FACE analysis of starch structure
Electrophoresis 1998, 19, 2603-261 1
Mathew K. Morell'
Michael S. Samuel'
Michael G. O'Shea'*
'CSIRO Plant Industry,
Canberra, Australia
'Cooperative Research Centre for
Plant Science, Canberra, Australia
2603
Analysis of starch structure using fluorophore-assisted
carbohydrate electrophoresis
The analysis of the fine structure of starches is important to the investigation of
linkages between starch structure and function and to the investigation of the
properties and roles of starch biosynthetic, modifying and degradation enzymes.
Fluorophore-assisted carbohydrate electrophoresis has recently been introduced as
a method for the analysis of the oligosaccharide populations released by the
enzymatic digestion of starches, which has advantages in resolution and sensitivity
over previously used methods, and provides the capacity for the facile analysis of
oligosaccharide populations on either a molar or mass basis. The use of
fluorophore-assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of
labeling and separation techniques. Examples of separations using slab gel
electrophoresis, DNA sequencer analysis and capillary electrophoresis are
presented and we conclude that on the basis of resolution and reproducibility,
capillary electrophoresis is the method of choice for the separation of
oligosaccharides of degree of polymerization from 1 to 100. Examples of
isoamylase-debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through
the analysis of oligosaccharides released by the debranching of limit dextrins
derived from starches and glycogens is introduced as a useful diagnostic of starch
structure. The potential for future development of novel diagnostics for starch
structure using fluorophore-assisted carbohydrate electrophoresis is discussed.
1 Introduction
Despite the fact that carbohydrates are the most abundant
carbon-containing class of compounds on earth and are an
important renewable resource, they have been underrepresented in the array of straightforward separation and
detection systems introduced into chemical and biological
laboratories over the past two decades. High resolution
systems for the analysis of proteins and nucleic acids have
developed rapidly to the point where endeavours such as the
various genome sequencing, expressed sequence tag, and
protein expression characterisation projects are rapidly
achieving the sequence analysis of entire genomes and the
characterisation of the full complement of genes expressed
in specific tissues. The development of comparable
carbohydrate separation and analysis technology has not
reached as universally into biological research laboratories
as might be expected given their biological significance.
Two reasons can be advanced. First, the uncharged nature
of carbohydrates at all but extreme high pH prevents
separation by the high-resolution electrophoretic systems
developed over the past two decades. Secondly, the
majority of carbohydrates lack appropriate groups which
facilitate sensitive detection and quantification using
straightforward techniques such as spectrophotometric
Correspondence: Dr. M. K. Morell, CSRO Plant Industry, GPO Box
1600, Canberra, ACT 2601, Australia (Tel: +61-2-6246-5074; Fax: +61-26246-5000; E-mail: [email protected])
Abbreviations: ANTS, 8-arnino-1,3,6-naphthalenetrisulphonicacid;
APTS, 8-amino- 1,3,6,pyrenetrisulfonic acid; DP, degree of polymerisation; FACE, fluorophore-assisted carbohydrate electrophoresis; FACECE, FACE utilising capillary electrophoresis;FACE-DSA, FACE utilising
DNA sequencer analysis; FACE-SGE, FACE utilising slab gel electrophoresis
Keywords: Starch / Capillary electrophoresis / Oligosaccharide /
Fluorophore-assisted carbohydrate electrophoresis
detection. Among carbohydrates, the analysis of starches
presents an especially formidable challenge. Starches are
deceptively simple in composition, containing a single
monomeric unit, glucose, polymerised via just two linkage
types, a-1,4 and a-1,6. However, starches are complex in
structure given their high degree of polymerisation,
polydispersity, and complexity of chain arrangement. There
are few distinguishing features or motifs which readily
facilitate structural analysis.
Starches are traditionally described as being composed of
two populations of molecules: amylose and amylopectin.
Amylose denotes a population of molecules with a degree
of polymerisation typically less than 5 000, which contain
fewer than approximately 10 a-1,6 branch points per
molecule. In contrast, amylopectins have a degree of
polymerisation of up to a million and contain approximately
one a-1,6 linkage for every 20 a-1,4 linkages. One broad
measure of starch structure is to measure the amylose-toamylopectin ratio by either size exclusion HPLC [ 11, iodine
binding [2, 31, differential scanning calorimetry [4], or a
selective precipitation procedure utilising concanavalin A
[5-71. While the amylose-to-amylopectin ratio is an
important measure and is predictive of some starch
properties, more detailed measurements of starch structure
are required in order to relate structure to function and to
understand the intricacies of the starch biosynthetic process.
Direct measurements of a-1,6 frequency and chain length
distribution are important descriptors of starch structure.
