Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1975 0-(2-hydroxyethyl)-amylose as the substrate of porcine pancreatic [alpha]-amylase action: structural analysis of 0-(2-hydroxyethyl)- maltooligosaccharides Yuk-charn Chan Iowa State University
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CHAN, Yuk-charn, 1940- 0"C2-HYDR0XYETHYL)-AMYL0SE AS THE SUBSTRATE OF PORCINE PANCREATIC ((-AMYLASE ACTION: STRUCTURAL ANALYSIS OF 0-(2-HYDR0XYETHYL)- MALT00LI60SACCHARIDES.
Iowa State University, Ph.D., 1975 Chemistry, biological
Xerox University M!0r0f!!ms, Xnn Arbor, Michigan 48106
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. 0-(2-hydroxyethyl)-amylo8e as the substrate of porcine
pancreatic «-amylase action: structural analysis of
0-(2-hydroxyethyl)-maltooligosaccharides
by
Yuk-charn Chan
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Biochemistry and Biophysics Major; Biochemistry
Approved:
Signature was redacted for privacy.
In Charge of Major Work
Signature was redacted for privacy.
For the Major Department
Signature was redacted for privacy. For the Gradufetffe College
Iowa State University Ames, Iowa 1975 11
TABLE OF CONTENTS
Page
DEDICATION iv
ABBREVIATIONS v
SYMBOLS AND NOMENCLATURE OF SUGARS vl
Symbols vl
Nomenclature vl
I. INTRODUCTION 1
II. LITERATURE REVIEW 10
A. Porcine Pancreatic a-Amylase 10
B. Amylase Action on Modified Maltoollgosaccharldes, Amylose, and Starch 13
C. Gas Chromatography of Carbohydrates 24
III. MATERIALS AND METHODS 25
A. Materials 25
B. Methods 28
IV. EXPERIMENTAL 34
A. Complete Acid Hydrolysis of 0-(2-Hydroxyethyl)- amylose 34
B. Porcine Pancreatic a-Amylase Action on 0- (2-Hydroxyethyl)-amylose 34 ill
IV. EXPERIMENTAL (Continued)
C. Identification of the Reducing Ends of Oligosaccharides in the a-Amylase Digest of 0-(2-Hydroxyethyl)amylose 34
D. Preliminary Separation of Porcine Pancreatic a-amylase Digest of 0-(2-Hydroxyethyl)amylose by Using a Charcoal Column 40
E. Glucoamylase I Action on 0-(2-Hydroxyethyl)starch 40
F. Glucoamylase Action on 3^-0-methylmaltose 48
G. Glucoamylase Treatment of Carbohydrate Fractions from Charcoal Column Chromatrography and the Original a- amylase Digest 48
H. Further Separation of Fractions 4 and 5 from Charcoal Column to Subfractions by Preparative Paper Chromatography and Sephadex G-15 Column Chromatography 48
I. Structural Analysis of 4IS2 53
J. Structural Analysis of 4III 53
K. Structural Analysis of 5III 82
L. Structural Analysis of 5IV 97
M. Further Porcine Pancreatic a-amylolysis of 5IV 111
N. Attempted g-amylolysis of 5IV 133
V. DISCUSSION AND CONCLUSION 134
A, Discussion of Results 134
B. Conclusion 153
VI. BIBLIOGRAPHY 162
VII. ACKNOWLEDGMENTS 172 iv
DEDICATION
To my parents:
I owe them more than I can say V
ABBREVIATIONS
GA glucoamylase g.I.e. gas liquid chromatography
HE 2-hydroxyethyl
Me methyl
PPA porcine pancreatic a-amylase t.l.c. thin layer chromatography vi
SYMBOLS AND NOMENCLATURE OF SUGARS
Symbols
glucose or o anhydro glucose unit a-1,4 link
reducing end glucose unit
maltose or G^ OO0 maltotriose or G^ OOÔ0 ïaaltotetraose or anhydro 2-0-(2-hydroxyethyl)-D-glucose unit R HHE HE. 'b anhydro 6-0-(2-hydro%yethyl)-D-glucose unit 9 anhydro 3-0-(2-hydroxyethyl)-D-glucose unit Nomenclature
For substituted maitooligosaccharidss, vs vise the following nomen clature system: •U a -0-(substituent)maltooligosaccharide a: The numeral indicates the position of the oxygen through
which the substituent group attaches to the glucose unit, b; The superscript numeral indicates the serial number of the
glucose unit on which the substituent group is attached
through an oxygen. The superscript numeral for the reducing
end glucose unit is always 1. vii
O For example: 6 -0-(2-hydroxyethyl)maltriose indicates that 2- hydroxyêchyi gi'cup attaches through an oxygen which is connected to the
C6 carbon atom of glucose unit number 3 (nonreducing end) in a malto- triose molecule. 1
I. INTRODUCTION
It is well known that enzymes are proteins which perform catalytic
reactions by first forming a complex with the substrate. Porcine pancre
atic a-amylase (PPA) is an enzyme, one of the «-amylases, that catalyzes
the hydrolysis of the a-1,4- glucosidic linkages in starch and glycogen.
The specific action of this enzyme has been well studied in our laboratory
(1-7)5 and a model consisting of a five glucose unit binding site has
been proposed (4). The catalytic groups of this enzyme are assumed to be
between the second and third glucose unit binding subsites from the re
ducing end (Figure 1). This enzyme which attacks starch and glycogen has
little or no action on other natural polysaccharides. Certainly, its
specificity depends on the a-1,4- glucosidic linkage in starch and glycogen,
but so far whether the specificity is also dependent on the conformation of
the glucose units in the polysaccharides and the arrangement of the hydroxyl
groups is not clear. In other words, the following questions have been
raised.
1. How does the substrate bind on the active site of the enzyme in
the enzyme-substrate complex?
2. Do the hydroxyl groups in the polysaccharide chain play important
roles in the enzyme-substrate ccisplex?
3. What are the orientation and conformation of the glucose units
on the active site in the transition state, before, and after the transition
state?
Obviously, the solution to these questions can give some useful Figure 1. Some known properties of porcine pancreatic a-amylase (8-10). A model of the enzyme containing five glucose binding sites is also shown in the figure. The indentations in the block and the Roman numerals I to V represent glucosyl binding subsites of the enzyme (4) 3
Porcine pancreatic alpha-amylase
1. = 52,600 - 1,200 daltons
2. Two active sites per 50,000 daltons
3. Catalytic groups; carboxylate and imidazolium ions
4. Five glucose binding sits per active site
SUBSTRATE
ENZYME 4
information on the sterlc-flt requirements for the enzyme and its mechanism of action.
In the present work, sparsely substituted 0-(2-hydroxyethyl)amylose
(D.S. between 0.1 and 0.2) was used as the substrate for porcine pancreatic o-amylase action. Monosubstituted disaccharides, trisaccharldes and tetrasaccharides were isolated from the enzymatic digest for structural analysis. The results were used to interpret the binding of the substrate on the enzyme by using the proposed five glucose binding site model (see
Chapter V, Section B, Conclusion).
The reasons why sparsely substituted 0-(2-hydroxyethyl)-amylose
(Figures 2 and 3) was chosen as the substrate are as follows:
1. The substituted amylose has the same number of hydroxyl groups as the original unsubstituted amylose. The only difference is that the hydroxyl groups from the substitution are attached to the second carbon 1 2 atom of the ethylene group (-0- CH^ CHg-) instead of directly to the carbons of the pyranose ring (Figures 2 and 3).
2. The 6-0-(2-hydrosyethyl) glucose unit in the polysaccharide chain can be considered as a single glucosyl branch point as in some model
branched maltodextrlns (6,7). Thus its influence upon the cleavage of
adjacent glucosidic bonds can be compared with branched structures with single glucose side chain.
3. The additional hydroxysthy1 groups in the amylase chain change the conformation of amylose, and thus increase amylose solubility in water.
Finally, a homogeneous aqueous medium is available for enzymatic reaction. Figure 2. Schematic drawing of monohydroxyethylated glucose units in the amylose chain. Each pyranose ring has a chair form conformation with all hydroxyl and 0-(2-hydroxyethyl)-groups in equatorial positions. For simplicity the pyranose ring can be represented by a circle and the position of the substitution S is indicated by a bar pointed to the corresponding direction as shown in the figure.
1. 6-0-(2-hydroxyethyl)-D-glucose
2. 3-0-(2-hydroxyethyl)-D-glucose
3. 2-0-(2-hydroxyethyl)-D-glucose
The simplified structures will be used in this dissertation and HE will be used to represent the 0-(2-hydroxyethyl)-group s
ÇHgOH
M ."P
f* u mu
i»
S = = CH2CH20H " HE Figure 3. 0-(2-hydroxyethyl)amylose (0.1 - 0.2 D.S.) in
1. Haworth formula 2. simplified structural formula CHgOH OCH^CHgOH ÇHgOH
CHgOH CHgOH
ÇCI^CHgOH
CHgOH UMgVn rw nw
\ ZA /\ i OH OCHgCHgOH OH
uc
"CAIMD- jZ)-0—O-C^O" HE HE 9
4. Action of porcine pancreatic a-amylase on 0-(2-hydroxyethyl)- amylose gives simpler enzymatic products in comparison with 0-(2-hydroxy- ethy1)starch. The possibilities to obtain a-limit dextrin due to the branching in the starch molecule are eliminated.
5. 0-(2-hydroxyethyl)amyloses are commercially available.
6. The successful characterization of 2-0-, 3-0-, and 6-0-(2- hydryxyethyl)glucoses by gas liquid chromatographic method (11) showed many advantages over column and paper chromatography in the characterization of the same isomeric compounds. Three inner glucosides of 2-0-(2-hydroxyethyl)- glucose which cannot be visualized by alkaline AgNO^ dip on paper chromatograms were also identified by gas chromatography (12). This pro vides a ready means to determine the monomeric units in the hydroxy- ethylated oligosaccharide produced by the action of porcine pancreatic a- araylase on 0-(2-hydroxyethyl)amylose. 10
II. LITERATURE REVIEW
A. Porcine Pancreatic a-Amylase
Several excellent reviews dealing with o-amylase including porcine
pancreatic a-ainylase have been published (13-19). Porcine pancreatic a-
anylase was first isolated in crystalline form by Meyer et al. in 1946
(12,20). The optimum activity of the enzyme solution is between pH 6.5
and 6.9 (12); a monovalent anion is required as an allosteric activator
(21). Ten millimolar concentration of chloride ion gives optimal activa
tion: bromide and other anions are less effective. It is a calcium
metalloenzyme with at least 1 gram atom of very firmly bound calcium per
mole of enzyme (22). The calcium ion functions as a stabilizer against
denaturation and proteolysis to maintain the enzyme in the proper configura
tion for biological activity (23,24) and acts as a cc-factor necessary for
amylolytic activity (16). Improved methods for the preparation of
crystalline enzyme were given by Fischer and Bernfeld (25), Caldwell et al.
(26) and Loyter and Schramm (27). This enzyme was previously reported as a
homogeneous enzyme by free boundary electrophoresis and paper electrophoresis
(12,20,26) with a molecular weight of 45,000 daltons from sedimentation and
diffusion measurements (28). However, two isoenzymes were isolated by DEAE-
cellulose chromatography and disc gel electrophoresis (29,30). a-Amylase I
has five aspartic acids and one asparagine more than a-amylase II (10). Both
multiforms of the amylase are physically distinct but structurally related
with common active sites (30). Amino acid composition analysis of
crystalline enzyme gave a molecular weight of 51,300 + 450 (31). The enzyme 11
also contains five disulfide links to maintain its tertiary structure
(32). Apparently, each isozyme has at least two independent binding sites for the formation of a multimolecular complex with glycogen (11).
Measurement by equilibrium dialysis showed that 1 mole of enzyme specifi
cally bound 2 moles of maltotriose (11).
By difference spectrophotemetric and kinetic studies, it was assumed that a tryptophanyl side chain may be involved in the binding site of the enzyme (33). Preparative ultracentrifugation and perturbation experiments showed that oi-amylase binds 3 moles of B-cyclodextrln per mole of enzyme but only one $-cyclodextrin molecule interacts with the tryptophanyl side chain of the amylase (34). The radius of gyration of the enzyme was found to be 26.9 % by small angle x-ray scattering investigation, and a model containing a trough-like active site In the enzyme molecule was proposed
(35). Porcine pancreatic a-anylase contains two sulfhydryl groups (24), but both of them are not essential for enzyme activity (36).
The action pattern of porcine pancreatic a-amylase displays a very high degree of multiple attack. By comparing the blue value and reducing value plots for different a-amylases, it was originally suggested that the differences in the plots were due to the different a-amylases hydrolyzing amylose into various chain length (37). In 1966, Abdullah et al. (1) used ycyclodestrin as the substrate for porcine pancreatic a-amylase action in the presence of 6-amylase. A large amount of maltotriose was detected in the enzymatic digest during initial stage of the reaction. This indicates the possibility of multiple attack of the a-amylase. Additional evidence from paper chromatographic analysis of human salivary a-amylase and porcine 12
pancreatic a-amylase digests showed that these two enzymes produce very similar products, therefore their wide differences in blue value-reducing valve curve are due to the different degrees of multiple attack (3).
Porcine pancreatic a-amylase has a degree of multiple attack of 8 per effective encounter by kinetic studies, three times that of human salivary a-amylase. The direction of multiple attack is toward the non- reducing end (5). The action of porcine pancreatic a-amylase on individual reducing-end labeled maltodextrines (G^-Gg) was studied by Robyt and
French (4). In the initial stage, maltopentaose was cleaved only at the second glucosidic linkage from the reducing end. Higher maltooiiogo- saccharides had more than one cleavage point, but neither glucosidic bonds at the reducing ends nor the nonreducing terminal bonds were cleaved. Glucose was produced exclusively from G^ and G^. From these investigations, a five glucose binding site (binding subsites I, II, III,
IV, and V in Figure 1) has been proposed with the catalytic group positioned at Lhe second bond from the reducing end of the enzyme (Figure
1) (4).
