (2) Cohen, A. S.; Najarían, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.; Karger, B. L. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9660-9663. (3) Bente, P. F.; Myertson, J. U. S. Pat. 4,810,456, March 7, 1989. (4) Yin, H.-F.; Lux, J. A.; Schomburg, G. J. High Resolut. Chromatogr. 1990,13, 624-627. (5) Dolntk, V.; Cobb, K. A.; Novotny, M. J. Microcolumn Sep. 1991, 3, 155-159. (6) Baba, Y.; Matsuura, T.; Wakamoto, K.; Morita, Y.; Nishitsu, Y.; Tsuhako, M. Anal. Chem. 1992, 64, 1221-1225. (7) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990,516, 33-48. (8) Grossman, P. D.; Soane, D. S. Biopolymers 1991, 31, 1221-1228. (9) Nathakamkitkool, S.; Oefner, P. J.; Bartsch, G.; Chin, . A.; Bonn, G. K. Electrophoresis 1992,13, 18-31. (10) Paulus, A.; Gassmann, E.; Field, M. J. Electrophoresis 1990,11, 702-708. (11) Drossman, H.; Luckey, J. A.; Kostichka, A. J.; D’Cunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900-903. (12) Swerdlow, H.; Wu, S.; Harke, H.; Dovichi, N. J. J. Chromatogr. 1990, 516, 61-67. (13) Liu, J.; Shirota, O.; Novotny, M. J. Chromatogr. 1991,559, 223-
(16) Fasman, G. D., Ed. Poly-a-amino Acids; Marcel Dekker, Inc.: York 1967 (17) Yang, J.-T.; Wu, C.-S.; Martinez, . M. Methods Ezymol. 1986, 130, 208-269. (18) Song, S.; Asher, S. A. J. Am. Chem. Soc. 1989, 111, 4295-4305. (19) Schechter, B.; Wilchek, M.; Arnon, R. Int. J. Cancer 1987, 39, 409-413. (20) Kaloyanides, G. J.; Ramsammy, L. In Nephrotoxicity; Bach, P. H.; Ed.; Marcel Dekker, Inc.: New York, 1991; pp 117-125. (21) Ramsammy, L. S.; Josepovitz, C.; Lane, B. P.; Kaloyanides, G. J.
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J. Pharmacol. Exp. Ther. 1989, 250,149-153. (22) Lekchiri, A.; Castellano, A.; Morcellet, J.; Morcellet, M. Eur. Polym. J. 1991, 27, 1271-1278. (23) Little, B. J.; Sikes. C. S. ACS Symp. Ser. 1991, No. 444,263-279. (24) Morgan, D. M. L.; Larvin, V. L.; Pearson, J. D. J. Cell Sci. 1989,
94 553-559. (25) Kohanski, R. A. J. Biol. Chem. 1989, 264, 20 984-20 991. (26) Langeland, N.; Moore, L. J.; Holmsen, H.; Haarr, L. J. Gen. Virol. 1988, 69, 1137-1145. (27) Ryser, H. J.-P.; Shen, W. C. In Targeting of Drug with Synthetic Systems; Gregoriadis, G., Senior, J., Poste, G., Eds.; Plenum Publishing Co.: New York, 1986; pp 103-121. (28) Tung, L. H. In Encyclopedia of Polymer Science and Technology; Mark, H. F., Bikales, N. M.; Overberger, C. G., Menges, G., Kroshwitz, J. I., Eds.; John Wiley & Sons: New York, 1987; Vol. 7, pp 298-327. (29) Cooper, A. R. In Encyclopedia of Polymer Science and Technology; Mark, H. F., Bikales, N. M., Overberger, C. G., Menges, G., Kroschwitz, J. I., Eds.; John Wiley & Sons: New York, 1987; Vol. 10, pp 1-19.
235.