Total branch frequency can be measured by debranching
with isoamylase, and measuring branch point concentration
by reducing sugar assay as a function of total glucose. In
order to characterise the chain length distribution resulting
from total debranching, various types of gel permeation
*Current address: Bureau of Sugar Experiment Stations, PO Box 86,
Indooroopilly, Queensland, Australia 4068
2604
M. K. Morell, M. S. Samuel and M. G.OShea
Electrophoresis 1998, 19, 2603-261 1
chromatography using soft gels or HPLC with mass-based
detection by refractive index or direct chemical assay have
been employed [8-lo]. However, while a distribution is
obtained, resolution is poor. More recently, anion exchange
chromatography at high pH using an HPLC and pulsed
amperometric detector system has been used in order to
resolve individual oligosaccharides released from the
debranching of starches [ 11-13].
In this paper we describe the use of fluorophore-assisted
carbohydrate electrophoresis (FACE) as a method for starch
structural analysis. Starch structures can be dissected by
digestion with a range of specific enzymes and the released
oligosaccharides analysed using FACE techniques f14, 151.
The labelling and separation techniques are described and
the future development of the technology is discussed.
resulting P-limit dextrin was shown not release maltose on
further incubation with P-amylase. The P-limit dextrin was
debranched as described [14].
2.3 Reductive amination with APTS
Aliquots of carbohydrate were labelled in 200 pL microfuge
tubes by the addition of 5 pL of a solution of 0.2 M APTS in
15% aqueous glaciae acetic acid and 5 pL of freshly
prepared 1 M aqueous sodium cyanoborohydride. Standard
reactions contained between 5 and 20 nmol of reducing
ends. The reaction mixture was incubated at 37OC for 15 h
and diluted to a total volume of 100 ~.LLwith an
electrophoresis sample buffer consisting of 90 m~ Tris,
90 m borate and 6 M urea. Labelled oligosaccharides were
stored at -7OOC until required.
2 Materials and methods
2.1 Materials
Isoamylase, pullulanase, and P-amylase were obtained from
Megazyme International Ireland (County Wicklow, Ireland). Glucose, maltose, and maltooligosaccharides from
degree of polymerisation (DP) 3 to 7 were obtained from
Boehringer Mannheim (Mannheim, Germany). 8-Amino1,3,6-pyrenetrisulfonic acid (APTS) was obtained from
Lambda Fluoeszenztechnologie(Graz, Austria) and sodium
cyanoborohydride from Sigma (St. Louis, MO, USA).
Bovine liver glycogen and potato amylose were from
Sigma.
2.4 Electrophoresis of APTS labelled oligosaccharides
in polyacrylamide slab gels
Gels contained either 20% polyacrylamide (with a 19:l
ratio of acrylamide to the crosslinker N,N'-methylenebisacrylamide) and buffer containing 89 m~ Tris-borate (pH
8.0), 2 mM EDTA, and 8 M urea. Gels were 32 cm in length
and 0.5 mm thick. Slab electrophoresis was conducted on a
Macrophor electrophoresis unit (Pharmacia Biotech,
Uppsala, Sweden) using a running buffer containing
89 m Tris-borate containing 2 m~ EDTA. Electrophoresis
was performed at 8°C at a constant power of 75 W until the
unreacted APTS reached the bottom of the gel (approximately 4 h).
2.2 Enzyme treatments
Starches and glycogens were debranched using isoamylase
as previously described [14]. The P-limit dextrins of
glycogens and starches were prepared by dialysing a
mixture of P-amylase and the target glucan overnight
against sodium acetate buffer, pH 4.5. The reaction was
boiled for 5 min and the polymeric glucan collected by
precipitation following the addition of three volumes of
ethanol, and centrifugation at 5 000 rpm for 10 min. The
y G H-Y
HO
OH
2.5 Fluorescent imaging of polyacrylamide slab gels
Slab gels were imaged in a Molecular Dynamics Fluorimager using low intensity argon-ion laser excitation
(488 nm). Data were collected in the normal sensitivity
mode without the use of a filter and processed using
ImageQuantm software Version 4.2 (Molecular Dynamics,
Sunnyvale, CA, USA).
"9
- -7GIH
-0 NH,-X
HO
OH
NaCNBH3
Y
HO
OH
HO
OH
NH2 S03H
I
I
H03S
8-amino-l,3,6-pyrene
trisulfonic acid
APTS
8-amino-l.3.6-na~hthalene
trisulfonic acid
ANTS
Figure 1. Reductive amination is used to
covalently link a charged fluorophore (X) to
the reducing end of an oligosaccharide.
Only the reducing terminal sugar is shown,
the remainder of the oligosaccharide is
unaltered by the reaction and is denoted
by (Y).Thestructure of two frequently used
charged fluorophores, APTS and ANTS, are
also shown.
