bk-1999-0731.ch021

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
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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
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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 .
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4
5
6
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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
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ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
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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
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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
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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
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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,
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Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0731.ch021
I
6
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5
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r" P G G - G l u c a n
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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
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4
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- PGG-Glucan "
P G G
G l u c a n
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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.
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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.
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