The mechanism of action of pancreatic a-amylase was studied by Waklm,
Robinson and Thoma (38). They proposed a double displacement mechanism which involves protonation of the glycosidlc bond by an imidazolium ion, attack on the anomeric carbon atom by a caïboxyl group to form a hypothetical glycosyl-enzyme ester intermediate and hydrolysis of the ester by water.
This mechanism interprets the unusual polymerization (glycosylation) of a-D-glucosyl fluoride catalysed by this enzyme (39), the pH-actlvity curve, the glycosidlc bond cleavage with retention of anomeric configuration, the 13
polarity of multiple attack and inhibition by maltobionic acid lactone
(40,41,19).
B. Amylase Action on Modified Maltooligosaccharides, Amylose, and Starch
In studies of the mechanism of enzyme action, there are two general
approaches (42);
1. To study enzyme protein itself, determining which amino acid
residues are important in the binding and catalytic process.
2. To study the nature of those groups in the substrate that seem
to be critical to the binding and catalysis.
Since the second approach is used in this investigation, the modifica
tion of maltooligosaccharides, amylose and starch have been of great
interest in addition to the action pattern of amylase on these modified
substrates.
1. Modified maltooligasaccharides
The synthesis of particular glucosidic linkages is very difficult by
chemical methods. Disaccharides such as maltose (43), trehalose (44),
sucrose (45), nigerose (46,47), kojibiose (48) and isomaltose (49) were
successfully synthesized; however, the syntheses of tri-saccharides and
higher oligosaccharides with special interest are far beyond the straight
forward chemical techniques which we can handle at the present time. 4-o,
6-3-Bis-D-glucopyranosido-D-glucose was prepared by Goldstein and Lindberg
(50), but the molecule is too small for a- or 3-amylase action.
The research of solid phase synthesis of oligosaccharides was stimulated 14
by Merrlfleld's successful total synthesis of the protein rlbonuclease
(51). The process of sequential synthesis of oligosaccharides on a solid support has many attractive features, especially in the preparation of modified oligosaccharides which can be used as ideal substrates for enzyme action. The proper insertion of a modified monomer into an expected position along the oligomer chain of the substrate could tremendously simplify the final analysis of the enzymatic digest. Unfortunately, this technique is still at the stage of preliminary investigation without fruitful results (52-55). Solid-phase synthesis of maltooligosaccharldes requires the use of a glucose derivative with a reactive leaving group at
C-1, the C-4 hydroxyl group protected by a readily removable blocking group, the remaining hydroxyl groups protected by stable groups, and a resin which can be taken off easily from the formed oligesaccharide de rivative (52). The chemistry also involves the complicated steric control of the glucosidic linkage formation (53,54).
Although the synthesis of a-(1,4)-glucosidic linkages is difficult
by the usual chemical means, the synthesis of some monosubstituted
maltooligosaccharides by enzyme techniques is possible. D-enzyme incubated with methyl-a-D-glucoside and amylopectin gave a series of nonreducing
methyl maltooligosaccharldes. Other carbohydrates such as D-xylose, sucrose,
etc., were also shown to be acceptors (56). Transglycosylase obtained
from Bacillus macerans effects a transfer of the glucosyl units of cyclo-
hexaamylose to different acceptors; followed by redistribution reactions, homologous series of glycosides are produced (57). Pazur et al. used this
enzyme to prepare methyl glycosides of maltooligosaccharides as substrates 15
for yeast maltase, fungal amyloglucosldase and crystalline salivary
amylase action (58). Methyl glycosides of D-xylose and 6-0-methyl-D-
glucose were successfully used as acceptors in the same reaction (59).
The resultant oligosaccharides were subjected to 3-amylase digestion (60).
There are some disadvantages of using these enzyme-made oligo
saccharides for a-amylase actions. Besides the limited number of acceptors which can be used in the syntheses, the structural features of the oligo saccharides being made can only have modified glucose units at the re
ducing ends or very close to the reducing ends. Therefore, complete action
patterns cannot be obtained when these oligosaccharides are used as
substrates.
Homologous oligosaccharides with single a-l,6-linked glucose unit
"stubs" at the nonreducing end of the oligosaccharide chain were prepared
by Kainuma and French (6). Waxy maize starch was partially hydrolysed in
0.08 N HCl to a degree of 0.27. After mild treatment by Diazyme (a crude
preparation of glucoamylase), the hydrolysate was further treated with
pullulanase to remove the unwanted side chains without acting on the stubs-
Chromatography in the first direction of the 2-dimensional paper chroma
tography revealed the presence of glucose, maltose and a series of non-
reducing end stubbed oligosaccharides in the preparation. Following treat
ment with porcine pancreatic a-asylase directly on the paper chromatogram
and the chromatography in the second direction, it was found that
stubbed pentasaccharide (6^-a-glucosylmaltotetrose) and higher components
were cleaved to give stubbed tetrasaccharide (6 -a-glucosylmaltotriose),
but the stubbed tri- and tetrasaccharide were resistant to attylase action 16
(Figure 4a) (6). In the preparation of maltoollgosaccharides containing a single a-l,6-linked glucose unit at position 3 from the reducing ends, coupling reaction was performed by j|. macerans transglycosylase action on 3 cyclohexaamylose with 6 -o-glucoayImaltotriose (B^) as acceptor (7). The coupled products were debranched by pullulanase to give a mixture of homologous single stubbed oligosaccharides (B^, Bg, Bg, B^,...) and maltoollgosaccharides. This reaction mixture was subjected to porcine
pancreatic a-anylase action and analyzed by 2-dimensional paper chroma
tography as mentioned in the preceding investigation. There was no detectable hydrolysis of or B^. Eg was partly hydrolyzed to Gg and B^.
By and Bg were completely hydrolysed to give B^ and B^ respectively, plus
Gj (Figure 4b) (7). The high degree of position specificity of pancreatic
amylase acting on these oligosaccharides indicated that both the single
glucose stub and the nonreducing end of the oligosaccharides were critical
in positioning the substrate at the binding site of the enzyme. It was
concluded that in the five glucose binding sites of the enzyme (Figure 1)
(4), stubs or branches were permitted afc subsites I or V but prohibited at
III and IV. A stub or branch at subsite II restricted, but did not block
the action (7).
2. Modified sar;lcs£s and starches
The polymerization of 2,3,6-tri-(N-phenyl carbainate)-D-glueopyranose
using phosphorus pentoxide in dimethyl sulfoxide and chloroform gave a (1-4)
linked gluean with degree of polymerization about 60 and most of the gluco-
sidic linkages in ^-configuration (61a). Obviously, the steric outcome of Figure 4a. Position specificity and products of actions of porcine pancreatic alpha-aiaylase: on nonreduclng end stubbed oligosaccharides (6)
Figure 4b. Position specificity and products of actions of porcine pancreatic alpha-asylase: on Bu-coupled stubbed oligosaccharides (7)
a-1,6 link
a-1,4 link
glucose
glucose unit
reducing end glucose unit
enzymic cleavage point 18
A OO0 + ^000 'P
^ooo^ 005& + OjZ) t
^ooo- OO0 + O-0
B
B, OO0 •
no reaction
-î_ - ? 00000 O0 OO0
Q B. 000600 OO0 + ÔO0 o B,8 0000^00 OO0 + OOOÇi 19
glucoside-forming reaction could not be controlled by the usual chemical means. Therefore, natural occurring amyloses and starches are used to prepare sparsely-substituted amyloses and starches in our studies of amylase action. General procedures for starch modifications can be found in "Methods in Carbohydrate Chemistry," Volume IV, edited by Whistler et al., 1974 (61b).
All native starches contain bound phosphorus, but most of the phosphorus is chemically bound to the amylopectln component of starch (62-63). After potato starch was treated with crystalline salivary a-amylase, a maltotetraose containing a glucose phosphate unit was obtained (64,65). Parrlsh and Whelan (65,66) used a sensitive
perlodate erosion technique to characterize this limit dextrin and found
the phosphate group esterifled onto the third glucose unit from the re ducing end. Further studies on the a-llmlt dextrlns obtained from a-
araylolysls of potato starch by dephosphorylation gave maltotrlose,
maltotetraose and sîâltoperiîiaosê (67). Hypothetical structures of the 3 original phospho a-limit dextrlns were suggested, including 6 -phospho-
maltotrlose.
A partially methylated amylose was first used by llusemann and Lindemann
in amylase studies (68), but no information was given to the location of the
methyl ether groups. The distribution of methyl groups in starch partially
methylated (D.S. 0.34) by liquid ammonia and methyl iodide was determined by
quantitative paper chromatography after hydrolysis (69). The analysis gave
31.5% of 2-0-Me glucose and 21.5% of 6-0-Me glucose; no 3-0-Me glucose was
found. The attempt of Bines and Whelan (70) to prepare a partly methylated 20
amylose selectively methylated at C-2 position in their enzyme specificity
studies by a previously patented method proved unsuccessful. 6-0-methyla-
ted amylose,(D.S. 0.2) was prepared by methylation of 2,3-di-O-benzoyl-
amylose (42,71). Both Bacillus subtilis and porcine pancreatic a-amylase
caused significant hydrolysis of low D.S. 6-0-methylamylose. However,
there was no evidence of the cleavage of any 6~0-methyl-D-glucosyl bond;
at least, 6-0-methyl glucose was not detected as an amylolytic product by
t.l.c. analysis (42).
Saier and Ballou isolated a 6-0-methyglucose-containing polysaccharide
from acetone dried Mycobacterium phlei (72). This polysaccharide has a
3-0-methylglucose unit at the nonreducing end linked a-1,4 to at least four
a-1,4 glucose units. When the polysaccharide was digested with porcine
pancreatic a-amylase, a disaccharide, a trisaccharide and a tetrasaccharide
containing 3-0-methylglucose were obtained. Structural analysis showed 2 3 4 that they are 3 -O-methylmaltose, 3 -O-methylmaltotriose and 3 -O-methyl-
!Saltotetr«ose. Further a-anwlolysis of the tri- and tetrasaccharide gave 2 3 -O-Esthylmaltoss, glucose and maltose.
The procedures for the syntheses of authentic monomethylated glucoses
can be found in a review article written by Bourne and Peat (73).
In the studies of a-amylase specificities, partially modified azyloses
such as 6-iodo-6-deoxy- and 6-amino-6-deoxyanylose were also prepared as
substrates for Bacillus subtilis liquefying amylase action (74,75). 6-Iodo-
6-deoxyamylose (D.S. 0.037) was obtained by reaction of 6-tosyl-2,3-
diacetylamylose (76) with sodium iodide in N,N-dlinethylformaaiide (74). A
monosubstituted trisaccharide with the 6-0-iodo-6-deoxyglucose unit at the 21
center was isolated (74,77). ô-amino-ô-deoxy-anqrlose was first prepared by Whistler and Medcalf (78). The tosyl groups of 6-tosyl-2,3-diphenyl- carbamoyl any lose were displaced with azide ion; then reduction with lithium aluminum hydride gave 6-amino-6-deoxyanylose (D.S. 0.33). After treatment with Bacillus subtills liquefying amylase at pH 5.7, 6^-amino- 1 2 2 6 -deoxy maltose and 6 -amlno-6 -deoxymaltotriose were isolated and identified as their peracetates by mass spectrometry (75,77).
In the present investigation sparsely substituted 0-(2-hydroxyethyl)- amylose was used as the substrate for porcine pancreatic a-amylase action.
Hydroxyethyl and hydroxypropylstarches can be prepared by reaction of alkaline starch with ethylene and propylene oxide and then neutralized with dilute hydrochloric acid (79). The determination of hydroxylalkyl groups in low substituted starch ethers can be achieved by hot, constant-boiling hydriodic acid treatment (30,81). The ether linkages are cleavaged and the hydroxyalkyl groups decompose quantitatively into ethyl iodide and ethylene which may be determined volumetrically in standard solutions of silver nitrate and bromine. Because the terminal methyl of the hydroxy-
propyl group appears as a distinct doublet in the nuclear magnetic
resonance spectrum, it has been utilized as a basis of quantitation for hydroxypropylamylose (82).
A study tfas made of the digestibility of hydroxypropyl starches by a
large amount of pancreatin (83) and the digestibility was estimated from
the reducing value of the digests. It was found that the digestibility
decreased quasi-sxponsntially with increasing degree of substitution.
Statistical analysis showed that the reducing power of the pancreatic 22
digests is a function of 5.3(1 - D.S.) (84). Two hydroxypropylated 2 2 dlsaccharides, 2 -0-(2-hydroxypropyl)- and 6 -0-(2-hydroxypropyl)malto8e were isolated and identified as their peracètate derivatives by mass and n.m.r. spectrometry from the feces of rats fed with O-(hydroxypropyl)- starch (D.S. < 0.11) (85-87). Acid hydrolysis of a low-substituted hydroxypropyl starch (D.S. 0.07) followed by peracetylation and n.m.r. study showed that over 80% of the hydroxypropyl groups were attached to
0-2 of the glucose moiety, about 7% to 0-6, and a negligible amount to 0-3
(88).
Hydroxyethyl starches with D.S. 0.1 to 0.2 are particularly useful in
the textile and paper industries. In comparison to the unmodified starches,
they have superior properties such as lower gelatlnization temperature, im
proved paste and filming characteristics, and absence of rétrogradation and
gelling. Distribution o£ hydroxyethyl groups in commercial hydroxyethyl
starch with D.S. 0.1 has been thoroughly studied by Srlvastava et al.