Electrophoresis 1998, 19, 2603-261 1
FACE analysis of starch structure
2.6 Electrophoresis of APTS labelled oligosaccharides
using a DNA sequencer
Details of the separation of APTS labelled oligosaccharides
using either an Applied Biosystems 373A or 377 DNA
sequencer (Perkin-Elmer Corporation, Applied Biosystems
Division, Foster City, CA, USA) have previously been
reported [14, 151. Fluorescence data was collected and
analysed using Genescan (Perkin Elmer Corp., Foster City,
CA, USA) software (version 2.1).
2.7 Capillary electrophoresis of APTS labelled glucans
Separation was performed on a P/ACE System 5010
instrument with an argon-ion laser-induced fluorescence
detector (P/ACE System Laser Module 488) supplied by
Beckman Instruments, Fullerton, CA, USA. The capillary
used was a 50 pm diameter eCAPTM neutral coated
capillary with the supplied carbohydrate separation gel
buffer. The capillary was cut to a length of 47 cm and the
sample was introduced by pressure injection of the
reductive amination reaction mixture, typically 1-5 s at
0.5 psi. Separation was achieved using an applied voltage of
23.5 kV (current 15 PA) at a temperature of 25OC.
amination reaction. The introduction of a single label at the
reducing end allows quantification on a molar basis, with
ready conversion to a mass basis if individual oligosaccharides are baseline resolved and their molecular mass is
known.
The choice of label is driven by the nature of the detection
system being used. A wide variety of labels can be
considered if UV or visible detection is used, whereas a
fluorophore must suit both the emission and absorption
characteristics of the detector employed. While a variety
of aromatic primary amines have been used [15-181
polysulfonic acid derivatives of monoamino-substituted
aromatic compounds, such as 8 amino 1,3,6-naphthalenetrisulfonic acid ANTS [19-211 and APTS [14,, 15, 22, 231
have been extensively used (Fig. 1). APTS has found
particular application because its absorption and emission
characteristics are ideally suited to the argon-ion laser
systems used in many standard LIF detectors, for example,
those employed on DNA sequencers and capillary electrophoresis instruments.
.2
4000
0
2000
c
E0
Table 1. Reproducibility of fluorophore labelling by reductive amination
% Standard deviation
Normalised
fluorescenced,e,
~
2a'
3b'
3"'
4"'
5a'
6"'
7a'
Total
6.07
8.32
4.90
6.29
5.93
6.01
5.72
a
v)
Reductive amination is widely used as a straightforward
means of derivatizing a reducing sugar. The primary amine
of the label forms a Schiff s base with the hemiacetal of the
reducing sugar. The imine so formed is reduced using
sodium cyanoborohydride, covalently linking the label to
the sugar (Fig. 1). The key features of the label are that it
must contain a primary m i n e group to allow reductive
amination, be capable of spectrophotometric or fluorescence detection, and be charged in order to provide
electrophoretic mobility. The carbohydrate must contain a
single reducing end and be stable under the generally acidic
conditions and elevated temperatures used for the reductive
Fluorescencec.e,
c
8
3.1 The labelling reaction and choice of label
DP
DP7
6000
5al
3 Results
2605
2.54
3.53
2.57
2.41
2.46
2.31
2.47
6.32
a) Linear a-1,4 maltooligosaccharides
b) Arabinotriose
c) % Standard deviation calculated directly from arbitrary fluorescence
units
d) % Standard deviation calculated after normalisation of the data. To
normalise the data, the sum of the fluorescence of the DP 2-7 peaks for
each electrophoresisrun was summed. The fluorescence of each DP was
subsequently calculated as a percentage of that sum.
e) n=6 for each DP
6000
i?
.-
4000
2000
40000
-
roo0
1500
2d00
2doo
Scan Number
II D P 7 1 .
C
.g 30000
.-
5
20000
C
0
al
p
10000
9
s
o
ii 150000
i?
E
g 100000
4
50000
5
___(
10
15
20
25
30
Time (min)
Figure 2. Analysis of isoamylase-debranchedwheat starch and glycogen
by (a), (b) DNA sequencer analysis and (c), (d) capillary electrophoresis.
The debranching, labelling and electrophoresis of each of the samples on
each of the separation formats is described in Sections 2.2-2.4.
Fluorescence eluting before the arrows in panel ( a x d ) derives from
unreacted APTS or fluorescent impurities in the APTS reagent. The
stepwise appearance of the peaks in the capillary electrophoresis traces in
panels (c) and (d) was induced by the software used to prepare the figure
and is not apparent in the original data.