(14) Liu, J.; Shirota, O.; Novotny, M. Anal. Chem. 1992,64, 973-975. (15) Liu, J.; Dolntk, V.; Hsieh, Y.-Z.; Novotny, M. Anal. Chem. 1992,
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As demonstrated in this work, CE with gel-filled capillaries oligomeric components of various poly(amino acid) preparations so long as sufficiently concentrated gels are employed. While the component peaks were ordinarily detected here by UV absorbance at 220 nm, sensitivity for certain poly(amino acids) could further be enhanced by fluorescent labeling30 at the terminal amino group, followed by detection through laser-induced fluorescence. The gel composition and operating conditions were investigated to provide optimum separations of polyaspartate, polyglutamate, and polylysine oligomeric mixtures. Polyornithine and polyarginine were also included for the sake of comparison. can resolve successfully the
EXPERIMENTAL SECTION Apparatus and Chemicals Used. Most electrophoretic separations were carried out with a Model 3850 capillary electropherograph (1SCO, Inc., Lincoln, NE) equipped with an
electromigration injection control accessory. The instrument slightly modified to work with short capillaries. Another instrument employed was a previously described home-built instrument30 featuring a laser-induced fluorescence detector. A model 543 argon ion laser (Omnichrom, Chino, CA), operating at 457 nm, was used as the excitation source. Fluorescence emission with the maximum at 552 nm was collected and recorded as previously described.31 HydroLink Long Ranger (50%) was received from AT Biochem (Malvern, PA). All samples of poly (amino acids) were purchased from Sigma (St. Louis, MO) and Polyscience, Inc. (Warrington, PA). Penta-a-L-lysine, tri-a-L-glutamate, and di-a-L-glutamate, used as the only available retention standards, were purchased from Research Plus (Bayonne, NJ). Vinylmagnesium bromide (1 M solution in tetrahydrofuran) and tetrahydrofuran were obtained from Aldrich (Milwaukee, WI). Fused-silica capillaries (50 Mm and 75 #¿m, i.d., 360 jum, o.d.) were received from J&W Scientific (Folsom, CA). Preparation and Evaluation of Gel-Filled Capillaries. Before the gels were polymerized inside fused-silica capillaries, the capillary inner surface was modified by a previously described method32 featuring first the attachment of vinyl groups to silica, followed by a coat of linear polyacrylamide. The original method was slightly modified to permit the treatment of very long capillaries at one time. The isotachophoretic method enabling a sequential gel polymerization was generally described in our previous communication.5 However, preparation of highly concentrated gels requires more attention to detail. The mixtures to be polymerized, containing an appropriate concentration of acrylamide/ Bis or of HydroLink (T = 10-50%) and the appropriate concentration of triethanolamine hydrochloride (typically, 50200 mM), were first degassed by evacuation. Pieces (50 cm) of a coated capillary were subsequently filled with a polymerization medium, with both ends dipped in the respective 2-mL electrode vials containing 1.5 mL of fresh 0.5 M ammonium persulfate (cathodic vial) and 1.5 mL of polymerization mixture (anodic vial). The voltage of 120-200 V was applied during 12-48 h, depending on the monomer concentration. The resulting electric current was checked by using a Keithley 485 autoranging picoammeter. After the polymerization process is finished, the prepared capillaries are visually checked under a microscope for possible voids (“vacuum bubbles”) before equilibrating the formed gels with operational electrolytes. Gel-filled capillaries were connected to the electrode vials containing an operational electrolyte (typically, 0.1-0.2 M Tris-Tricine), while the voltage was gradually increased up to 5 kV during 20-30 h. The capillaries were once again checked for the occurrence of voids and stored was
(30) Liu, J.; Hsieh, Y.-Z.; Wiesler, D.; Novotny, M. Anal. Chem. 1991, 63, 408-412. (31) Liu, J.; Shirota, O.; Wiesler, D.; Novotny, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2302-2306. (32) Cobb, K. A.; Dolnfk, V.; Novotny, M. Anal. Chem. 1990,62,24782483.
