Chapter 21 Application of Multi-Detector SEC with a Post Column Reaction System: Conformational Characterization of PGG-Glucans 1 Y. A . Guo , J. T. Park, A . S. Magee, and G. R. Ostroff Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 Alpha-Beta Technology, Inc., One Innovation Drive, Worcester, M A 01605 Multi-detector Size Exclusion Chromatography (SEC) with several post column reaction systems has been used to characterize the conformation of PGG-Glucans. PGG-Glucans are β(1—>3) glucans isolated from the yeast cell wall (saccharomyces cerevisiae). Three conformational aspects of PGG-Glucans were characterized: 1) Single Chain (SC) conformation, 2) Triple Helical (TH) conformation, and 3) Triple Helical aggregates. PGG-Glucans were separated by SEC and analyzed using the refractive index, multi-angle laser light scattering, fluorescence, and polarimeter in combination with the post column reaction system. The Single Chain conformation was detected and characterized using SEC followed by Aniline Blue (AB) post column reaction system and using SEC coupled with polarimetric detection. The Triple Helical conformation was characterized using SEC coupled with polarimetry and sodium hydroxide post column reaction system. The Triple Helical aggregate was characterized using multi-detector SEC with and without sodium hydroxide post column reaction system. The Aggregate Number Distribution (AND) of PGG-Glucans across the entire molecular weight range was determined. The A N D for Triple Helical PGG-Glucan ranged from 3 to over 10. These results indicate that PGG-Glucan forms aggregate of triple helical structure in aqueous solution. PGG-Glucan, soluble β(1—>6) branched β( 1 —>3) glucan, is an immunomodulator that can enhance the host defenses by selectively priming neutrophil and monocyte/macrophage microbicidal activities without directly inducing leukocyte activation or stimulating the production of pro-inflammatory cytokines ' . PGG1 2 1 Current address: GelTex Pharmaceuticals, Inc., Nine Fourth Avenue, Waltham, MA 02154. © 1999 American Chemical Society Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 311 312 Glucan is isolated from the yeast cell wall of saccharomyces cerevisiae. The conformation of PGG-Glucan strongly affects its biological activities . Deslande, Marchessault and Sarko studied the conformation of curdlan, an unbranched B(l—>3) glucan, by X-ray diffraction and concluded that curdlan forms Triple Helical conformation. Thistlethwaite, Porter and Evans studied Aniline Blue binding properties to β(1->3) glucan and concluded that Aniline Blue binds to β(1—>3) glucan in NaOH aqueous solution. Our previous study showed that Aniline Blue binds specifically to Single Chain conformation . Itou, Teramato, Matsuo and Suga studied the optical rotation of Triple Helical schizophyllan and concluded that the specific rotation was +75 deg*cm / g*dm in aqueous solution at the wavelength of 350 nm and 20°C. Hara, Kiho, Tanaka and Ukai reported a value of the specific rotation of + 19 deg*cm / g*dm for Triple Helical β(1—>3) glucan at the wavelength of 586 nm and 20°C. Our previous study showed that PGG-Glucan gave negative optical rotation under alkaline condition . 2 3 4 5 6 3 7 Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 3 5 Many other researchers also showed that β(1—>3) glucans form T H conformation in solutions " . We observed that the formation of TH was dependent on its single chain length. When the chain length is sufficiently long, polymer molecules are able to interact with each other via inter-chain hydrogen bonding, therefore, form T H or T H aggregate. However, when the polymer chain length is too short, it is incapable of forming strong inter-chain interactions, therefore, it remains in the SC conformation. The objective of this study was to develop methods to detect and characterize the SC, T H and T H aggregate conformations in soluble PGGGlucans. In this study, PGG-glucan conformers were separated in aqueous solution under pH 7 condition. The SC conformer was detected and characterized using a multi-detector SEC with a post column A B reaction system and a polarimeter. The T H conformer was characterized using SEC technique with a DRI and a polarimeter as the detectors. The aggregate state of T H conformation was determined through an Aggregate Number Distribution measurement using multi-detector SEC with a post column NaOH reaction system. 8 10 Experimental Section Materials. Unfractionated soluble PGG-Glucan (Alpha-Beta Technology, Inc. Worcester, MA) was isolated from the yeast cell wall of saccharomyces cerevisiae. The purified T H and SC PGG-Glucan conformers were fractionated from the unfractionated soluble PGG-Glucan using a preparative SEC. Aniline Blue was purchased from Polyscience, Inc. Sodium nitrate (NaN0 ), HC1, and NaOH were purchased from E M Science. 