2606
Electrophoresis 1998, 19, 2603-261 1
M. K. Morel], M. S . Samuel and M. G. OShea
The critical parameters for the reductive amination reaction
for monomeric sugars using APTS have been defined to be
the concentration, temperature and the pK, of the acid
catalyst [15, 24-26] and the concentration of AF'TS. The
efficiency of labelling short oligosaccharides with a range
of primary amines has been reported to be both high (>95%)
and consistenat [19, 241. O'Shea et al. [15] demonstrated
that standard deviations of labelling over a range of
maltooligosaccharides from glucose to a pool with an
average DP of 135 ranged from 0.8% to 7.7%, with a mean
variance of 2.4%.
10 0
75
75
s
s
50
50
25
25
00
00
75
75
50
50
25
25
00
Table 1 shows the reproducibility obtained when a mixture
of oligosaccharides was labelled with APTS and analysed
using capillary electrophoresis and LIF detection. The %
standard deviation obtained for the sum of the distribution
was 6.3% and there was no difference in reproducibility
with chain length over this chain length distribution. The
total variation observed includes variations introduced
during the labelling reaction (see above), during injection
(determined to be 2 f 0.5% for the instrument used), and
through detection, peak integration and analysis of the data
by the capillary electrophoresis instrument and software.
Overall labelling efficiency and injection volume errors can
be accounted for by normalising each distribution, revealing
the underlying variation in labelling and detection for each
individual oligosaccharide. Table 1 shows that for a pool
containing linear maltooligosaccharidesfrom DP 2 to 7, and
arabinotriose, standard deviations for individual oligosaccharides ranged from 2.4% to 3.5%. Similar levels of
reproducibility have been obtained for debranched glycogen
and amylopectin chain length distributions [ 151. In order to
investigate the dependence of labelling on the ratio of APTS
to reducing ends, we labelled aliquots of debranched wheat
starch (containing a range of amounts of reducing ends from
5 nmol to 200 nmol) in the presence of 1 pmol of APTS
(data not shown). Comparison of distributions showed that
total labelling efficiency was constant over the range from
5 to 100 nmol, and that the normalised distributions were
stable over this range. For DP 6-35, relative standard
deviations (n=5) ranged from 1 to 2% per oligosaccharide,
and remained under 5% for oligosaccharidesof up to DP 45.
Standard deviations of greater than 10% were observed at
higher DP if standard labelling reactions containing either
less than 5 nmol or greater than 100 nmol were included in
the analysis. The lowest standard errors were observed in
labelling reactions containing between 10 and 50 nmols of
reducing ends.
7.5
s
00
1
Potato
I
High Amylose Maize
17.5
s
0 10 20 i0 '40' sb ' 60 ' $0 ' dOb ' YO 20
'
'
3b '40 i0 ' 6 0
'
'
?O ' 80
DP
DP
Figure 3. Capillary electrophoresis analysis of isoamylase-debranched
glycogen and starches. The substrates were dispersed, debranched using
Pseudomonas sp. isoarnylase, labelled with AWS, and analysed by
capillary electrophoresis as described in Sections 2.2-2.4. Fluorescent
peaks eluting between a detection time of 5.6 min (APTS-labelled glucose)
and 45 min (APTS-labelled DP 85) were integrated and normalised by
expressing fluorescence as a percentage of the total fluorescence eluting
over this time period.
Few studies have examined the labelling efficiency of
longer oligosaccharides, presumably because of the technical difficulty in obtaining sufficient amounts of individual
oligosaccharides. For example, individual maltooligosaccharides up to only maltoheptaose are commercially
available. OShea et al. [ 151 used anion-exchange chromatography in order to quantify the percentage of labelled and
unlabelled oligosaccharides following the reductive amination procedure. While glucose (95%) and maltose (88%)
were labelled to higher extents, the labelling efficiency
from DP 3 to DP 135 for a series of linear maltooligosaccharides was essentially identical, at about 80%.
3.2 Separation and detection methods for starchderived oligosaccharides
Three methods for the analysis of fluorophore-labelled
oligosaccharides derived from starch have been developed:
slab gel electrophoresis through polyacrylamide gels
Table 2. Parameters calculated from the isoamylase-debranchedstarch chain length distributions determined
by capillary electrophoresis
Glycogen
Wheat
Potato
Maize
Maize (wx)
Maize (ae)
s
DP
3-17
Molar
DP
18-30
DP
>30
DP
3-17
Mass
DP
18-30
DP
>30
%BT)
Abs.
valueb)
84.2
60.5
34.3
57.1
62.9
34.7
14.6
30.5
39.7
26.3
29.7
38.9
1.2
9.0
26.0
16.6
7.4
26.4
68.9
41.9
18.2
34.4
45.4
18.1
21.6
38.3
34.8
29.1
38.4
33.7
3.5
19.8
46.9
36.6
16.2
48.2
8.67
5.57
3.88
4.93
5.77
3.82
50.4
n.a.