Table I. Homopolymers Analyzed by Capillary Gel Electrophoresis1 LALLS data viscosity data poly-(a,/3)-D,L-aspartate Na+ poly-L-aspartate Na+ poly-(a,/8)-D,L-aspartate Na+ poly-L-aspartate Na+ poly-L-glutamate Na+ poly-L-glutamate Na+ poly-L-arginine HBr poly-L-lysine HBr poly-L-lysine HBr poly-D,L-lysine HBr poly-L-lysine HBr poly-L-lysine HBr6
poly-L-ornithine HBr
10~3MW
DP5
8.2 13.0 26.0 28.8 10.6 18.1 11.6 3.97 3.5 5.8 22.8
60 95 190 210 70 120 60 19 17
10"3
MW
DP6
5.4 9.6 22.9 31.7 6.2 12.1 7.0 3.0
39 70 167 231 41
4.8
80 49 14
28 109
17.2
23 82
60
14.5
74
40-60.0 11.7
All samples
were diluted to 100 mg/mL concentration. 6 DP = of degree polymerization.6 Purchased from Polyscience; the method used to determine molecular weight was not indicated. “
room temperature with their ends dipped in the appropriate operational electrolyte. Capillaries could be used for up to 7 days while a working electric field strength of 200 V/cm was employed. The shelf-life of the gel-filled capillaries ranged between 1 and 2 months. The detection cell was made just before setting it in the instrument’s capillary holder. Two sharp, equidistant cuts (ca. 2 mm) were made by a razor blade on the whole perimeter of the capillary, and the polyimide overcoating between the cuts was removed. The detection cell was further cleansed with acetone. Because of the need for sensitive UV detection, most of the capillaries used were 75 Mm, i.d. Electric fields of 100-200 V/cm were applied to the capillaries at their effective length, typically 30 cm. UV absorption at 220 nm was used as the detection principle for all poly(amino acid) mixtures. Electromigration (5-60 s at 5 kV) was used as the sampling procedure. Aliquots (40 mL) of some polymer solutions (100 g/L) were mixed with 10 mL of 20 mM KCN and 5 mL of 10 mM 3-(4carboxybenzoyl)-2-quinolinecarboxaldehyde for fluorescent la30 beling. The mixture was subsequently allowed to stand at room temperature for 1 h prior to sample introduction.
at
RESULTS AND DISCUSSION Resolution of Various Poly(amino acid) Specimens. Depending on the method of polymerization and the nature of the monomers, various oligopeptide homopolymers can be synthesized. For the purpose of our experiments, highly charged homopolymers such as polyglutamate, polyaspartate (both polyanions), polylysine, polyarginine, and polyomithine (polycations) were utilized. Their molecular weight ranges and properties are listed in Table I. Although their electrophoretic properties had not been reported previously, we expected these highly charged species to migrate with reasonable velocities, given a suitable buffer medium, under typical CE conditions. In an open tube, the homopolymers listed in Table I migrate as a broad but nearly Gaussian peak, regardless of their polymerization degree (results not shown). This situation is reminiscent of the problems encountered with oligonucleotides33 and borate-complexed oligosaccharides31 which, like the poly (amino acids), possess unfavorable mass-to-charge ratios. Clearly, the sieving effects of gel media were required for the separation of these uniformly charged polyions. A series of gel-filled capillaries were further evaluated for separation of the poly (amino acids). The low-concentration polyacrylamide gels, which are now effectively utilized in the (33) Dolník, V.; Liu, J.; Banks, J. F., Jr.; Novotny, . V.; Boíek, P. J. Chromatogr. 1989, 480, 321-329.