3 Multi-detector SEC with a Post Column Reaction System. As shown in Figure 1, the multi-detector SEC with a post column reaction system consists of a pump (L6000, Hitachi Instruments Inc.), an autosampler (AS-4000, Hitachi Instruments, Inc.), SEC columns (two KB804 and one KB803, Shodex), a post column mixing tee and a reaction coil (Upchurch). A post-column pump (L-6000, Hitachi Instrument Inc.) was Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 313 SEC Columns Mobiie V. Pump Phase J Injector 3 Fluorescence Waste • DRI U - H MALLS Poiarimeter Figure 1. Block diagram of a multi-detector SEC with post-column reaction systems. Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 314 used to deliver the post-column reagents. A DRI detector (Sonntek) was used to determine the polymer concentration. A fluorescence detector (1046A, Hewlett Packard Co.) was used to detect Aniline Blue-SC complex. A polarimeter (AutoPol IV, Roudolph, Inc.) was used to measure the optical rotation of PGG-Glucans. A multi-angle laser light scattering detector (miniDAWN, Wyatt Technology Corp.) was used for the molecular weight determination. The mobile phase was 1.0 Ν NaNO For SC detection, the post column mobile phase contained 1.0 mg/ml Aniline Blue. For T H aggregate number distribution determination, the post column mobile phase contained 866 m M NaOH. The flow rate was 0.5 ml/min for both mobile phase and the post column mobile phase. After mixing with NaN0 , the final NaOH concentration that PGG-Glucans came in contact with was 433 mM. This condition is called pH 13 condition. The condition without post column reaction system is called pH 7 condition. pH 13 condition is fully disaggregated condition for PGG-Glucans and pH 7 condition is aggregate condition for PGG-Glucans . v Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 3 5 Aniline Blue Binding. Commercial Aniline Blue was dissolved in water at 1 mg/mL. This Aniline Blue solution was activated by adjusting the pH to 12 for 1 hour using 1 Ν NaOH. Then, the activated Aniline Blue solution was neutralized to pH 7 using 1 Ν HC1. Upon activation and neutralization, much more Aniline Blue fluorophore was generated . 5 Molecular Weight and Molecular Weight Distribution. The molecular weight and the molecular weight distribution of PGG-Glucans were determined using the above laser light scattering detector and the above column separation system. An Astra® software version 4.2 was used for the molecular weight calculation. The refractive index increment (dn/dc) of PGG-Glucan was measured by Wyatt Technology, Corp., they are 0.143 ml/g and 0.145 ml/g at 633 nm under pH 7 and pH 13 conditions, respectively. Aggregate Number Distribution (AND). PGG-Glucans were separated under pH 7 condition and detected under both pH 7 and pH 13 conditions. The aggregate number of PGG-Glucan was calculated using the pH 7 molecular weight divided by the pH 13 molecular weight for the same fraction in the DRI chromatogram. The A N D is the distribution of the aggregate number across the entire molecular weight range of the SEC peak. Results A n d Discussions Detection of Single Chain PGG-Glucan Conformation. The Single Chain PGGGlucan was detected using SEC with the post column Aniline Blue reaction system. The purified T H and SC PGG-Glucan conformers were injected into the SEC column and separated by size under pH 7. The Aniline Blue was delivered and mixed with the separated species through a post-column reaction system, and the fluorescence intensity was detected at the excitation and emission wavelength of 400 nm and 490 nm, respectively. Figure 2 shows the DRI and the fluorescence chromatograms. In Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 315 Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 Triple Helical •7? i? î5 Single Chain »? m %t m «· b) 1? J9 Î5 i? »? Time (min.) *2 M *· Figure 2. SEC chromatograms of SC and T H PGG-Glucan conformers obtained from a) DRI detector and, b)fluorescencedetector with a post column Aniline Blue reaction system. Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 316 Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 Figure 2, the fluorescence intensity of the SC PGG-Glucan is much higher than that for the T H PGG-Glucan, indicating that the SC PGG-Glucan forms a specific fluorescent complex with Aniline Blue. Detection of the Ordered Triple Helical PGG-Glucan Conformation. The ordered conformation of T H PGG-Glucan and disordered conformation of SC PGG-Glucan were characterized using a multi-detector (polarimeter, DRI detector and multi-angle laser light scattering) SEC system. Figure 3 shows the DRI and the polarimetric chromatograms for the unfractionated soluble PGG-Glucan. In Figure 3, the SC PGGGlucan fraction gave negative optical rotation. This is due to the chiral center of the β(1—>3) backbone linkage in the absence of physical aggregation. In contrast, the T H PGG-Glucan fraction gave positive optical rotation indicating an ordered conformation. The molecular weight distribution of unfractionated soluble PGGGlucan ranged from 5,000 to 2 million g/mol. The results indicate that the conformational transition from SC to