54.6
35.5
10.4
56.8
a) Percentage of cr-1,6 bonds of the oligosaccharides eluting between DP 3 and 83; BP, branch points
b) Absolute value - the addition of the sum of differences between each of the starches and wheat starch for
each oligosaccharide, after converting negative differences to positive values.
2607
FACE analysis of starch shucture
Electrophoresis 1998, 19, 2603-261 1
I
I
I
1
Glycogen
?
go
Q)
s
44
I
-2
-4
J U
6
I
10
.
,
20
.
I
30
’
I
40
.
I
50
.
l
60
.
I
70
’
I
I
80
Degree of Polymerisation
Figure 4. Comparison of the chain length distributions of isoamylasedebranched glycogen and various starches. The normalised distributions
were analysed by subtracting the wheat starch distribution from (0)
glycogen, (X) potato starch, (0)maize, (+) waxy maize and ( 0 )amylose
extender maize.
(FACE-SGE) [l5, 191, polyacrylamide gel electrophoresis
using a DNA sequencer (FACE-DSA) [14], and capillary
electrophoresis (FACE-CE) [151. Examples of the analysis
of isoamylase-debranched glycogen and wheat starch using
the DNA sequencer and capillary electrophoresis are shown
in Fig. 2.
Illlllllrrlllll
Waxy Maize
4
3
2
1
40
High Arnylose Maize
3.2.1 Slab gel electrophoresis
A simple method for the analysis of labelled carbohydrates
involves the use of standard polyacrylamide gels which are
run for a fixed length of time and subsequently analysed
using a flat-bed scanner or photographic system. In an early
demonstration of the technique, Jackson [181 separated
ANTS-labelled oligosaccharides on standard polyacrylamide gel electrophoresis systems and identified and
quantified bands using a charge-coupled device [19]. This
system is capable of excellent separation and quantification
of maltooligosaccharides from glucose to DP 15; however,
baseline resolution of the full range of oligosaccharides
obtained from the debranching of glycogen or amylopectin
is not achieved.
3.2.2 DNA sequencer analysis
This technique has advantages over traditional slab gel
electrophoresis, where a gel is run and then imaged, in that
the labelled species move past a fixed window, maintaining
resolution between peaks over much greater chain length
ranges than the traditional format. This difference in
resolution is important when dealing with chain lengths
above 15, such as those found in oligosaccharide populations released from debranched glycogens and amylopectins. Panels a and b of Fig. 2 show the analysis of
debranched glycogen and wheat starch, respectively, by
DNA sequencer. Baseline separation of the oligosaccharides from DP 3 to 75 can be achieved using this technique
and the sensitivity of detection is high, providing detection
2
1
0
Degree of Polymerisation
Figure 5. Capillary electrophoresis analysis of debranched P-limit
dextrins. Preparation of the 0-limit dextrins, debranching, APTS labelling
and capillary electrophoresis were as described in Sections 2.2-2.4. The
data were normalised as described in Fig. 3. Minor peaks eluting between
the linear oligosaccharideunits are branched oligosaccharideswhich can be
eliminated by pullulanase digestion.
down to one femtomale per oligosaccharide [14]. The main
limitation of the technique is that the precise tracking of gel
lanes is problematic and, while allowing levels of accuracy
sufficient for the qualitative assignment of sequences or
fragment sizes, the reproducibility of the technique is not
sufficient for the confident resolution of subtle differences
in profiles [15].
3.2.3 Capillary electrophoresis
Capillary electrophoresis provides a highly resolving and
highly sensitive alternative to gel electrophoresis. Buffer
systems containing entangled polymer matrices provide a
highly effective molecular sieve to allow separation on the
2608
M. K. Morel], M. S. Samuel and M. G. OShea
basis of size and shape. Panels c and d of Fig. 2 show an
analysis of debranched glycogen and wheat starch,
respectively, by capillary electrophoresis. Reproducible
baseline resolution is obtained by FACE-CE analysis of
linear oligosaccharides over the range obtained from DP 3
to DP 85 in a 45 min separation, encompassing the range of
oligosaccharides obtained from glycogens and amylopectins. The range over which baseline resolution is obtained is
superior to standard high pressure anion exchange chromatography (HPAEC) techniques; however, a comparable
resolution range has been reported for HPAEC chromatography using a very shallow gradient involving a 3 h
separation time [111.