(89,90). Employing Smith degradation (91,92), which involves periodate
oxidation, sodium borohydride reduction, acid hydrolysis of the resulting
polyol, and quantitative fractionation of the hydrolysate on a carbon-
celite column, it was shown that in the commercial sample of hydroxyethyl
starch, 84% of the hydroxyethyl groups were substituted at 0-2 of the
anhyùrûglueose units, the remaining ether groups being located mainly at
the 0-6 position with only negligible substitution at 0-3 (89). Another
method utilizing yeast feraentation to remove glucose from the acid
hydrolysate or the hydroxyethyl com starch and quantitative chromatographic
separation gave similar results (90). In addition to 2-0-, 3-0-, and 23
6-0-(2-hydroxyethyl)-D-glucoses, three isomeric anhydro derivatives of
2-0-(2-hydroxyethyl)-D-glucose were Isolated from the acid hydrolysate
of 0-2-hydroxyethyl)starch and Identified as 1,2-0-ethylene-a-D-gluco-
furanose, 1,2-0-ethylene-g-D-glucopyranose and 1,2-0-ethylene-a-D-
glucopyranose (93). The formation of these 1,2-0-ethylene-D-glucoses took
place by intramolecular glucosidation of 2-0-(2-hydroxyethyl)-D-glucose,
and were easily separated by gas chromatography as their TMS-derivatives
(12). 2-0-s 3-0-, and 6-0-(2-hydroxyethyl)-D-glucoses were synthesized
by reduction of the corresponding carboxymethyl derivatives with lithium
aluminum hydride (94). 2-0-, 3-0-, and 6-0-carboxymethyl derivatives (95)
were first synthesized through carboxymethylation of methyl 3;5;6-tri-0-
benzyl-a,0-D-glucofuranoside, 1,2 ;5,6-di-O-isopropylidene-D-glucofuranose
(96,97), and 1,2;3,5-di-O-methylene-D-glucofuranose (98,99) respectively,
followed by removal of the blocking groups (95). 1,2-0-ethylene-D-
glucoses were directly synthesized from 2-chloroethyl-D-glucofuranoslde
(100,101). An improved method using ethylene carbonate as the hydroxyethyla-
tion agent has also been reported (102,103). So far two papers related to
the investigation of porcine pancreatic a-amylase action on 0-(2-hydroxy-
ethyl)-starches have been published by other researchers (104,105). It
has been shewn that the amylolysis rates decreased as the D.S. increased,
aad 2-0-(2-uyuroxyethyl) groups induced higher resistance to amylolysis of
the starch than 6-0-(2-hydroxyethyl) groups. No detailed investigation has
been performed.
Sparsely modified deoxyamylose could be a very interesting substrate
for amylase action to investigate the roles of hydrogen bonding between
c- 24
hydroxyl groups of the glucose unit and the enzyme in the binding and catalytic process. In a preliminary investigation, 6-deoxyamylose (D.S.
0.9, prepared by reduction of 2,3-di-0-benzoyl-6-iodoamylose with LiAlH^) on digestion by salivary a-amylase gave a sugar with the paper chroma tographic mobility of 6-deoxyglucose (106). An improved synthesis of 6- deoxy-cyclodextrins and amy lose has been developed (107,108).
C. Gas Chromatography of Carbohydrates
The first report on gas liquid chromatography of carbohydrate was published In 1958 (109). The major difficulty for the rapid preparation of volatile derivatives of the polyhydroxy compounds in gas chromatography was overcome by Sweeley and his coworkers by the application of trimethyl- silyl derivatives (110).
An excellent review article of employing gas chromatography in carbohydrate chemistry was published by Button, which contains 732 entries from the year of 1958 to 1972, and 13 very useful tables (111). In recent years, the application of the gas chromatograph-mass spectrometer combina tion has been widely utilized in the structure determination in carbohydrate chemistry; a review article which consists of 29 entries for carbohydrates was published in 1972 (112).
Gas chromatographic analyeta of trimethylsilylated hydrcxyethyl stylose hydrolyzate was reported by Brobst and Lott (113). 0-(2-hydroxyethyl)- glucoses and the inner glucosides of 2-0-(2-hydroxyethyl)glucose were identified by using a SE-30 column (8,3). 25
III. MATERIALS AND METHODS
A. Matetials
1. Enzymes
a. Porcine pancreatic a-amylase (g-l,4-glucan-4-glvcanohydrolase,
3.2.1.1) Crystalline porcine pancreatic a-amylase, prepared by a modification of the method of Caldwell et (26), was obtained from
Worthington Biochemical Corporation, Freehold, New Jersey. A 10-ml stock solution was made containing 4000 units of enzyme in 10 mM NaCl and 20 mM sodium glycerophosphate buffer at pH 6.9. Both crystalline enzyme and stock enzyme solution were used in the enzymatic digestions.
b. Glucoamylase (Amylo-l,4:1,6-Blucosidase, 3.2.1.3) Gluco- amylase from a selected strain of Rhlzopus nlveus was purchased from Miles
Laboratories, Elkhart, Indiana. This twice recrystalllzed enzyme did not have any a-amylase activity and was prepared by Seikaku Kogyo Co., Tokyo,
Japan. A stock dilution of this enzyme containing 42 units jper ml of
0.05 M sodium acetate buffer (pK 4.3) wss =cde for îsost of the glucor- amylolysls. Occasionally, glucoamylase I of Aspergillus nlger obtained from Takamine Laboratory, Inc., Clifton, New Jersey, was used. Gluco-
anylases from both sources were checked and found to have the same specificities.
c. Sweet potato p-amylass (a-l,A-glucan «altohydrolase. 3.2.1.2)
Sweet potato 6-amylase was also purchased from Worthington Biochemical
Corporation. Dilutions with 0.05 M sodium acetate buffer (pH 4.5) were
made freshly before use in each experiment. 26
2. Substrate
a. Superlose 500 Superlose 500, a sparsely 0-2-hydroxyethylated amylose with D.S. between 0.1 and 0.2, was obtained from Stein, Hall &
Co., Net? York, New York. Complete acid hydrolysis of this substituted amy lose followed by g.l.c. analysis showed that predominantly the C-2 position of the glucose unit was etherified. Most of the remaining substitution was located at C-6 and there was only a very small quantity of C-3 hydroxyethylated glucose.
b. Hydroxyethyl com starch Sparsely 0-2-hydroxyethylated corn starch with 0.1 D.S. was a gift from Penick and Ford, Ltd., Cedar Rapids,
Iowa. The distribution of hydroxyethyl groups in this starch was found to be similar to Superlose 500.
3. Standards
2-0- and 3-0-(2-Hydroxyethyl-D-glucoses were gifts from Dr. H. C.
Srivastava, Ahmedabad Textile Industry's Research Association, Ahmedabad,
India.
6-ô-(2-Hydroxyethyl)giucose was prepared through the synthesis of l.2:3.5-di-0-methylene-a-D-glucose (98,99). The anion of this intermediate,
generated by the action of NaH-DMSO on this compound was reacted with
ethylene carbonate. After fractionation on s silica gel column to obtain
the hydroxyethyl derivative» the blocking groupa were removed to give
6-0-(2-hydroxyethyl)-D-glucose (102).
3^-0-Methylmaltose was obtained by deacetylation of 1,2,6,2',3',4',6'-
hepta-O-acetyl-3-O-methyl-G-maltose (114), which was a gift from Dr. W. E.
Dick of Northern Regional Research Laboratory, Peoria, Illinois. The 27
acetate (16 mg) was deacetylated in 0.05 N methanollc NaOCH^ (1 ml) at
0°C for 18 hours. The syrup obtained after neutralization with 1 M acetic acid and evaporation under reduced pressure was desalted by passing through a G-15 Sephadex column. Paper chromatography showed that it con tained mostly 3^-0-methyltnaltose but also a trace amount of maltose.
4. Gas chromatography
A Packard Model 409 gas chromatograph (Packard Instrument Company,
Downers Grove, Illinois) equipped with flame ionization detectors was
used in the investigation. Column packings and stainless steel tubing were purchased from Applied Science Laboratories, State College. Pennsyl
vania. Trimethylsilylation reagents such as Tri Sil 'Z', pyridine, N,N- dimethyl formamide, hexamethyIdisilazane, and trlmethylchlorosilane were obtained from Pierce Chemical Company, Rockford, Illinois. Tri Sil 'Z' is formulated with N-trimethylsilyl-imidazole in dry pyridine.
5. Miscellaneous
Darco G-60 activated carbon was purchased from Atlas Chemicai In
dustries, Wilmington, Delaware. Celite 560 was from Johns-Manville,
Lompoc, California. Blue dextran 2000, Sephadex G=15, and a gel filtra
tion column K16-100 (1.6 x 100 cm) equipped with an A16 flow adapter were
obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey. A
Chromatronix R6031 3v valve attached to the inlet of the column was from
Chromatronlx Incorporated, Berkeley, California, Polystaltic Pumps were
from Bachlsr Instruments, Fort Lee, New Jersey. Silica Gel G for thin
layer chromatography was obtained from E. Merck, Darmstadt, Germany. 28
Orcinol for total carbohydrate determination was purchased from J. T.
Baker Chemical Co., Phillipsburg, New Jersey. High purity maltose for preparing various concentrations of standard solutions in total carbo hydrate and reducing value determinations was obtained from Hayashibara
Co., Ltd., Okayama, Japan, or a preparation in our laboratory.
B. Methods
1. Paper chromato graphy
Whatman 3MM papers were used for both preparative and analytical paper chromatography. Three solvent systems were commonly used:
A: (1-butanol-pyridine-water, 6:4:3, v/v)
B: (1-propanol-ethyl acetate-water, 6:1:2, v/v)
C: (Ethyl acetate-acetic acid-water, 9:2:2, v/v).
Both solvent systems (A) and vS) were utilized in the ascending technique at 65*C (115). Descending paper chromatography was performed by solvent systems (C) at room temperature (93,94). Alkaline AgNO^ was used in the detection of the reducing carbohydrates (116).
Filter papers were previously washed with 2% NH^OH for two days before they were used in the ascending preparative paper chromatography (72). For a size 13" % 17" filter paper, 10 - 15 mg. of carbohydrate can be applied aiiu gives a good séparation^ After the chromatogram was irrigated three times at 65°C with solvent system (A), the guide strips wsrs cut off and developed to locate zones of carbohydrates on the chromatogram. Each in dividual zotte containing carbohydrate was excised and the carbohydrate was eluted out with distilled water. The final solution was concentrated 29
under reduced pressure at a temperature below 40*C.
Occasionally, solvent systems such as:
D; (methyl ethyl ketone-acetic acid-boric acid saturated in water,
9:1:1, v/v) (117) and
E: (nitromethane-acetic acid-ethanol-water saturated with boric
acid, 8:1:1:1, v/v) (118) 2 were used to separate glucose from 3 -0-(2-hydroKyethyl)-maltose by the descending technique at 40*C. The compounds were detected by the AgNO^
dip with 0.5 M sodium hydroxide in 80% ethanol containing 4% pentaerythritol
(119).
2. Charcoal column chromatography
One kilogram of Darco G-60 activated carbon and an equal amount of
Celitc 560 were mixed together. The mixture was slurried with 1 N HCl overnight. The slurry was filtered by suction and washed with water until
the water was neutral and a 5% aqueous solution of n-butyl alcohol was
used to remove remaining HCl in the charcoal (120). After it was washed
several times with water, the column was packed and further eluted with
water uatll no n=SuOH was in the charcoal. The column was 52 cm long and
with a diameter of 10 cm. Gradient elutlon was used for the separation
(see experiment).
3. Gel filtration chromatography
Column A (1.5 x 100 cm) and Column B, a K16-100 column equipped with
an A16 flow adaptor and a Chromatronix RéOSl Sv valve, were packed with
58.7 and 80 g. (dry weights) of Sephadex G-15, respectively. The weighed 30
gel for each column was previously washed with 300-inl portions of 0.05 N
NaOH and 0.05 N HCl twice. Alternatively, four 300-ml portions of 1 N propanoic acid can be used for the washing instead of the diluted acid and base. The gel was washed again with water until neutral and the columns were packed.
Column A was used to purify oligosaccharides isolated from prepara tive paper chromatography, and to determine their relative elution volumes.
One-ml fractions were collected with a fraction collector at a flow rate of 12 ml per hour.
The outlet of the Column B was directly connected to the AutoAnalyzer for determining the relative total carbohydrate contents of individual components eluted from the column. A sample loop of 0.175 ml capacity was attached to the Chromatronix valve at the inlet of the column. The reverse flow method was used in this continuous assay, and the same flow rate was maintained as for Column A.
Columns were calibrated using G^, G^, G^, G^, and G^ and used for de termination of the molecular weights of various subfractions. Foiystaltic
pumps were utilized to pump the eluant (water) through the columns.
4. Thin layer chromatography
A suspension of Silica Gel G was prepared by dispersing 35 g. of the solid mixture in 100 ml chloroform-methanol (2:1, v/v). After stirring the dispersion mixture for one minute, a sandwich of two microscope slides (7.6 X 2.5 cm) was dipped into the suspension and slowly withdrawn.
The solvent quickly evaporated, and the two plates were then separated. The plates were activated at 110°C for ten minutes before use (121). 31
For preparing a bigger plate, a suspension of Silica Gel G was prepared by dispersing one portion of the solid mixture to two portions of water (w/w). The glass plates coated with this suspension were dried at 110*C for ten minutes before use.
Five to thirty micrograms of the carbohydrate in an aqueous solution was spotted on the plate and dried with a stream of dry nitrogen. After irrigating with solvent system F, (1-butanol-acetlc-ether-water,
9:6:3:1, v/v), the spots were located by spraying with 25% aqueous HgSO^ and heating over a hot plate.
5. Total carbohydrate analysis
Total carbohydrate was determined by the orclnol sulfuric acid pro cedure adapted for use with the Technicon AutoAnalyzer (122,123). A reagent solution contairiing 0.5 g. of orclnol and 40 mg of sodium borate per liter of 70% sulfuric acid was prepared to replace conc. sulfuric acid and 2% phenol solution in the phenol-sulfuric acid method (124). In addi tion, the sample solution was mixed with the reagent in the manifold and then pasted through the heating bath at 90°C instead of a cooling coil as in the procedure of using phenol-sulfuric kcid.