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c)
a)
10 %
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Figure 1. Capillary electrophoresis of poly-(a,/3)-D,L-aspartate, 5400, In HydroLink gels of different concentrations: (a) 10% HydroLink gel (conditions: total capillary length, 49 cm; effective length, 30 cm; Inner capillary diameter, 75 Mm; outer capillary diameter, 360 Mm; voltage, 9.8 kV; sampling, 20 s at 5 kV); (b) 15% HydroLink gel (conditions: total capillary length, 45 cm; effective capillary length, 27 cm; Inner capillary diameter, 75 Mm; outer capillary diameter, 360 Mm; voltage, 9.0 kV; sampling, 30 s at 5 kV); (c) 20% HydroLink gel (conditions: total capillary length, 46 cm; effective capillary length, 30 cm; Inner capillary diameter, 75 Mm; outer capillary diameter, 360 Mm; voltage, 9.2 kV; sampling 30 s at 5 kV).
separation of nucleotides up to approximately 440 bases4·6’34 and in DNA sequencing up to 700 bases,35 were also unsuccessful in facilitating adequate resolution of the homopolymers tested. However, highly concentrated polyacrylamide and HydroLink gels proved more useful, as seen in Figure l(a-c), producing progressively improved resolution of a 5400 (average molecular weight) polyaspartate for 10%, 15%, and 20% gels. Resolution could further be increased by preparing the columns with 25 % HydroLink matrix (data not shown), but at a considerable increase in the analysis time (from 60 min for 20% gel to over 90 min for 25% gel). HydroLink gels (30%) provided no further improvement, while the total migration time increased further, to 120 min. At the same applied electric field strength (200 V/cm), the current values dropped by approximately 20% when we proceeded from 20 % to 30 % gel concentration. Similar trends were observed with all tested samples of polyaspartate and polyglutamate. When the samples of poly-L-glutamate in various gel-filled capillaries were analyzed, certain unusual effects were observed. Figure 2 exemplifies two anomalies of the recorded polyglutamate profiles: (a) a bimodal distribution of the oligomeric “envelope” and (b) peak splitting observed with certain profile constituents. The oligomer distribution appears to be an inherent characteristic of this particular sample. It may have its origin in a particular synthetic procedure. While exact quantitation of the individual profile regions cannot be carried out without appropriate standards due to the nature of the (clearly discriminating) electromigration sampling technique, the bimodal distribution is still obvious and unique to the polyglutamate sample. Clearly, this type of analytical information would be unavailable with the other commonly used measurement methods. A word of caution is, however, in order. When a sample is injected by electromigration into a highly concentrated gel, smaller (and faster) oligomers are being preferentially introduced into the system. This is evidenced in Figure 3 (a vs b) comparing roughly the areas under the first and second envelope. (34)
J&W Scientific Products Catalog & Reference Guide 1992-1993;
J&W Scientific: Folsom, CA, 1992; p 299. (35) Mao, D. J&W Scientific, private communication.
Figure 2. Capillary electrophoresis of poly-L-glutamate, 6200, In a 20% HydroLink gel. Separation conditions are the same as In Figure 1c.
The peak splitting observed with the polyglutamate in Figure 2 is reminiscent of a similar phenomenon observed with synthetic polynucleotides,36 but our interpretation of its cause is very different. In the case of polynucleotides,36 partially dephosphorylated structures were identified as the cause. Since the polyglutamate profiles featuring peak splitting were found to depend strongly on various experimental conditions (sample size, temperature, buffer composition, etc.), we tentatively attribute the peak splitting effect to the random coil/helix conformational changes. (Because aspartic acid does not prefer -helix as much as glutamic acid,16·37 the polyaspartate samples did not exhibit similar behavior.) Additional experiments are needed to verify these assumptions. Separation of polycationic polymers in gel-filled capillaries provided additional information on behavior of the model poly(amino acids). Once again, highly concentrated gels were (36) Dubrow, R. S. Am. Lab. 1991, 23, No. 5, 64-67. (37) Prevelige, P., Jr.; Fasman, G. D. In Prediction of Protein Structure and the Principles of Protein Conformation; Fasman, G. D., Ed.; Plenum Press: New York and London, 1989; pp 391-416.