Other advantages offered by capillary electrophoresis are
automated sample injection, precise control of electrophoresis parameters, data collection, and analysis. Samples
can be monitored a short time after injection, in contrast to
the DNA sequencer where a run of many hours is required
before any information is obtained. Capillary electrophoresis instruments can be set up to monitor either UV-visible
absorption or fluorescence; although UV-visible detection is
less expensive than LIF detection, however, the sensitivity
of detection is reduced by several orders of magnitude. The
ability to utilise an in-line detector in capillary electrophoresis rather than a gel imaging system in the DNA
sequencer provides considerable advantages in the ease and
accuracy of detection of the eluting species. Using the
instruments available to us (see [14], [15] and Section 2.7),
we have found capillary electrophoresis separation of
APTS-labelled oligosaccharides to be both higher resolving
and a more reproducible method than DNA sequencer
analysis [15]. This difference is illustrated in Fig. 2 where
the resolution of oligosaccharides by capillary electrophoresis (panels c and d) is compared to that obtained by
DNA seauencer (uanels a and b). The clear baseline
separation' of oligoiaccharides by capillary electrophoresis
allows for more accurate and reproducible peak integration
and, therefore, greater reproducibility of the overall
distribution. The flexibility of separation formats, capillaries, and matrices provides the prospect for developing
improved and novel separations applicable to the analysis of
starches and starch-derived oligo- or polysaccharides.
Eiecrrophoresis 1998, 29, 2603-261 1
261 and can be readily separated from unreacted AFTS by
capillary electrophoresis [26]. A standard curve using
known glucose concentrations can be established and used
to provide accurate quantification of glucose concentration.
This procedure allows the total amount of starch to be
accurately determined and is especially valuable when the
amount of starch available is so limited that chemical or
enzymatic techniques are insufficiently sensitive.
3.3.2 Debranching with isoamylase
The a-1,6 bonds in starch are specifically cleaved by
isoamylase. In a typical starch, about 5% of the glycosidic
linkages are a-1,6 linkages derived from amylopectin. In a
starch containing 20-30% amylose by weight, with an
average chain length of between 250 and 500 [27] and a
ratio of branched to unbranched molecules of 0.2-0.8 by
mole [28] fewer than 0.2% of the bonds in that starch can be
a-1,6 bonds derived from amylose. Isoamylase digestion of
glycogens and starch produces populations of linear
oligosaccharides with degrees of polymerisation between
3 and 85 which can be resolved on capillary electrophoresis
[I51 (Fig. 3) or DNA sequencer instruments [14]. Because
there is a single fluorophore per oligosaccharide and the
distribution is composed only of linear oligosaccharides,
this molar-based distribution can be readily converted to a
mass-based distribution. In addition, this method gives the
percentage of branch points in amylopectin (Table 2).
Longer chain lengths in the sample, such as those derived
from amylose, may be labelled but do not reach the detector
in either FACE-CE or FACE-DSA under the conditions
described here. Therefore, the analysis of a debranched
starch by capillary electrophoresis or DNA sequencer is
essentially equivalent to the analysis of the amylopectin and
the prior separation of amylopectin from amylose is not
necessary.
The data shown in Fig. 3 are consistent with distributions
obtained previously for these polysaccharides; however,
note that these distributions are expressed on a molar rather
2o
,
.
,
'
$0
'
io
Ill,
The size and complexity of the intact amylose and
amylopectin molecules does not favour the use of
techniques such as FACE where data collection is on a
molar, rather than a mass, basis. However, the enzymatic
digestion of starch using highly purified and specific
enzymes produces diagnostic oligosaccharide populations
which can be readily labelled and resolved by electrophoresis. Four types of enzymatic digestions are discussed
here which provide informative monomer or oligosaccharide populations.
'
Starch can be completely depolymerised to yield glucose
using a cocktail of amyloglucosidase and a-amylase.
Glucose is labelled with high efficiency with APTS [15,
I
I
3.3 Enzymatic digestion coupled with FACE as a tool
for starch analysis
3.3.1 Total depolymerisation
'
lb
.
io
. $0 ' i o
io
Degree of Polymerisation
'
'
Figure 6. Capillary electrophoresisanalysis of branching enzyme I action.
Potato amylose (1 mg in 1.0 mL of sodium citrate buffer, pH 7.0) was
incubated with 2 pg purified wheat branching enzyme I at 25°C overnight,
debranched with isoamylase, AF'TS-labelled, and analysed by capillary
electrophoresis. The data were normalised and plotted against DP.