6. Reducing values
Reducing values of carbohydrates were measured by the alkaline ferrlcyanlde-cyanide method with the Technicon AutoAnalyzer (125).
7. Sodium boeohydriuë réduction of oligosaccharides
In a small test tube (1 x 7.5 cm), 0.1 to 0.2 ml of the solution 32
containing 0.4 to 2 mg of the saccharide was added with 0.5 to 1 ml of freshly prepared 1% NaBH^ in distilled water. After 4 hours at room temperature, excess NaBH^ was decomposed by adding Amberllte IR-120 ion exchange resin until the solution reached pH 6. The supernatant solution was dried under reduced pressure, and methanol (3x1 ml) was added to remove methyl borate (126). The residue was dissolved in 0.1 ml of distilled water for further hydrolysis.
8. Complete acid hydrolysis of di-. trl- and oligosaccharides
About 100 - 600 pg of the saccharide In 0.1 ml of aqueous solution was transferred by a Hamilton syringe to a small glass tube (0.3 x 8 cm) which had already been sealed at one end. The same volume of 2 N HgSO^ was then added to obtain a solution containing 1 N HgSO^. After passing nitrogen througjh the solution, the other end of the tube was sealed and the tube was submerged in an oil bath which was maintained at 110*C for four hours. The tube was then opened, and the hydrolysate was transferred to a small test tube (i x 7.5 cm). A solution (0.2 N) was added drop by drop until the pH reached 7, then barium sulfate was removed by csntrifugation at 10,000 rpm for 20 minutes « The final solution was dried
under reduced pressure and again 0.1 ml of distilled water was added. The solution was transferred to a small Reacti-Vlal (0.3 ml container. Pierce) and was evaporated for analysis.
9. Gas chromato graphy
A stainless steel column (1/8 In. x 6 ft.) which had been packed with
3% SE-30 (w/w) on 100/120 mesh Gas Chrom Q was operated isothermally at 180°C 33
for analysis of TMS-monosaccharldes. The flow rate of nitrogen was 40 ml per minute with Injector temperature at 300*C and detector temperature at 235°C. Flow rates of air and hydrogen were 250 and 30 ml per minute, respectively. The 0.1 ml of the hydrolyzate obtained from acid hydrolysis
In the Reactl-Vlal was dried with a stream of dry nitrogen, and then trlmethylsllylated with Trl-Sll 'Z'. The concentration of the sugar In the solution was 1%. When the N,N-dlmethylformamlde, hexamethyldlsllazane, and trlmethylchlorosilane system was used, the dried hydrolyate was dissolved In N,N-dlmethylformamlde, then hexamethyldlsllazane and trl methylchlorosilane were added to obtain a reaction mixture containing 1% of carbohydrate. The final proportion of N,N-dlmethylformamlde, hexa methyldlsllazane , and trlmethylchlorosilane was 5:5:3 (126). In both cases, the reaction mixtures were warmed up to 70*C for 10 minutes to
Insure complete trlmethyl sllylation. One to two microliter allqucts of the samples were Injected Into the column. Alternatively, 300 to 600 yg of dry powdered monosaccharide was used for trlmethyl sllylation.
As for the disaccharides, the conditions were maintained the same as for the monosaccharides except the column temperature was raised to 220*C, and the same procedure for trlmethylsilylation was used. In each round of the experiments, authentic compounds were Injected to check the retention times. In the mynosaeeharlds analysis, for convenience, an acid hydrolysate of 0-(2-hydroxyethyl)amylose was often used as a reference mixture. 34
IV. EXPERIMENTAL
A. Complete Acid Hydrolysis of 0-(2-Hydroxyethyl)amylose
0-(2-Hydroxyethy1)amylose (2.84 g.) was hydrolyzed with 6.7 ml of conc. HgSO^ (D = 1.84) in 200 ml of water. After the reaction mixture was refluxed for 7 hours, the hydrolyzate was neutralized with solid BaCO^ and filtered. The filtrate was deionized by adding 1% polyethylene glycol treated MB-3 Amberlite resin. The final deionized solution was concen trated under reduced pressure to a syrup which was further dried in a vacuum oven at 35*C overnight. The monosaccharides in the final product were analyzed by paper and gas chromatography (Table 1 and Figure 5).
B. Porcine Pancreatic a-Amylase Action on 0-(2-Hydroxyethyl)-amylose
Tt-jenty grams of Superlose 500 was dissolved in 198 ml of 20 mM sodium glycerol phosphate buffer (pH 6.9) in 10 mM sodium chloride solution. Final clear solution was maintained at 39*C, and 2 ml of porcine pancreatic a- amylase (10,500 units/ml) was added. After 3 days, 1 mi of the above digest was deionized and a solution containing approximately 50 mg/ml of the sugar mixture was prepared. Paper chromatographic analysis of this enzymatic digest Is shown in Figure 6.
C, Identification of the Reducing Eads of Oligosaccharides in the a-Amylase Digest of 0-(2-Hydroxyethyl)amylose
The original enzymatic digest of 0-(2-hydroxyethyl)amylose was reduced by sodium borohydride and the reduced digest was further subjected to acid hydrolysis. Gas chromatographic analysis showed that sorbitol was the only 35
Table 1. and values of 0-(2-hydroxyethyl)-D-glucoses and the reference compounds, to
Sugar R^^ R^^
2-0-(2-hydroxyethyl)- D-glucose 0.864 1.13 0.880 1.16
3-0-(2-hydroxyethyl)- D-glucose 0.864 1.13 0.880 1.16
6-0-(2-hydroxyethyl)- D-glucose 0,818 1.07 0.824 1.09
0.766 1 0.756
Gg 0.597 0.78 0.528 0.70
0.435 0.56 0.339 0.45
G, 0.286 0.37 0.189 0.25 4
G^ 0.182 0.24 0.098 0.13
^Solvent system À, 1-butanol-pyridine-water (6:4:3, v/v); three ascents at 65°C.
^Solvent system Bs l-propanol-ethyl acetate-water (6:1:2, v/v); three ascents at bS'C. Figure 5. Gas chromaCogram of O-C2-hydroxyethyl)amylose after acid hydrolysis with 1.2 N sulfuric acid at 100°C for 7 hours. The trimethylsilyl derivatives are identified as
a, a-D-glucopyranose
b, 1;2-0-ethylene-a-D-glucopyranose
c, 3-D-glucopyranose
d,g. 3-0-(2-hydroxyethyl)-D-glucopyranose
e,h. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,i. 6-0-(2-hydroxyethyl)-D-glucopyranose
Broken line represents gas chroiaatograta obtained at lower attenuation (1/32) DETECTOR RESPONSE Figure 6. Paper chromatogram showing porcine pancreatic a-amylase action on 0-(2-hydroxyethy1)amylose.
rhe assay contained 100 mg of substrate plus 105 units of enzyme in 1 ml of 20 mM sodium glycerol phosphate buffer (pH 6.9) and 10 mM sodium chloride at 39°C for 3 days.
The paper chroma.togram was irrigated with solvent system B, three ascents at 65*C. G _ shows the standards of glucose to maltoheptaose. All 1, 2, and 3 are the same enzymatic digest of 0-(2-hydroxyethyl)- amylose. The amount of digest spotted on the paper was decreased with increasing order 39 40
alditol in the acid hydrolyzate (Figure 7).
D. Preliminary Separation of Porcine Pancreatic a-amylase Digest of 0-(2-Hydroxyethyl)amylose by Using a Charcoal Column
One hundred milliliters of the a-amylase digest (10 g.) was applied on the charcoal column. The column was eluted subsequently with 4 liters each of the 1%, 2%, 3%, 4%, 5%, 7% and 9% tert-BuOH solutions. The mixing vessel had a capacity of 4 liters, and was filled up with distilled water at the beginning. The outlet of the column was connected to an automatic polarimeter followed by a fraction collector. Twenty milliliters of the eluate was collected In each tube. Totally, 1520 tubes were used. One sample out of every 10 tubes was checked by paper chromatography. The tubes containing similar carbohydrates were pooled together to give seven fractions (Figure 8). Each fraction was concentrated under reduced pressure at 35®C. Total carbohydrate in each fraction was determined by the orcinol- sulfuric acid method adapted with a Technicon AutoAnalyzer (Table 2).
E. Glucoamylase I Action on 0-(2-HydrcKysthyl)starch
Two hundred milligrams of 0-(2-hydroxyethyl)starch were dissolved in
10 ml of citrate buffer (0.05 M, pH 4.8). One-half ml of the starch solu tion was mixed with 0.5 ml of the enzyme solution (13.8 units per ml) at room temperature and one drop of toluene was added to prevent mold growth.
Aliquots of 0.1 ml were pipetted at different time intervals and were heated in a boiling water bath to stop enzyme activity. Paper chromatography (sol vent system A) showed no production of 0-(2-hydroxyethyl)-D-glucose (Figure
9). Reducing value showed that there was 38.5% conversion to glucose after
1 week. Figure 7. Gas chromatogram of the original enzymatic digest which had been reduced by sodium borohydride followed by acid hydrolysis then trimethylsilylated.
ajC. D-glucopyranose
b. sorbitol
d,g. 3-0-(2-hydroxyethyl)-D-glucopyranose
e,h. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,i. 6-0-(2-hydroxyethyl)-D-glucopyranose 42
abc
Reduced PPA Digest
II V,I M
lA
10 15 20
TIME (MINUTES) Figure 8. Paper chromatograms of porcine pancreatic a-amylase digest of 0-(2-hydroxyethyl)amylose fractionated by the charcoal column. The irrigating solvent in (A) was solvent system A, and in (B) was solvent system B. Both chromatograms were three ascents at 65°C.
G^_y: Standards, glucose to maltoheptaose
1 to 7: Fractions from charcoal column
Subfractions were designated in sequence from the top to each fraction from charcoal column by Roman numerals. For example, the subtractions of fractions 4 and 5 were designated as:
41, 411, 4III, 4IV,...
51, 511, 5III, 5IV,... 44 45
Table 2. Charcoal chromatography of a-amylase digest of 0-(2- hydroxyethyl)amylose
Fractions Tube No. Concentrated Concentra- Total carbo- volume (ml) tion (mg/ml) hydrate (mg)
1 100-400 15 132 1980
2 401-620 10 138 1380
3 621-740 8 23.7 189.6
4 760-830 6 48.9 293.4
5 831-1170 15 117.6 1764
6 1171-1350 8 75.9 607.2 Figure 9. Paper chromatogiram of glucoamylase action on 0.1 D.S. 0-(2-hydroxyethyl)starch Three ascents irrigated with solvent system A at 65°C. HESAH represents the acid hydrolyzats of 0-(2-hydroxyethyl)starch which was used as a mixture of standards. indicates glucose to maltoheptaose 8 24 48 iEiours 48
F. Glucoamylase Action on 3^-0-methylmaltose
Fifty microliters of the aqueous solution containing 400 yg of 3^-0- methylmaltose was mixed with 2 units of glucoamylase (R. niveus) in 50 yl of 0.05 M sodium acetate buffer at pH 4.5. After incubation at 39°C for
2 days, the enzymatic reaction was stopped by heating in a boiling water bath. Paper chromatography showed no cleavage of this compound, but maltose was completely converted to glucose under the same condition
(Figure 10).
G. Glucoamylase Treatment of Carbohydrate Fractions from Charcoal Column Chromatography and the Original Or-amylase Digest
Two units of glucoamylase I (A. nlger) was used in each assay for 2 mg of the substrates. Since the original glucoamylase solutions were highly buffered around pH 4.5, the assays were performed by mixing the enzyme solution, water and the aqueous solution of each substrate to a final
volume of 500 Pi. After 48 hours at room temperature, the reaction mix
tures were heated in a boiling water hfith !'of 'j Hit nut t a
enzyme. The resultant solutions were deionized by Âmberlite MB-3 and then concentrated under reduced pressure. The results are shown in Figure 11.
K. Further Separation of Fractions 4 and 5 from Charcoal Column to Subfractions by Preparative Paper Chromatography aud Sephadex G=15 Colusn Chromatography
Subfractions 41, 411, 4III, 4IV, 51, 511, 5III, and 5IV were prepared
by preparative paper chromatography (solvent system A, 3 ascents at 65°C).
Arabic numbers indicate the numbered fractions from charcoal column and
Roman numbers indicate the numbered subfractions obtained from preparative Figure 10. Paper chromatogram of 3^-()-methylinaltose after treatment of glueoamylase {see text). One ascent irrigated with solvent system B at 65°C.
1. 3^-0-methylnaltose after glueoamylase treatment
2. 3^-0-methylmaltose after glueoamylase treatment and deionized
3. original 3^-0-methylmaltose
4. 3-0-methyl-D-glucose
5. maltose after glueoamylase treatment (control)
6. mailtose after glueoamylase treatment and deionized (control) 50 Figure 11. Glucoamylase action on 0-(2-hydroxyethyl)-limit dextrine from '%-amylolysis.
1 to 7. Fractions from charcoal column s IGA to 7GA. Fractions from charcoal column treated with glucoanylase
0. Original a-amylase digest
OGA. Original a-amylase digest treated with glucoamylase
Chromatographic, condition,; Solvent system B, 3 ascents at 65°C vso VDi «» vss '•-h '•-h «D» TO: VSft 53
paper chromatography in the order of decreasing mobilities (Figure 8).
After each subfraction was eluted from the paper and concentrated under reduced pressure, a maximum of 12 mg of the subfraction in 0.25 to 1 ml
0.2% blue dextran solution was applied on the Sephadex G-15 column (column
A). The 1-ml fractions were checked by orcinol-sulfuric acid method with a Technicon AutoAnalyzer. Both elution profiles of 41 and 51 showed that each of them was further resolved into two components, namely, 4IS1,
4IS2, and 5IS1, 5IS2 in that order of decreasing relative elution volumes
(Figures 12 and 13), where S stands for Sephadex column chromatography.