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Figure 5. Electrophoresis of (a) poly-L-lyslne, 3500, and (b) poly-o.Llyslne, 4800, In 25% HydroLink gel. Separation conditions are the same as In Figure 4. Figure 3. Capillary electrophoresis of poly-L-glutamate, 6200, In HydroLink gel: (a) 10% HydroLink gel (separation conditions are the same as Figure 1a); (b) 15% HydroLink gel (separation conditions are the same as Figure 1b).
50
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110
min.
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b)
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40
Figure 4. Electrophoresis of poly-L-lysine of different molecular weight ranges In 25% HydroUnk gel: (a) 17 200; (b) 40 000-60 000. Conditions: total capillary length, 49 cm; effective capillary length, 30 cm; Inner diameter, 75 µ ; outer diameter, 360 µ ; voltage, 9.8 kV; sampling, 60 s at 5 kV.
necessary to obtain adequate resolution. Examples of the
polylysine profiles obtained with different molecular weight ranges in 25 % HydroLink gels are shown in Figure 4. Similar results were also achieved with polyarginine and polyomithine specimens (data not shown). Interesting results were obtained while comparing poly-L-lysine and poly-D,L-lysine of a similar average molecular range (Figure 5). The lack of resolution with poly-D,L-lysine may have been caused by the expected heterogeneity within this synthesis product, although distribution of the two enantiomeric forms within the formed polypeptide may not be entirely random.38 It is also worth pointing out that samples of poly-(a,/S)-D,L-aspartate were fully separated (Figure lc).
Conformation of the amino acid homopolymers is expected to be dependent on pH. While uncharged forms are thought to develop -helices, fully dissociated poly (amino acids) should predominantly exist as the random coils.16 In contrast, polyL-lysine perchlorate is reported to form an «-helix even at a neutral pH in highly concentrated solutions.39 Unlike the samples of polyglutamate and polyaspartate, both of which easily formed relatively sharp peaks during capillary electrophoresis, we experienced some difficulties in separating polycations. When a new capillary was used to analyze polylysine, polyomithine, or polyarginine specimens, the first two runs were usually irreproducible, with the peaks being small and poorly resolved. After “priming” the capillaries with two or three cationic samples, resolution improved considerably. A tight sorption of positively charged solutes to the residual (presumably, negative) column sites is the likely explanation for this effect. After all, L-lysine and its polymerization products were reported to adsorb on polymethacrylate gels.40 Comparison of Polyacrylamide and HydroLink Gels. HydroLink is a commercial product of a polyamide nature, but its exact composition has not been disclosed. It has been used predominantly in slab gel electrophoresis of DNA fragments, with an extended reading ability (compared to polyacrylamide) of over 600 nucleotides.41 In making gelfilled capillaries, we found only minor differences between the two gel types. At 20% T, polyacrylamide (C = 5% Bis) may feature slightly smaller pores than HydroLink of the same total concentration. This observation on the poly (amino acids) is in agreement with electrophoresis of DNA fragments in 5% gels (V. Dolník and M. Novotny, unpublished experiments). The successes in preparation of void-free capillaries and the lifetimes of columns are roughly comparable. The main advantage of HydroLink appears to be a substantial lifetime for its concentrated monomer. After 1 year of storage in the refrigerator, it can still be used with confidence.
Quantitative Aspects and Fluorescent Labeling of Poly(amino acids). Detecting the oligopeptides separated
in acrylamide-based gels presents some difficulties. Judicious column conditioning with the operating electrolyte was found to be essential. A detector wavelength of 220 nm was found to be the best compromise between high blank absorbance values and the detection response to the poly(amino acids) studied in this work. (38) Gratzer, W. B. In Poly-a-amino Acids. Protein Models for Conformational Studies·, Fasman, G. D., Ed.; Marcel Dekker, Inc.: New York, 1967; pp 177-238. (39) Bello, J. Biopolymers 1992, 32, 185-188. (40) Seno, M.; Lin, M. L.; Iwamoto, K. Colloid Polym. Sci. 1991,269,
873-879. (41) Gelfi, C.; Canal!, A.; Righetti, P. C.; Vezzoni, P.; Smith, C.; Mellon, M.; Jain, T.; Shorr, R. Electrophoresis 1990, 11, 595-600.