Electrophoresis 1998, 19, 2603-261 1
than a mass basis [ l l , 141. Bovine liver glycogen has a peak
at DP 7 and a smooth trailing distribution to higher chain
lengths, consistent with the simple glycogen elongation and
branching systems operating in bacteria and animals. In
comparison, oyster glycogen has a peak at DP 6 [29];
however, glycogen structures are variable according to the
source and physiological conditions. Wheat and barley
samples show typical distributions for debranched amylopectins of starches with “A” type crystallinity and there is
clear evidence of the periodicity thought to derive from the
action of different isoforms of starch biosynthetic enzymes
[ l l , 301. Waxy maize shows a reduced number of chains of
DP 30 or greater (Fig. 3, Table 2) consistent with the lack of
granule-bound starch synthase in this mutant and the
proposed involvement of granule-bound starch synthase in
amylopectin synthesis [31] in addition to its primary role in
amylose biosynthesis. High amylose maize showed a shift
to a longer chain length distribution (Fig. 3, Table 2)
reflecting the deficiency of branching enzyme I1 activity in
this mutant [32]. Interestingly, this distribution is similar to
that of potato starch and is consistent with the observation
that starches giving “B” and “C” type X-ray diffraction
patterns have longer chain length distributions [ l l , 331. The
differences in profiles shown here indicate that the analysis
of chain length distributions provides signatures which are
diagnostic for the presence or absence of particular isoforms
of starch biosynthetic enzymes during starch deposition.
In order to compare distributions, normalisation of the data
over a common chain length distribution provides the
percentage contribution of each oligosaccharide to the total
distribution and a common basis for further treatment of the
data. One simple means of comparing distributions is to
sum the percentages over different size ranges: for
amylopectins and glycogens we find DP 3-17, DP 18-30
and DP 30-85 pools to be informative (Table 2). Another
powerful method of comparison is to compare samples
against a reference sample by subtracting a reference
sample from each other sample in the comparison set. The
difference value for each chain length can be readily plotted
[1 1, 141 and provides an easily interpreted visual impression
of the differences between distributions (Fig. 4). This
treatment of the data illustrates differences between
distributions which are frequently not readily apparent by
visual comparison of the primary distributions. While
summation of the differences between two distributions
by definition gives zero, converting the sign of negative
difference to positive sign to give the absolute value, and
then summing the differences, gives a useful parameter
describing the total difference between distributions
(Table 2). The numerical parameters derived from this
treatment of the data provide an effective means of testing
for the statistical significance of differences between chain
length distributions and are especially valuable where the
differences are subtle.
FACE analysis of starch svucture
2609
had an odd or even number of residues. When starch is the
substrate, both amylopectin and amylose will be subject to
P-amylolytic attack and the resulting distribution of chains
will, therefore, contain linear oligosaccharides derived from
both polymer types, although they will predominantly arise
from amylopectin. Examples of the analysis of the
oligosaccharides derived from isoamylase debranching of
@-limit dextrins derived from glycogen, potato starch,
normal maize starch, waxy maize starch and high amylose
maize starch are given in Fig. 5. Note that glucose and
maltotriose released by P-amylase treatment have been
removed by precipitation and the chain length distribution
shown is the product of the action of isoamylase on the
isolated P-limit dextrin. Approximately 5% of the oligosaccharides have intermediate detection times between
those found for linear maltooligosaccharides and these
oligosaccharides are thought to contain a-1,6 bonds bearing
a single maltosyl or maltotriosyl unit. No relationship
between individual intermediate species and specific
oligosaccharide structures has yet been made. Inclusion of
pullulanase in the reaction eliminates these species (data not
shown). While the P-limit dextrin of glycogen has the
expected shorter chain length distribution than P-limit
dextrins of starches, interesting differences are observed
among the starches. The normal and waxy maize distributions are similar, differing from glycogen by the presence of
a trailing distribution from DP 20 to 45. The normal and
waxy maize distributions differ from potato and high
amylose maize in that these latter distributions have a lower
percentage of the overall distribution in the range from DP 4
to 20, but a pronounced enrichment in the distribution from
DP 25 to 35. This difference correlates with the X-ray
diffraction patterns and Naegli dextrin structures of these
starches [33].
3.3.4 Limit a-amylase digestion
Limit digestion of starches with a-amylase generates
populations of linear and branched oligosaccharides which
are informative about starch structure. The majority of the
starch is digested to linear oligosaccharides from glucose to
DP 5, which arise from the a-amylase digestion of
unbranched regions of starch and subsequent condensation
reactions catalysed by a-amylase. However, because aamylase is unable to cleave a-1,6 linkages, or a-1,4
linkages of a substituted chain where there is a spacing of
just one unsubstituted monomer between 2 a-1,6 substituted
monomers, oligosaccharides containing single and multiple
a-1,6 branch points are obtained [34] which give information on the branch spacing of the amylopectin which can be
related to functional properties [35]. FACE-CE techniques
can be used to characterise these populations (I. L. Batey,
CSIRO-Plant Industry, personal communication).