All other subfractions from preparative paper chromatography gave single peaks in the gel filtration. Relative elution volumes of the subfractions containing 0-(2-hydroxyethyl) sugars are shown in Table 3. Thin layer chromatographic data of 4IS2 and 4III are shown in Table 4.
I. Structural Analysis of 4IS2
4IS2 and an aliquot of the same subfraction which had been reduced by sodium borohydride were hydrolyzed ia 1 N After triscthylsilylation^ both acid hydrolyzates were subjected to gas chromatographic analysis
(Figures 14 and 15).
J. Structural Analysis of 4III
1. Acid hydrolysis of 4III and reduced 4IZI
Subfraction 4III was hydrolyzed by 1 N H^SO^ to check the monosaccharide components in the hydrolyzate (Figure 16). After original 4III was reduced with sodium borohydride, it was also acid hydrolyzed and then analyzed by
gas chromatography (Figure 17). Figure 12. Chromatography of 41 on Sephadex G-15. Chromatographic conditions; Sephadex G-15 column A (1.4 x 100 cm); eluant (water) flow rate at 12 ml per hour; room temperature; one-ml fractions were collected 55
41
'1200
4IS2
•1000
- 800
0I
1 600
O
i r 400
h 200
JL
60 70 80 90 100
TUBES Figure 13. Chromatography of 51 on Sephadex G-15. Chromatographic conditions were the same as in Figure 8 57
51 5IS1
1600
1400 t5
800
600
400
5IS2
200
60 70 80 90 100
TUBES 58
Table 3. Relative elutlon volumes (V^/V ) of Important 0-(2-hydroxyethyl)- maltooligosaccharides determined by Sephadex G-15 column chromatography
M.W. M.W. (exp.)^
«1 1 1.77 180.16
0.70 1.61 342.30 S
0.45 1.46 504.46 S
«4 0.25 1.39 666.62
0.13 1.29 828.78 S
4IS2 0.96 1.58 347.7
4III 0.57 1.42 580.7
5IS2 0.96 1.57 359.0
5I1I 0.58 1.46 510.9
5IV 0.33 1.34 750.2
"Solvent system B, three ascents ac ô5°C.
^Column A.
^M.W. estimated from their relative reduced volumes. 59
Table 4. and Rg values of certain carbohydrates on thin layer chromato- grams. Solvent system F, 1-butanol-acetic acid-ether-water (9:6:3:1, v/v), one ascent under room temperature
0.28 1
Gg 0.17 0.59
G^ 0.08 0.29
G^ 0.0034 0.12
2-0-(2-hydroxyethyl)-D-glucose 0.25 0.90
3-0-(2-hydroxyethyl)-D-glucose 0.29 1.02
4IS2 0.17 0.59
4III 0.057 0.20 Figure 14. Gas chromatogram of 4IS2 after acid hydrolysis and triaetiiylsilylatior.
a,b. D-glucopyranose
c,d. 3-0-(2-hydroxyethyl)-D-glucopyranose 61
a
TMS-G
b 4IS2
A TMS-3-0-HEG \r § S i 10 15 20
TIME (MINUTES) Figure 15. Gas chromatogram of 4IS2 which had been reduced by sodium borohydride followed by acid hydrolysis then trimethylsilylated
a. sorbitol
b,c. 3-0-(2-hydroxyethyl)-D-glucose DETECTOR RESPONSE
*». H CO to N3 » O Figure 16. Gas chromatogram of acid hydrolyzate of 4III. Trimethylsilyl derivatives are identified as:
a. 1,2-0-ethylene-a-D-glucofuranose
b.d. D-glucopyranose
c. 1,2-0-ethylene-a-D-glucopyranose
e,f. 2-0-(2-hydroxyethyl)-D-glucopyranose
! { i
I 65
4III
III
e I» \ il ^ H t
10 15 20
TIME (MINUTES) Figure 17. Gas chromatogram of reduced 4III after acid hydrolysis and triraethylsilylation.
a. 1,2-0-ethylene-a-D-glucofuranose
b.e. D-glucopyranose
c. 1,2-0-3thylene-a-D-glucopyranose
d. sorbitol
f,g. 2-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE 6»
2. Glucoam^lase treatment of 4III and Isolation of the subfractions
After 20 mg of purified 4III was incubated with 22 units of gluco-
amylase (R. niveus) in 1 ml of 0.05 M sodium acetate buffer (pH 4.5) at
39*C for 2 days, paper chromatography showed three spots, 4IIIGA1, 4IIIGA2,
and 4IIIGA3 in the order of decreasing mobilities (Figure 18). GA stands
for glucoamylase action. These subfractions were isolated by preparative
paper chromatography and further purified by passing through a Sephadex
G-15 column (column A)(Table 5). Gas chromatographic analyses of 4IXIGA2
and 4IIIGA3 after acid hydrolysis and trimethylsilylation are shown in
Figures 19 and 20.
Table 5. Carbohydrate subfractions isolated from glucoamylolysis of 4III. Chromatographic conditions are the same as in Table 3
M.W. (exp.) %G
4IIIGA1 1.00 1.75 201.6
4IIIGA2 0.83 M •^ J 40S.2
4IIIGA3 0.53 1.47 494.7
3. Relative total carbohydrate of 4IIIGA1. 4IIIGA2, and 4IIIGA3 la the glucoamylase digest of 4III
An assay containing 2.4 mg of 4III and 2 units of glucoamylase (R.
niveus) in a total volume of 0.2 ml sodium acetate buffer (pH 4.5) was pre
pared under the same experimental condition as previously mentioned. After
the enzymatic reaction was stopped by heating in a boiling water bath for Figure 18. Paper chromatogram showing the action of glucoamylase on 4III.
y. glucose to maltoheptaose standards
4IIIGA. glucoamylase digest of 4III; 4IIIGA1, 4IIIGA2 and 4IIIGA3 are numbered in se quence from the top.
Chromatographic conditions: Solvent system B, three ascents at 65"C 70
4IIIGA 4III Figure 19. Gas chromatogram of 4I1IGÀ2 after acid hydrolysis and trimethylsilylation
a. 1,2-Q-ethylene-a-D-glucofuranose
b. 1,2-0-ethylene-8-D-glucopyranose
c.e. D-glucopyranose
d. 1,2-0-ethylene-a-D-glucopyranose
f,g. 2-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE t:
•u H H H g to Figure 20. Gas chromatogram of 4IIIGA3 after acid hydrolysis and trimethylsilylation.
a. 1,2-0-ethylene-a-D-glucofuranose
b. d. D-glucopyranose
c. 1,2-0-ethylene-a-D-glucopyranose
e,h. 3-0-(2-hydroxyethyl)-D-glucopyranose
f.i. 2-0-(2-uydroxysthyl)-D-glucopyranose
g,j. 6-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE 75
5 minutes, it was dried by a stream of dry nitrogen. The residue was redissolved in 0.2 ml of 0.2% blue dextran solution, and 0.175 ml of this final solution was applied on Sephadex G-15 column (column B) for chromatography. The elution profile is shown in Figure 21.
4. Sodium borohydride reduction of 4IIIGA.3 and partial acid hydrolysis of the reduced compound
4IXIGA3 (1.12 mg) was reduced with 10 mg of NaBH^ in 1 ml of distilled water for 4 hours under room temperature. The reaction mixture was neutralized with 2 M acetic acid and the reducing value of the neutralized solution was checked by the alkaline ferricyanide method to test for complete reduction. After rencval of the borate and purification with
G-15 Sephadex column chromatography, the final reduced compound was dissolved in 0.2 ml of 0.1 N HgSO^. The solution was transferred into a sealed glass tube (0.3 x 8 cm) and maintained at 110°C in an oil bath for
3 hours. The tube was then opened, and the content was transferred to a
Binall teat tube. After neutralization with 0.2 N Ba(0H)2, the supernatant solution was dried under reduced pressure. The residue was dissolved in
0.2 ml of distilled water for G-15 column chromatography (Figure 22). The disaccharide and monosaccharide fractions were pooled separately and dried under reduced pressure. Both fractions were trimethylsllylated. In the disaccharide fraction, gas chromatography shewed the presence of 3 -0-(2- hydroxyethyl)maltitol and maltltol (Figure 23a). Maltose, 2-0-(2-hydrosy- 2 ethyDnaltose (4IIIGA2) and 3 -0-(2-hydroxyethyl)maltose (43:82) and their reduced forms were used as standards. In the monosacchsride fraction, all the 0-(2-hydroxyethyl)-D-glucoses were found in addition to sorbitol and Figure 21. Chromatography of glucoamylase digest of 4III on Sephadex G-rl5 column. Chromatographic conditions: Column B (1,6 x 100 cm); reverse flow method at a flow rate of 12 ml/hour; at room temperature. The amounts of carbohydrate in the subfractions 4ÎÏIGÂ1, 4IIIGA2, and 4IIIGA3 were estimated by integration between c - d, b - c, and a - b, respectively. The base line with negative total carbohydrate values is due to the base line shift of the recorder 77
270 4IIIGA2 4IIIGA
230
190
.150 4IIIGA1
J.10
70
tl 1 .. 4IIIGA3
30
240 300 360 420 480
TIME (MINUTES) Figure 22. Sephadex G-15 chromatography of 4IIIGA3 after sodium borohydride reduction and partial acid hydrolysis with 0.1 N H^SO^ at 110°C for 3 hours. Trisaccharide alditol was pooled between a and b; disaccharide and disaccharide alditol were pooled between b and c; monosaccharide and alditol were pooled between c and d
! TOTAL CARBOHYDRATE JIG/ML
M o\ 00 O N5 o o O O
> 4S m H R. M m w > U> a /-s M PO o vx M N (U 0 H rt rt m H- P) H-* H- Figure 23a. Gas chrojnatogi:am of TMS-disaccharides and TMS-disaccharide alcohols obtained from partial hydrolysis of reduced 4IIIGA3 and trimethyl- silylatloD.
a. ?
b. maltitol
c. ? 2 d. 3 -0-(2-hydroxyethyl)maltitol
e. ? DETECTOR RESPONSE
M O
% Ul w s H a C3 H M NJ CO o
N3 Ln
W o
CO Ul
18 82
D-glucose (Figure 23b). The relative amounts of 0-(2-hydroxyethyl)-D- glucoses were in the order of 6-0- > 2-0-> 3-0-.
5. Porcine pancreatic a-amylolysls of 4IIIGA3
4IIIGA3 (260 yg) was treated with porcine pancreatic a-amylase (20 units) in 0.2 ml of 10 mM sodium glycerol phosphate buffer (pH 6.9) in
5 mM NaCl solution at 39°C for 2 days. Maltotriose was used as the control.
The enzymatic digest was subjected to paper chromatography, and the result is shown in Figure 24.
K. Structural Analysis of 5III
1. Acid hydrolysis of 5III and reduced 5III
5III and sodium borohydrlde-reduced 5III were subjected to acid hydrolysis. Figures 25 and 26 show the gas chromatograms of the acid hydrolyzates.
2. Glucoaatylase treatment of 5III
Purified 5III (11.3 mg) was treated with glucoamylase (11 units)
(R. nlveus) under the same condition as for glucoamylolysis of 4III. Pre parative paper chromatography gave subfractions 5ÏIIGÂ1, 5IIIGA.2, and
5IIIGA3 with the same mobilities as 4IIIGA1, 4IIIGA2, and 4IIIGA3, respectively (Figure 27). After purification by gel filtration chroma tography on column A (Table 6), 5ÎIIGA2 and 5IIIGA3 were hydrolyzed for gas chromatographic analysis (Figures 28 and 29). Figure 23b. Gas chromatogram of TMS-monosaccharides and TMS-alditols obtained from partial hydrolysis of reduced 4IIIGA3 and trimethylsilylation.
a,c. D-glucopyranose
b. sorbitol
d,g. 3-0-(2-hydroxyethyl)-D-glucopyranose
e,h. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,i. 6-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE
S
ui
•3 H §
§ ca U1
NJ Figure 24. Paper chroiaatogram of 4IIIGA3 after further treatment of porcine pancreatic a-aoylase.
^. Standard mixture of glucose to maltoheptaose
1. maltotriose
2. maltoticiose after a-amylolysis (control)
3. 4IIIGA3
4. 4IIIGA3 after a-amylolysis
Chromatographic conditions: Solvent system B, three ascents at 65°C 86 Figure 25. Gas chromatogram of 5III after acid hydrolysis and trimethylsilylation
a. B-D-glucofuranose
b.d. D-glucopyranose
c. 1,2-0-ethylene-a-D-glucopyranose
e.g. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,h. 6-0-(2-hydroxyethyl)-D-glucopyranose 88
b d
5III
ft
W
5 10 15
TIME (MINUTES) Figure 26. Gas chromatogram of 5III which had been reduced with sodium borchydride followed by acid hydrolysis and then trimethylsilylated.
a,d. D-glucopyranose
b. 1,2-0-ethylene-a-D-glucopyranose
c- sorbitol
e,g. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,h. 6-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE
oi
M O
:> 0 c. m U1 (O H H H ? » Ni o ( / Figure 27. Paper chromato^;ram of subfractions 5III and 5IV after treatment of glucoamylase.