ordered T H conformation occurred at the molecular weight around 15,000 daltons. Aggregate State of Triple Helical PGG-Glucan. The aggregate state of the T H PGG-Glucan was studied using a novel multi-detector SEC with a post column delivery system". PGG-Glucans were separated under pH 7 or in the aggregated state. Sodium hydroxide was delivered after the column and mixed in-line with PGG-Glucan fractions to disaggregate the ordered PGG-Glucan conformer. The molecular weight was determined under pH 7 and pH 13 conditions. Figure 4 shows the molecular weight and molecular weight distribution for the TH and SC PGG-Glucan conformers. The SC conformation is confirmed by the similar value of the molecular weight under pH 7 and pH 13 conditions. This coincides with the results obtained from the Aniline Blue fluorescence and the polarimetry experiments. In contrast, T H PGG-Glucan showed evidence of ordered aggregation as indicated by the difference in the molecular weight under pH 7 and pH 13 conditions. The aggregate number for the T H and the SC PGG-Glucan conformers was calculated and presented in Figure 5. The aggregate number was determined to be one for the SC PGG-Glucan conformer and ranged from 3 to over 10 for the T H PGG-Glucan conformer and its aggregate. These results strongly indicate that PGG-Glucan isolated from the cell walls of yeast can form aggregate of triple helical structures. Many researchers reported that β-glucans (Scleroglucan, Lentinan, Schizophyllan) isolated from other sources form a single triple helix '". 9 Conclusions 1. PGG-Glucan can exist in single chain, triple helical and triple helical aggregate conformations depending on its single chain molecular weight. The aggregate number is one for the SC conformer and ranges from three to over ten for the T H or T H aggregate conformers. Schematic representation of the possible conformations of PGG-Glucan in aqueous solution is shown in Figure 6. 2. The T H conformer exists in an ordered conformation as indicated by the positive Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 317 7 1.0x10, Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 I 6 1.0x10| a) 5 1.0x10L 4 1.0x10L- 3 1.0x10' c ο ο ÙL ,·32 40 •48 56 64 le ο Triple Helical . Sing e Chain α ο PGG-Glucan \ r" P G G - G l u c a n ι Elution Time b) (min) Figure 3. Detection of SC and T H PGG-Glucan conformers in unfractionated PGG-Glucan using multi-detector (DRI, polarimetry, and MALLS) SEC system, a) The chromatogram was obtained from DRI detector and the molecular weight distribution was obtained from both DRI and M A L L S detectors, b) The optical rotation chromatogram was obtained from a polarimetric detector. Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 318 S i n 1 n y i n 6 1.0x10 c 1.0x10 5 1.0x10 4 Triple Helical 9 ' e Chain . - PGG-Glucan " P G G G l u c a n Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 ω ο 3 1.0x10 ' 18.0 20.0 22.0 24.0 26.0 28.0 Volume (mL) Figure 4. Molecular weight distribution of SC and T H PGG-Glucan conformers under pH 7 and pH 13 conditions, determined using multi-detector (DRI and M A L L S ) SEC with a post column NaOH reaction system for pH 13 condition and without a post-column reaction for pH 7 condition. The chromatograms were obtained from DRI detection. \ Triple Helical * PGG-Glucan * \ ^ Single Chain PGG-Glucan Elution Time (min) Figure 5. Plot of aggregate number distribution (AND) versus elution volume for the SC and T H PGG-Glucan conformers in neutral aqueous solution, determined by the multi-detector SEC with a post column NaOH reaction system. Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 Figure 6. Schematic representation of possible conformations of PGG-Glucans aqueous solution, isolated from the yeast cell wall of saccharomyces cerevisiae. Provder; Chromatography of Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1999. 320 optical rotation. In contrast, the optical rotation of the SC conformer is negative. The conformation transition occurred at the single chain molecular weight around 15,000 g/mol. 3. Multi-detector SEC with a post column reaction system is a powerful technique to study the aggregate number distribution of unfractionated PGG-Glucan across the entire molecular weight range. Acknowledgments Author acknowledge Roudolph, Inc. for lending the polarimeter model AutoPol IV. Downloaded by GRIFFITH UNIV on October 25, 2017 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Bleicher, P.; Mackin, W. J. Biotechnology inHealthcare1995, 2, 207-222. Jamas, S. B.; Easson, D. D., Jr.; Ostroff, G. R. and Onderdonk, A. B. Polymeric Drugs and Delivery Systems; ACS Proceedings; Edited by Dunn, R. L. and Ottenbrite, R. M., ACS: Washington D.C. 1991, Serial No. 469, pp 44. Deslandes, A. B.; Marchessault, C. D.; and Sarko. Macromolecules 1980, 13 (6), 1466-1471. Thistlethwaite, P.; Porter, I. and Evans, N. Journal of Physical Chemistry 1980, 90(21), 5058-5063. Park, J.T.; Guo Y.A. and Magee, A.S. 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