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When U V absorbance detection is used, the molar response values increase with molecular weight due to the increasing number of peptide bonds. In practical terms, this means that a lower detection sensitivity experienced by the fastestmigrating peaks in the overall electropherogram compensates somewhat for the sampling bias due to the electromigration sample introduction discussed above. This assumption was briefly tested by inserting a 2-cm segment of a coated32 capillary, which was filled with the sample previously diluted in the operational electrolyte. While higher oligomers now seemed to be introduced in somewhat higher proportions, the resolution of components was seriously impaired. While some preconcentration can be expected to occur at the solution-gel boundary, the starting sample zone was found to be too wide. The problem could potentially be solved by the use of isotachophoretic preconcentration.42 However, due to the presence of gel, such a capillary could be used just once.
Due to the presence of primary amino groups in the poly(amino acids), fluorescent labeling can enhance detection sensitivity. 3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde, developed in this laboratory for the laser-induced fluorescence detection of primary amines,30 was evaluated for this purpose. Suitable results were obtained in derivatization of the polyglutamate and polyaspartate samples at their N-termini. Due to the ill-defined (and, apparently, incomplete) tagging at the side chains of polylysine, “humps” indicating unresolved components were observed with this homopolymer. Figure 6 illustrates the profile of6200 MW poly-L-glutamate after fluorogenic derivatization. At least 2 orders of magnitude greater sensitivity is feasible with the use of laserinduced fluorescence. Here, the resulting fluorescence signal is primarily due to the number of free amino groups, and for larger oligomers, the molar response shall decrease. This situation is somewhat reminiscent of our previously reported results with a fragmented poly(galacturonic acid),14 where only the terminal group of an oligosaccharide was available for fluorescent labeling. In Figure 6, a bimodal distribution of the polyglutamate oligomers is still observed. The electropherogram now appears to feature single peaks. (The peak with the migration time of 47.1 min is attributable to the reagent blank.) This is presumably due to a smaller injected sample, one of the conditions suppressing the above-mentioned conformational forms observed with polyglutamate.
CONCLUSIONS Capillary gel electrophoresis has been demonstrated to separate various charged poly(amino acids) into their individual oligomers. Over 50 peaks can readily be distinguished in a single run. While cross-linked polyacrylamide and (42) Dolnlk, V.; Cobb, K. A.; Novotny, M. JMicrocolumn Sep. 1990, 2,127-129.
Figure 6. Capillary electrophoresis of poly-L-glutamate, 6200, In 20 % HydroLink gel with laser fluorescence detection. Conditions: total capillary length, 48 cm; effective capillary length, 30 cm; Inner diameter, 75 µ ; outer diameter, 360 µ ; voltage, 9.6 kV; sampling, 30 s at 5 kV.
HydroLink gels can be used for the purpose, a search for additional, less UV-absorbing matrices appears worthwhile. Although some matrix adsorption was observed with polycationic oligomers, these gels appear basically compatible with polypeptide samples; additional problems with protein migration remain to be elucidated. Just as with the negatively charged oligonucleotides2’4'6·7 and sugar-borate complexes,13 the negatively charged polyglutamate and polyaspartate provide nearly ideal electromigration behavior in such gels. Similarly to oligosaccharide analysis,13-16 highly concentrated gels appear essential to resolution of the charged poly (amino acids).
ACKNOWLEDGMENT This work was supported by Grant No. R01-GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services, and a grantin-aid from J&W Scientific, Folsom, CA. A gift of fusedsilica capillaries from J&W Scientific is also acknowledged. Received for review September November 24, 1992.
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