{glucose to maltoheptaose "1-7" 5IIIGA. filll after glucoamylase treatment; 5IIIGA1, 5IIIGA2, and 5IIIGA:l are numbered In sequence from the top
5IIIGA(D) Deionized glucoamylase digest of 5III
5IVGA. .'jIV after glucoamylase treatment; 5IVGA1, 5IVGA2, iind 5IVGA3 are numbered in sequence from the top
5IVGA(D). Deiolized glucoamylase digest of 5IV
Chromatographic conditions: Solvent system B, three ascents at 65°C 5, ^ SHIGA !>i:CI SHIGA 51 VGA 5IV 51 VGA G,.- (D) (D) Figure 28. Gas chromatogram of 5IIIGA2 after acid hydrolysis and trimethylsilylation.
a. 1,2-Q-sthylene-3-D-glucopyranose
b.d. D-glucopyranose
c. 1,2-0-ethylene-o-D-glucopyranose
e,f. 2-0-(2-hydroxyethyl)-D-glucopyranose 94
' 5IIIGA2
@ \ il II I
S S Ui II ^ î
10 15 20
TIME (MINUTES) Figure 29, Gas chromatogram of 5IIIGA3 after acid hydrolysis and trimethylsilylation.
a;b. D-glucopyranose
c,d. 6-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE
VO o\ 97
Table 6. Carbohydrate subfractions isolated from glucoamylolysis of 5III. Chromatographic conditions are the same as in Table 3
*G M.W.(exp.)
5IIIGA1 1.00 1.74 208.2
5I1IGA2 0.82 1.53 408.2
5IIIGA3 0,52 1.47 494.7
3, Relative total carbohydrate of 5XIIGA1, 5IIIGA2, and 5IIIGA3 in glucoamylase digest of 5III
Glucoamylase digest of 5III prepared as previously mentioned was applied with 0.2% blue dextran on Sephadex G-15 column (column B). The elution profile is shown in Figure 30.
4. Porcine pancreatic ot-amylolysis of 5III
The assay was performed the same as for the ci-amylolysis of AIIIGAS, except maltotetraose was used as control. 5III is resistant to further
amylolysls when G^ is completely converted to glucose and maltose,
L. Structural Analysis of 5IV
1. Acid hydrolysis of 5IV and reduced 51V
After acid hydrolysis they were analyzed by gas chromatography
(Figures 31 and 32). Chromatography of glucoamylase digest of 5III on Sephadex G-15 column. Chromatographic conditions; Column B (1,6 x 100 cm): reverse flow method at a flow rate of 12 ml/hour; room temperature. The amounts of carbohydrate in the subfractions 5IIIGA1, 5IIIGA2, and 5IIIGÂ3 were estimated by integration between c - d, b - c, and a - b, respectively. The base line with negative total carbohydrate values is due to the base line shift of the recorder 99
5IIIGA 250
210 5IIIGA2
^70
J.30 m 5IIIGA1 5IIIGA3
90
50
Blue dextran
240 300 360 420 480
TIME (MINUTES) Figure 31. Gas chromatogram of 5IV after acid hydrolysis and trimethylsilylatlon
a,c. D-glucopyranose
b. 1,2-0-ethylene-a-D-glucopyranose
d,g. 3-0-(2-hydroxyethyl)-D-glucopyranose
e,h. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,i. 6-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE
L Figure 32. Gas chromatogram of 5IV which had been reduced with sodium borohydride followed by acid hydrolysis and then trimethylsilylatlon
a y d • D-glucopyranose
b. 1,2-0-ethylene-a-D-glucopyranose
c. sorbitol
e,h. 3-0-(2-hydroxyethyl)-D-glucopyranose
f,i. 2-0-(2-hydroxyethyl)-D-glucopyranose
g,j. 6-0-(2-hydroxyethyl)-D-glucopyranose 103
a c d
! 5IV(R)
\ 11 ' i e
T h U A oA \ i
10 15
TIME (MINUTES) 104
2. Glucoamvlase action on 5IV
Twelve milligrams of 5IV was treated with glucoanylase (R. niveus) as previously mentioned. Subfraction 5IVGA1, 5IVGA2, and 5IVGA3 were isolated by preparative paper chromatography. Their mobilities on the paper chromatogram are similar to those for 5IIIGA1, 5IIIGA2, and
5IIIGA3, respectively (Figure 27). The relative elution volumes of these subfractions in Sephadex column chromatography (column A) are shown
In Table 7. The gas chromatograms of 5IVGA2 and 5IVGA3 after acid hydrolysis are also given in Figures 33 and 34.
Table 7. Carbohydrate subfractions isolated from glucoaaiylolysis of 5IV. Chromatographic conditions are the same as in Table 3
Rg Vg/V^ M.W. (exp.)
5IVGA1 1.00 1.75 201.6
5IVGA2 0.81 1.51 435.1
5IVGA3 0.52 1.42 580.7
3a Relative total carbohydrate in the alucoamylase digest of 5IV
After inactivation or the enzyme, an aliquot of the above digest con taining 0.2% blue dextran was applied on Sephadex G-15 column (column B).
The elution profile is shown in Figure 35. Figure 33. Gas chromatogram of 5IVGA2 after acid hydrolysis and trimethylsilylation
a. 1,2-0-ethylene-3-D-glucopyranose
b.d. D-glucopyranose
c. 1,2-0-ethylene-o-D-glucopyranose
e,f. 2-0-(2-hydroKyethyi)-D-glucopyranose DETECTOR RESPONSE
o o*
to Figure 34. Gas chromatogram of 5IVGA3 after acid hydrolysis and trimethylsilylation
a 5 b. D-glucopyranose
c,e. 3-0-(2-hydroxyethyl)-D-glucopyranose
d,f. 6-0-(2-hydroxyethyl)-D-glucopyranose 5IV6A3
f Figure 35. Chromatography of glucoamylase digest of 5IV on Sephadex G-15 column. Chromatographic conditions: Column B (1.6 x 100 cm); reverse flow method at a flow rate of 12 ml/hour; at room temperature. The amounts of carbohydrate in the subfractions 5IVGA1, 5IVGA2, and 5IVGA3 were estimated by integration between c - d, b - c, and a - b, respectively. The base line with negative total carbohydrate values is due to the base line shift of the recorder 110
5IVGA1
130 51VGA
110 5IVGA2 00
90
5IVGA3 70
Blue dextran 50
30
k J
240 300 360 420 480 TIME (MINUTES) Ill
M. Further Porcine Pancreatic a-amylolysis of 5IV
1. Porcine pancreatic a-amylolvsla of 5IV
Two milliliters of 51V (39.4 mg) was treated with 2 ml of the enzyme solution containing 1000 units of porcine pancreatic a-amylase In 20 mM sodium glycerol phosphate buffer and 10 mM NaCl at pH 6.9. One drop of
toluene was added Into the reaction mixture to prevent mold growth. The reaction mixture was maintained at 39*C for 4 days. Forty milligrams of maltotetraose was dissolved In 2 ml of water and the same amount of the enzyme solution was added under the same experimental condition as the control. Paper chromatographic analysis of the digest and the control is shown In Figure 36. Preparative paper chromatography gave four sub- fractions, 5IVA1, 5IVA2, 5IVA3, and 5IVA4 in the order of decreasing mobility (Figure 36), where A stands for a-amylase action. After purifica
tion by passing through Sephadex G-15 column (column A), 5IVA1 was resolved into two more subfractions, 5IVA1S1 and 5IVA1S2. 5IVA2, 5IVA3, and 5IVA4 gave single peaks on Sephades G-15 chrosstcgrass (Table 8).
Table 8. Carbohydrate subfractions Isolated from further o-amylolysis of 5IV. Chroaiatogsaphie conditions are the same as in Table 3
R- V_/V_ M.W.(exp.) O C KJ
SIvAlSl 1.00 1.75 195.3 5IVA1S2 0.98 1.55 382.8 5IVA2 0.73 1.62 305.5 5ÎVA3 0.60 1.42 580.8 5IVA4 0.36 1.33 774.9 Figure 26. Paper chronato{»ram of porcine pancreatic «-amylase digest of 5IV.
y. glucose to maltoheptaose
G^A. maltotetraose after a-amylolysis (control)
5IVA. 5IV after a-amylolysis; 5IVA1, 5IVA2, 5IVA3, and 5IVA4 are numbered in sequence from the top L-7 =4* °l-7 114
2. Acid hydrolysis of 5IVA1S2. 5IVA2. 51VA3. and 5IVA4 which were reduced with sodium borohydride
Procedures were the same as previously mentioned, the results are
shown in Figures 37, 38, 39 and 40.
3. Glucoamylolysis of 5IVA3 and 5IVA4
Six milligrams each of the saccharides were dissolved into 270 vl of
water and then diluted with the same volume of 0.1 M sodium acetate buffer
(pH 4.5). One hundred seventy microliters of the enzyme stock solution
(7 units, R. nlveua) in 0.05 M sodium acetate buffer (pH 4.5) was added.
The assay was maintained at 39'C for 2 days and maltoteteaose was used as
control. The results are shown in Figure 41. The resultant glucoamylolytic
digest of 5IVA3 was further separated by preparative paper chromatography
to 5IVA36A1, 5IVÂ3GA2, and 5IVA3GA3, in the order of decreasing mobilities.
All these subfractions gave single peaks in gel filtration column chroma
tography. Their mobilities on the paper chromatogram and their relative
élutioTi volîsss in Saphadex G-15 colv^ chromatography are very similar to
5IV6Â1, 5ÎVGÂ2, and 5IVGA3, respectively. The resultant glucoanxiolytic
digest of 5IVA4 was separated by using the same chromatographic techniques
to give 5IVA4GA1, 5IVA4GA2, and 5IVA4GA3 which have similar chromatographic
properties as 5IVA3GA1, 5IVA3GA2, and 5IVA3GA3; In addition, thêifè was a
negligible ssjount of 5XVA4GA4 which was resistant to glucoanylase action
(Figure 41). 5IVA3GÀ2, 5IVÀ3GA3, 51VA4GA2, and 5IVA4GA3 were reduced with
sodium borohydride and then acid hydrolyzed. The results are shown in
Figures 42-45. Figure 37. Gas chromatogram of 5IVA1S2 which had been reduced with sodium borohydride followed by acid hydrolysis and then trimethylsilylated
a. sorbitol
b;C: 3-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE
' 0)
ON Figure 38. Gas chromatogram of reduced 5IVA2 after acid hydrolysis and trimethylsilylation
a,c. D-glucopyranose
b. sorbitol DETECTOR RESPONSE Figure 39. Gas chromatogram of reduced 5IVA3 after acid hydrolysis and trimethylsilylation
a. 1,2-0-ethylene-3-D-glucopyranose
b;e, D-glucopyranose
c. 1,2-0-ethylena-a-D-glucopyrano8e
d. sorbitol
f, 3-0-(2-hydroxyethyl)-D-glucopyranose
g,i. 2-0-(2-hydroxyethy1)-D-glucopyranose
h,j. 6-0-(2-hyuroxy6thyl)=B-glucopyr3no8e
Figure 40. Gas chromatogram o£ reduced 5IVA4 after acid hydrolysis and trimethylsHylation
a.d. D-glucopyranose
b. 1,2-0-ethylene-a-D-glucopyranose
c. sorbitol
e,g. 3-0-(2-hydroxyethyl)-D-glucopyranose
f,h. 2-0-(2-hydroxyethyl)-D-glucopyranose 122
a c
d
5IVA4(R)
n f\ '^\j\ VJ
10 15
TIME (MINUTES) Figure 41. Paper chromatogram showing the action of glucoamylase on 5IVA3. and 5m.4.
glucose: to maltoheptaose
1. glucoauylase digest of maltotetraose (control)
2. glucoajiiylase digest of 5IVA3; 5I.VA.3GA1, 5IVA3GA2, and 5I'\'A3GA3 were numbered in sequence from the top
3. glucoasiylase digest of 5IVA4; 51VA4GA1, 5IVA4GA2, and 5IVA4GA3 were nunibered in sequence from the top 124 Figure 42. Gas chromâtogram of reduced 5IVA3GA2 after acid hydrolysis and trimethylsilylation
a. 1,2-0-ethylene-a-D-glucofuranose
b. 1,2-0-ethylene-e-D-glucopyranose
c. 1.2-0-ethylene-a-D-glucopyranose
d. sorbitol
e.f. 2-0-(2-hydroxyethyl)-D-glucopyranose 126
5IVA3GA2(R)
.5 10 15 20
TIME (MINUTES) r
Figure 43. Gas chromatogram of reduced 5IVA3GA3 after acid hydrolysis and trimethylsilylation
a,c. D-glucopyranose
b. sorbitol
d,g. 3-0-(2-hydroxyethyl)-D-glucopyranose
e,h. 2-0-(2-hydroxyethyl)-D-glucopyranose
f,i. 6-0-(2-hydroxyethyl)-D-glucopyranose DETECTOR RESPONSE '••J
Figure 44. Gas chromatogram of reduced 5IVA4GA2 after acid hydrolysis and triaathylsllylation
a. 1,2-0-ethylene-a-D-glucofuranose
b. 1,2-0-ethyiefte-g-D-glucopyranose
c. 1,2-0-ethylene-a-D-glucopyranose
d. sorbitol
e.f, 2-0-(2-hydroxyethyl)-D-glucopyranose H Figure 45. Gas chromatogram of reduced 5IVA4GA3 after acid hydrolysis and trimethylsilylation
a,c. D-glucopyranose
b. sorbitol
d,c. 3-0-(2-hydroxyeuhy1)-D-glucopyranose DETECTOR RESPONSE
on H
•u$ NJ O w PO 133
N. Attempted g-anylolyals of 5IV
One hundred fifty micrograms of 5IV was treated with 11.7 units of g-amylase In 0.1 ml of 0.05 M sodium acetate buffer (pH 4.5) at 30°C for
17 hours with maltotetraose as control. Paper chromatography showed no evidence of cleavage of 5IV by this enzyme. 134
V. DISCUSSION AND CONCLUSION
Â. Discussion of Results
0-(2-hydroxyethyl)amylose was acid hydrolyzed to its individual monomer components. Paper chromatographic and Rg values of 0-(2- hydroxyethyl)-D-glucoses are shown in Table 1. Inner glucosides formed by intramolecular glucosidation of the anomeric 2-0-(2-hydroxyethyl)-D- glucoses cannot be visualized by the alkaline-AgNO^ dip on paper chromato- grams. 2-0-(2-hydroxyethyl)-D-glucose has the same mobility as its 3-0- isomer no matter whether solvent system A or B is used for irrigation. All
0-(2-hydroxyethyl)-D-glucoses have higher mobilities than glucose, there fore, monohydroxyethylated maltose, maltotriose and higher oligosaccharides should have higher R^ or Rg values than the corresponding unsubstituted maltooligosaccharides. When the logarithm of a partition function, a', is plotted against molecular size, glucose, maltose, and homologous un substituted maltooligosaccharides lie on a straight line, when either solvent system A or iJ is utilized for irrigation (Figures 46 arid 47). a' is defined as R^/(1 - R^) and R^ is the true or single ascent value calculated from the apparent R^ value (127). The R^ values given in Table 1 are apparent R^ values for three ascents. As the apparent R^^ value of 2-0- or 3-0-(2-hydroxyethyl)-D-glucose is known, its a' can be calculated. Thus, a straight line parallel to the line consultuteu by glucose, inaltcse, and higher maltooligosaccharide can be drawn to estimate the mobilities of 2-0- and 3-0- monohydroxyethylated maltose, and maltooligosaccharides (127,72).