3.4 Analysis of starch-derived polysaccharides
3.3.3 P-Amylase digestion followed by isoamylase and
pullulanase debranching
Digestion of starch with P-amylase results in the progressive removal of maltose units from the nonreducing
ends, leaving a stub of either two or three glucose units at
the branch point, depending on whether the external chain
The examples of the use of FACE for the analysis of starch
structure given above are appropriate to the analysis of
oligosaccharides with a degree of polymerisation of less
than 100, which primarily originate in the amylopectin
fraction of starch. FACE technology has the potential to be
used for the analysis of longer glucans, such as those which
constitute the amylose fraction of starch; however, the
2610
M. K. Morell, M. S. Samuel and M. G. OShea
labelling and separation of such glucans are more problematic. We have used a wide range of slab gel electrophoresis
formats to separate longer glucans, with the intention of
separating the polymers which constitute the amylose
fraction of starch [15]. Above a DP of 100, separations
are no longer able to give resolution at the individual chain
length level. Such glucans do not migrate to the detection
window of a DNA sequencer of the detector of capillary
electrophoresis instrument within the period of even an
extended electrophoresis run time for the respective instruments. We have sought to side-step this limitation by using
a slab gel system using thin gels (0.4 mm) with low
polyacrylamide concentration (4%) to successfully charactense polysaccharide populations with chain lengths from
100 to about 250 for linear a-1,4 glucan and 100-1000 for
pullulan [ 151.
The creative exploration of polymer formats and buffer
systems using capillary electrophoresis may allow similar
or improved separations and should be explored; however,
there are three disadvantages of using FACE for the
analysis of long polysaccharides. First, the efficiency of
labelling with increasing chain length, and with the
introduction of branch points, has not been established.
Labelling efficiency may be substantially reduced if the
polymer is insoluble under the acidic conditions used for
labelling. Secondly, the restriction to a single label per
molecule progressively reduces the signal as a function of
mass as chain length increases. Third, the presence of
branch points within labelled molecules breaks the
straightforward relationship between electrophoretic mobility and mass.
3.5 Use of FACE to analyse starch synthesis or
modifying enzymes
The kinetic analysis of starch synthesis, degradation or
modifying enzymes is complex because unlike many other
enzymes, the substrate is rarely available as a single
molecular entity, and the product of one reaction cycle is
usually a substrate for further rounds of catalysis.
Furthermore, there is frequently interest in not only the
overall rate of the reaction, but also in the rate of reaction at
given positions within the substrate, for example, the rate of
reaction at a bond at a given distance from either the
reducing or nonreducing end of an oligosaccharide.Figure 6
shows the distribution of oligosaccharides obtained by
isoamylase debranching the product of the exhaustive
incubation of amylose with a highly purified preparation
of wheat branching enzyme I [36]. This distribution is
consistent with the distributions obtained after the incubation of maize branching enzyme I with amylose or
amylopectin [33, 371.
4 Concluding remarks
The application of FACE technologies to the analysis of
starch-derived oligosaccharides has demonstrated that the
technology provides a useful addition to the range of
techniques available to the starch chemist and is likely to
find wide application. Our conclusion from comparative
work using three separation and detection technologies is
that capillary electrophoresis coupled with LIF detection
Electrophoresis 1998, 19,2603-261 1
provides the highest resolution and reproducibility for
maltooligosaccharidesfrom DP 3 to 85, and is currently the
method of choice.
The advantages of the FACE analysis of starch are
particularly marked in situations where the amount of
starch available for analysis is limited. This is commonly
the case for genetic mutants where the amount of available
starch is limited. The sensitivity of the technique also
allows for the characterisation of starches from individual
seeds, such as are obtained through chemical mutagenesis
programs, germplasm screens or genetic engineering
programs.
The worldwide interest in starches as a renewable and
versatile biomaterial has focused increasing attention on the
relationships between the structure of starches and their
properties. The FACE techniques outlined in this paper
provide a methodology for analysing the oligosaccharide
distributions released by the enzymatic digestion of starch.
Differences between the profiles obtained from the starches
from different species, and starch mutants within species,
can be accurately and reproducibly determined using
FACE-CE. The reproducibility of the technique allows the
more subtle differences within species to be determined and
allows linkages between starch fine structure and function
to be investigated through genetic analysis. The creative
coupling of FACE separation and detection techniques with
existing or novel enzymatic approaches to the dissection of
starch structure of the polysaccharide provides scope to
further enhance the range of tools available to the starch
chemist.
The authors tank Dr. Rudi Appels for his advice and
comments and Dr. John Huppatz f o r his support f o r the
capillary electrophoresis aspects of this project. The
financial support of the Grains Research and Development
Corporation, Goodman Fielder Ltd. and Groupe Limagrain
is gratefully acknowledged.
Received April 24, 1998
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