The mobilities of 6-0-(2-hydroxyethyl)maltooligosaccharides can also be Figure 46. Estimation of monohydroxyethyl sugar mobilities on paper chromatogram irrigated by solvent system A. A plot of log Rf/(1 - Rf) against the number of monomer units in the sugar molecule.
x: designates maltooligosaccharides including glucose and maltose. The single ascent Rg values of to were calculated from the apparent Rr values in Table 1
«-vn; standard curve for glucose and unsubstituted maltooligosaccharides
A: 2-0- and 3-0-(2-hydroxyethy1)-D-glucose with known Rj value
Aî 2-0- and 3-0- monohydroxyethylated maltooligo saccharides. Their mobilities are estimated by the locations on the straight line which is extended from A and parallel to • <
•: 6-0-(2-hydroxyethyl)-D-glucose with known Rg value
o; 6-0- monohydEOjcyethylatsd saltcoligosaccharides. Their mobilities are estimated by the locations on the straight line extended from a and parallel to
hypothetical straight lines drawn to parallel 136
2-0- or 3-O-HEG,
s 6 -O'HEGj
"s 1
«XxX
_j e 1 2 3 4 NUMBER OF MONOMER UNITS Figure 47. EstiaaElon of laoaohydroxyethyl sugar mobilities on paper chromatogram irrigated by solvent system B. A plot of log Rg/(1 - R,) against the number of monomer units in the sugar molecule.
The Interpretation is the same as in Figure 46 138
^ 2-0- or 3-O-HEG, |90 6-O-HEG,
160 n\ \
•30
r
2
KISŒER OF MOBBMER IMS 139
estimated by the same approach.
From the results in Figures 46 and 47, it is considered that the mobility of a monosubstituted maltooligosaccharide with n monomer units lies between the mobilities of two unsubstituted oligosaccharides with
(n - 1) and n monomer units. For examples, a monosubstituted trisaccharide should have a mobility between and G^, and a monosubstituted tetra- saccharide should have a mobility between G^ and G^.
Figure 5 shows the gas chromatogram of 0-(2-hydroxyethyl)amy lose after complete acid hydrolysis. All important peaks have been identified. The relative amounts of the hydroxyethylated glucoses are in the order of
2—0— > 6—0— > 3—0—.
Preliminary investigation of porcine pancreatic a-amylase digestion of
0-(2-hydroxyethyl)amylose by paper chromatography shows two interesting spots on the paper chroiaatogrsa (Figure 6)» One of these has a mobility between and G^, and another has a mobility between G^ and G^. Possibly, they are monohydroxyethylated trisaccharides and tetrasaccharides, re spectively. There is no evidence of hydroxyethylated D-glucoses being produced under the present experimental conditions; at least no 0-(2- hydroxyethyl)-D-glucose has been detected on the paper chroatatogram.
In order to test whether aubslte III of the enzyme binding site permits substitution of. the hydroxyl groups at the binding glucose unit, the original enzymatic digest was reduced with sodium borohydride and then hydrolyzed by acid to the individual monomers for g.l.c. analysis. Figure 7 shows that the only reduced monosaccharide is sorbitol. Therefore, in the catalytic reaction, substituents are not permitted to replace the hydroxyl 140
groups of the anhydroglucose unit which binds on subsite III. The gas chromatogram in Figure 7 also confirms that no monohydroxyethyl glucose is produced in the original enzymatic digest. If there were any, it also would be detected as a substituted alditol on the gas chromatogram.
The original pancreatic amylase digest of 0-(2-hydroxyethyl)-amylose contains large quantities of glucose and maltose in addition to the a-limit dextrin (Figure 6). To obtain enough a-limit dextrins for the structural analysis and a detailed investigation on the components in the digest, char coal column chromatography was used to fractionate the original reaction mixture. Seven fractions were obtained, and the results are shown in
Table 2 and Figure 8. In Figure 8, those fractions from charcoal chroma tography are further separated into subfractions by paper chromatography with two different solvent systems, and both chromatograms have almost the same kind of separation pattern. Apart from glucose and maltose, 4III,
5III, and 5IV are the most intense spots on both of the chromatograms. 4III and 5III have mobilities between and G^, very similar to the tri- saccharide spot in Figure 6. Possibly they are monohydroxyethylated tri- saccharide or trisaccharide mixtures. 5IV has a nobility between G^ and G^, very similar to the tetrasaccharide spot in Figure 6. Possibly it is a monosubstituted tetrasaccharide or tetrasaccharide mixture,
Glucoamyiase is an exoeasyme which clcaves terminal a-l:4-glucosidic linkages to liberate glucose from the nonreducing ends of amylose or amylopectin. It also cleaves a-1,6- linkages, but at a slower rate. Gluco amyiase treatment of 0-2-(2-hydro:qrethyi)starch does not produce any hydroxyethyl glucose (Figure 9); therefore, glucoamyiase action cannot cross 141
over hydroxyethylated glucose units and the reaction is stopped at the point of substitution. It was reported that when 6^-0-methylinaltose was used as the substrate for glucoamylase action, glucose and 6-0-methyl- 2 glucose were produced (42), but 6 -O-methylmaltose was completely resistant 2 to glucoamylase action (72). Saier and Ballou showed that 3 -O-methyl- maltose was resistant to glucoamylase action (72). In our present in vestigation, 3^-0-methylinaltose was also found to be resistant to this enzyme (Figure 10). Presumably, glucoamylase would not attack an a-1,4- glucosidic linkage which connects directly to a 3-0-(2-hydroxyethyl)-D- glueose unit. For 2-0- substitution, it will be shown in a later part of 2 this thesis that when 2 -0-(2-hydroxyethyl)maltotriose is subjected to 2 glucoamylase action, glucose and 2 -0-(2-hydroxyethyl)maltose are produced.
2 2-0-(2-hydroxyethyl)-maltose is resistant to further glucoamylase treat
ment. The substrate specificities are summarized in Scheme 1.
GA <- 0 ME T Me Km
no reaction MeP0 00 Me Ms 00 GA
Scheme 1. Substrate Specificities of Glucoamylase 142
Figure 11 is the paper chromatogram which shows glucoamylase action on the fractions obtained by charcoal chromatography. After gluco- amylolysis, fractions 1, 2, and 3 give single glucose spots. It is con cluded that these fractions do not contain hydroxy3thylated oligosaccharide.
Fraction 4 and fraction 5 give three spots after glucoamylase treatment.
Beside the glucose spot, a second and a third spot with mobilities cor responding to monosubstituted di- and tri- saccharides are observed. For fractions 6 and 7, there are too many subfractions before and after gluco amylase treatment. Some of these subfractions might contain di- and tri- suuatituted oligosaccharides for which It would be very difficult to de
termine structures. Therefore in the present investigation, efforts are
concentrated on the isolation and identification of the subfractions con
taining 0-(2-hydroxyethyl) sugars in fractions 4 and 5. Presumably, they are monohydroxyethylated sugars.
Subfractions 41, 411, 4III, 4IV, 51, 511, 5III, and 5IV obtained by
preparative paper chromatography were subjected to Sephadex G-15 column
chromatography. All subfractions gave single peaks on Sephadex G-15
chromatograma except 41 and 51 which were further resolved into two compo
nents as 4IS1, 41S2 and 5IS1, 5IS2 (Figures 12 and 13). Among these sub-
fractions which were Isolated by the combinatioA of paper and Sephadex gal
chromatographies, 4IS2, 4III, 515%, 5111, and 517 are aubstiEuted sugars;
their chromatographic properties and their estimated molecular weights are
listed in Table 3. Figure 48 shows a plot of V^/V^ against log M.W. The
standards of to Gg give a straight line, and the molecular weight of
the substituted sugars are estimated by their relative elutlon volumes. Figure 48.. Calibration of Sephadex G-15 column (column A) and estimation of the molecular weights of 0-2-hydroxyethylated sugars.
V : elution volume of the substance e V ; void volume! of the column o V /V : relative eJlution volume e o As to Gg standards
0: 0-2-hydroxyethylated sugars
The molecular weights of 0-2-hydroxyethylated sugars are estimated by their relative elution volumes 1.7 X N
1.6 4IS2 5IS2
1.5 >• 5III 4III "1.4
5IV
1.3
2 4 5 6 7 8 145
On thin layer chromatography 4IS2 also shows as a single spot with the mobility of maltose (Table 4). Acid hydrolysis of 4IS2 gives glucose and 3-0-(2-hydroxyethyl)-D-glucose (Figure 14). After sodium borohydride reduction, acid hydrolysis of reduced 4IS2 gives sorbitol and 3-0-(2- 2 hydroxethyl)-D-glucose (Figure 15). This confirms that 4IS2 is 3 -0-(2- hydroxyethyl)-D-glucose, The same analytical procedures were applied on
51S2, and indicated that 51S2 is the same compound as 4IS2.
The estimated molecular weight of 4III in Table 3 shows that it is a trisaccharide. According to its paper chromatographic mobility» it is also a Moaosubstitutsd sugar. Thin layer chromatography of 4III gave a single spot with value between that of and (Table 4). However, acid hydrolysis of 4III gave glucose, 2-0-(2-hydroxyethyl)-D-glucose, and a small quantity of 3-0- and 6-0-(2-hydroxyethyl)-D-glucose (Figure 16).
This Indicates that 4III is a mixture of monohydroxyethylated trisaccharides with 2-0-hydroxyethylated trisaccharides as the major component. Because no substituted aldltol has been found in the acid hydrolysate of reduced
4III, all substitutions should be either at the middle glucose or at the nonreducing end glucose units of the trisaccharides (Figure 17).
Glucoamylase treatment of 4ÎII gives 4IIÏGA1, 4IÏIGA2 and 4IIIGA3 by paper chromatography (Figure 18)= Their chromatographic properties and estimated molecular weights are listed in Table 5.
4IIIGA1 is identified as glucose. 41ÎÎGÀ2 could be a monosubstituted disaccharide with an 0-(2-hydroxyethyl) group at the nonreducing end. Acid hydrolysis of 4IIIGA2 yields glucose and 2-0-(2-hydroxyethyl)-D-glucose, so 2 that it is confirmed to be 2 -0-(2-hydroxyethyl)maltose (19). The precursor 146
2 of this disaccharide is 2 -0-(2-hydroxyethyl)maltotriose which is cleaved by glucoamylase to give glucose and the disaccharide.
4IIIGA3 is resistant to glucoamylase and has the same chromatographic properties as the original 4III. Gas chromatographic analysis of the acid hydrolysate of 4IIIGÂ3 gave glucose, 6-0-, 3-0- and small quantities of
2-0-(2-hydroxyethyl)-D-glucose (Figure 20). The amount of 6~0-(2-hydroxy- ethyl)-D-glucose was slightly larger than its 3-0- isomers. A 6-0- hydroxyethylated trisaccharide which is resistant to glucoamylase action should have the substitution at the nonreducing end (Scheme 1). The iso-
lation of this 6"-0-(2-hydroayechyl)aaltotriose frcm a pancreatic amylase 3 digest of 0-(2-hydroxyethyl)amylose is not unexpected, as 6 -a-glucosyl- maltotriose was previously isolated from both pancreatic amylase digests of amylopectin and single "stub" oligosaccharides (7,128). The only structural 3 3 difference between 6 -0-(2-hydroxyethyl)-maltotriose and 6 -a-glucosyl- maltotriose is the replacement of a 6-a-glucosyl group by a 6-0-(2- hydrcxysthyl) siibstituent; The location of the 3-0- substitution in a 3-0- hydroKyechylated trisaccharide cannot be determined by its resistance to glucoamylase, except in the case the substitution is at the reducing end. •J The 2-0- substituted trisaccharide in 4IIIGA3 could be 2"'-0-(2-hydroxyethyl)-
-2 maltotriose, which is resistant to glucoamylase action, or 2 -0-(2- 2 hydroxyethyl)inaltQtrlose; due to the incomplete glucoamylolysis of this 2 -0-
trisaccharide. Partial acid hydrolysis of the reduced 4IIIGA3 followed by
pooling the mono- and disaccharide fractions from a Sephadex G-15 column
gave fractions which were subjected to gas chromatographic analysis (Figures
22, 23a and 23b). The following results were obtained: 147
2 1. The existence of maltitol and 3 -0-(2-hydroxyethyl)maltitol in 3 2 the disaccharide fraction shows the presence of 6 -0- and 3 -0-(2-
hydroxyethyl)maltotriose in 4IIIGA3 (Figure 23a).
2. In the monosaccharide fraction, besides sorbitol and glucose,
all 0-(2-hydroxyethyl)-D-glucoses are present (Figure 23b). As the partial
acid hydrolysate contains a large amount of unhydrolyzed trisaccharide
alcohols (Figure 22), these monosaccharides must have come from the non-
reducing ends of the trisaccharide molecules. The relative amounts of 0-
(2-hydroxyethyl)-D-glucoses are 6-0- > 2-0- > 3-0-, but in the acid
hydrolysate which is obtained from complete hydrolysis of 4III6À3, the order 3 3 turns out to be 6-0- > 2-0- > 3-=0" . This suggests that 6 -0-, 2 -0-, 2 3 3-0- and a small amount of 3 -0-(2-hydroxyethyl)maltotrio8es are present
in 4IIIGA3. 3 It is not surprising to find 3 -Q-(2-hydroxyethyl)-maltotriose in the
pancreatic amylase digest of 0-(2-hydroxyethyl)amylose. In the studies of 3 the polysaccharide from Mycobacterium phlei, 3 -0-methylmaltotriose, which
is analogous to 3 -0-(2"hydro%yethyl)maltotriose, was isolated (72). Fur
ther treatment of this 3-0-methylglucose-containing trisaccharide with por- 2 cine pancreatic a^amylase gave 3 -O-methylmaltose and glucose.
Further pancreatic a-amylosis of 4IIIGA3 gives three spots, one of 2 which has the «arae paper chromatographic mobility as 2 -0-(2-hydroxyethyl)-
maltose (Figure 24). This disaccharide could be produced by cleavage of the 3 glucosidic bond at the reducing end of 2 -0=(2=hydroxyethyl)!naltotriose.
The relative amounts of 4IIIGA1, 4III6A2, and 4IIIGA3 in the gluco-
amylase digest of 4III were estimated by integration of the areas between 148
c-d, b-c, and a-b of the elation profile of the enzymatic digest from
Sephadex G-15 column as shown in Figure 21. The mole percentages of
41IIGA1, 4IIIGA2, and 4IIIGA3 are calculated as 47.8, 48.5 and 3.7, respectively, so the ratio of 4IIIGA1 and 4IIIGA2 is very close to 1. 2 It is concluded that there is 93% of 2 -0-(2-hydroxyethyl)maltotriose, and only 7% of other trisaccharides in the subfraction 4III. This 7% of 3 2 3 trisaccharides consists of 6 -0-, 3 -0- and very small amounts of 2 -0- O and 3 -•0-(2-hydroxyethyl)maltotriose. The glucoamylase reaction of 4III is shown in Scheme 2.
OC^ GA. 0+00 HE
w 1
(y0
GA no reaction pO0 HE
QO0 J HI
Scheme 2. Glucoamylase Action on 4III
The structural analysis of 5III is somewhat essier. Based on the data in Table 3, Figures 25 and 26, this subfraction is a mixture of two mono- hydroxyethylated trisaccharides with either 2-0- or 6-0-(2-hydroxyethyl) groups attached to the middle or nonreduclng end glucose units. Glucoamylase 149
2 action on this subfraction gives glucose (5IIIGA1), 2 -0-(2-hydroxyethyl)- 3 maltose (5IIIGA2) and a trisaccharide, 6 -0-(2-hydroxyethyl)maltotriose which is resistant to glucoamylase action (Table 6, Figures 27-29). The relative mole percentages of 5IIIGA1, 5IIIGA2, and 5IIIGA3 are 42.2, 45.4, and 12.2, respectively (Figure 30), so the ratio of 5IIIGA1 to 5IIIGA2 is very close to 1. Subfraction 5III is resistant to further pancreatic 2 amylolysis. It is concluded that 5III contains 79% of 2 -0-(2-hydroxyethyl)- 3 maltotriose and 21% of 6 -0-(2-hydroxyethyl)maltotriose.
oq0 0 00 HE HE
HE HE 000 bo0
Scheme 3. Glucoamylase Action on 5III
According to the data in Table 3, 5Iv corresponds to monosubstltuted tetrasaccharide. Acid hydrolysis of 5IV gives glucose and all the 0-(2- hydroxyethyl)-B-giucoseSo Among the substituted glucoses, 2-0- and 3-0-
(2-hydroxyethyl)glucose are predominant (Figure 31). After acid hydrolysis of reduced 5IV, sorbitol is the only alditol in the hydrolysate (Figure 32).
In this case, substitution can be at any glucose unit in the trisaccharide
molecules except the reducing ends. Glucoamylase action on 5IV gives
5IVGA1, 5IVGA2, and 5IVGA3 with same mobilities as 5IIIGA1, 5IIIGA2 and
5IIIGA3, respectively (Figure 27). Table 7 gives their chromatographic 150
properties and their estimated molecular weights. 2 5IVGA1 is glucose. 5IVGA2 is identified as 2 -0-(2-hydroxyethyl)- maltose (Figure 33). Based on the specificity of glucoamylase, the pre- 2 cursor of this disaccharide should be 2 -0-(2-hydroxyethyl)maltotetraose.
Acid hydrolysis of 5IVGA3 gives glucose, 3-0- and a small amount of 6-0-
(2-hydroxyethyl)glucose (Figure 34), so that it is a mixture of 3-0- and
6-0- hydroxyethylated trisaccharides. As these two trisaccharides are 2 resistant to glucoamylase action, they should have the structures 3 -0- q and 6 -0-(2-hydroxyethyl)maltotriose. tforeover, the precursors of these two 2 3 trisaccharides must be 3 -0- and 6 -0-(2-hydroxyethyl)maltotetraoses.
The structural determination of the 3-0- substituted tri- and tetra- 2 saccharides is based on the argument that 3 -0-(2-hydroxyethyl)-maltotetraose is the only possible structure which is susceptible to glucoamylase to give glucose and a trisaccharide, because 3^-0= and 3^-0-(2-hydroxyethyl)malto- tetrase are resistant to glucoamylolysis (see Scheme 1, specificity of q glucoamylase). The isolation of 6 -0-(2-hydroxyethyl)maltotetraose is also 3 expected, because the structurally-related 6 -a-glucosylmaltotetraose has previously been detected in the pancreatic amylase digest of amylopectin and stubbed oligosaccharides (7,128).
The relative zoic percentage of 5IVGA1, 5IVGA2, and 5IVGA3 are 61.6,
27.7 and 10.7, respeetivaly (Figure 35)- These results are consistent with the calculated values in the glucoamylolysis of Eetrasaccharides; as two molecules of glucose will be produced in the formation of each disaccharide and only one molecule o£ glucose will be generated for each trisaccharide.
It is concluded that subfraction 5IV contains 72% of 151
J 2 3 2 -0-(2-hydroxyethyl)maltotetraose and 38% of 3 -0- and 6 -0-(2-hydroxy- 2 ethyl)-maltotetraoses in which 3 -0- tetrasaccharide is the major product.
ooc^
HE
Scheme 4. Glucoamylase Action on 5IV
Subfraction 5IV was further treated with porcine pancreatic a-amylase.
Paper chromatography of the enzymatic digest gave 5IVA1, 5IVA2, 5IVA3 and
5IVA4 (Figure 36), and the pattern of separation was very close to that of fraction 5 from charcoal chromatography (Figure 8). As expected, sub- fraction 5IVAi was resolved iuûù LwO coaponents, 5IVA1S1 and 5IVA1S2 by
Sephadex G-15 chromatography. The remaining subfractions 5IVA2, 5IVA3 and 5IVA4 gave single peaks on Sephadex G-15 column chromatograms. All
their estimated molecular weights and chromatographic properties are listed in Table 8.
SIVAISI is glucose. 5ÏVA1S2 could be the same compound as 4IS2 and
5IS2. Acid hydrolysis of reduced 5IVAIS2 gives sorbitol and 3-0-(2-hydroxy- 2 ethyl)-D-glucose. This confirms that 5IVA1S2 is 3 -0-(2-hydroxyethyl)-
maltose (Figure 37), 5IVA2 has the same chromatographic properties as G^. 152
Acid hydrolysis of reduced 5IVA2 gave sorbitol and glucose in 1:1 ratio
(Figure 38): this confirms that 5IVA2 is maltose.
5IVA3 could be a mixture of mono-substituted trisaccharides. Acid hydrolysis of reduced 5IVA3 gives sorbitol, glucose, 2-0-, and small quantities of 6-0- and 3-0-(2-hydroxyethyl)-D-glucose (Figure 39). Gluco- amylase digestion of 5IVA3 gave 5IVA3GA1, 5IVA3GA2 and 5IVA3GA3 (Figure 41) with the same chromatographic properties as 5IIIGA1, 5IIIGA2 and 5IIIGA3.
After reduction and acid hydrolysis, 5IVA3GA2 gave sorbitol and 2-0-(2- 2 hydroxyethy1)-D-glucose (Figure 42); therefore 5IVA3GA2 is 2 -0-(2- hydroxyethyl)maltose. Acid hydrolysis of reduced 5IVA3GA3 gave sorbitol, glucose, 6-0-, 3-0- and a small quantity of 2-0-(2-hydroxyethyl)-D-glucoses 3 (Figure 43). This indicates that 5IVA3GÂ3 contains 6 -0-(2-hydroxyethyl)- 2 maltotriose as the major component, some 3 -0-(2-hydroxyethyl)maltotriose 2 and small quantities of 2 =0=(2=hydrcxyethyl)!!ialtotriose due to the in complete glucoamylolysis of this compound. All these pieces of evidence 2 reflect that the original 5IVA3 contains a large quantity of 2 -0-(2- 2 2 hydroxyethyl)iaaltotriose, and a small amount of 6 -0- and 3 -0- isomers.
5IVA4 has the same chromatographic properties as 5IV. Acid hydrolysis of reduced 5ÎVÂ4 gave sorbitol, glucose, 2-0= and 3-0-(2-hydroxyethyl)-B- glucoses (Figure 40), so that there are two monohydroxyethylated tetra- saccharides in, this subfraction and they must be 2 2-0- and 3 2-0-(2-hydroxy- ethyl)maltotetrao8es.
Glucoamylase action on 5IVÂ4 gave 5ÎVA4GA1, 5IVA4GA2, and 5IVA4GA3 2 (Figure 41). They were identified as glucose, 2 -0-(2-hydroxyethyl)maltose, 2 and 3 -0-(2-hydroxyethyl)maltotrlo8e, respectively (Figures 44 and 45). In 153
the glucoamylase action on 5IVA4, there was a very small spot with the same paper chromatographic mobility as 5IVA4, and the compound was resistant to glucoamylase action. Because the quantity was negligible, no further analysis of this compound has been performed.
Porcine pancreatic o-amylase action on subfraction 5IV gives a double check on the identity of products which have been isolated from the amylase digest of 0-(2-hydroxyethyl)amylose, and the results are very satisfactory.
The reactions in a-anqrlolysis of 5IV are summarized in Scheme 5.
Scheme 5. Further Porcine Pancreatic a-amylolysis of 5IV
B. Conclusion
In the present investigation, seven monohydroxyathylated maltooligo-
saccharidss were isolated from a porcine pancreatic «-amylase digest of 0-
(2-hydroxyethyl)amylose. No substituted monosaccharide has been detected
in the enzyme digest. The structures of these 0-(2-hydroxyethyl) oligo
saccharides arc listed in Figure 49. Their percentages in the designated Figure 49. Structures of hydroxyethylated maltoollgosaccharides in simplified structural formula. These oligosaccha rides were isolated from porcine pancreatic a-amylase digest of 0-(2-hydroxyethyl)amylose. The percentages of sugars listed in this figure are the percentages in the designated subfractions
4IS2 2 3 -0-(2-hydroxyethyl)maltose 5IS2
p 4III 2 -0-(2-hydroxyethyl)maltotrlose 2 3 -0-(2-hydroxyethyl)maltotrlose 3 6 -0-(2-hydroxyethyl)maltotrio8e
2 5III 2 -0-(2-hydroxyethyl)maltotrlose 3 6 -0-(2-hydroxyethyl)maltotriose
2 5ÎV 2 -0-(2-hydroxyethyl)maltotstraose 2 3 -0-(2-hydroxyethyl)maltotetraose
6^'=0=(2~hydroxyathyi)!naltotetraose 155
Hydroxyethylated Maltooligosaccharides Isolated from Porcine Pancreatic Alpha-Amylolysis of 0-(2-Hydroxy- ethyl)amylose.
4IS2 5IS2 HE
4111 OKIHZ) 93% HE rv-TV-^
5 III O-Ch0 79% HE
21 %
72% ÎIE 5 iV O-O-O-0 1 HE KÉ }• 28* I 156
subfraction or subfractions are also indicated. Besides these oligo- 3 saccharides, trace amounts of 2"'-0- and 3 -0-(2-hydroxyethyl)maltotriose 3 were detected in the amylase digest. 2 -0-(2-hydroxyethyl)maltotriose
might come from the 2-0-hydroxyethylated nonreducing terminals of the 3 amylose chains. 3 -0-(2-hydroxyethyl)maltotriose was cleaved by extensive 2 porcine pancreatic a-amylolysis to 3 -0-(2-hydroxyethyl)maltose and glucose.
Sodium borohydride reduction of the total enzymatic digest followed
by complete acid hydrolysis gave no hydroxyethylated glucitols. This in
formation as well as the structures of the isolated hydro^qrethyl sugars in
dicates that hydroxyethyl a-limit dextrins cannot have substituents at their
reducing ends.
The action pattern of porcine pancreatic a-amylase on 0-(2-hydroxj'-
ethyl)amylose and monohydroxyethylated oligosaccharides is summarized in
Figure 50 and the binding specificities of the binding subsites are shown
in Figure 51.
From all these data we conclude that:
1. For the five glucose binding subsites in the binding site of the
enzyme molecule, 6-0- substituents are permitted on the glucose residues
at subsites I, II and V but prohibited at III and IV.
2. 2=0='substituents are permitted at I, IV and to a less extent at V,
but prouibitcd at II and III,
3. 3-0-substituents are permitted at I, II, IV and V, but prohibited
at III.
4. Complete prohibition of substituents at subsite III Indicates that
all hydroxyl groups on the glucose residue which binds at this subsite are Figure 50. Action pattern of porcine pancreatic a-amylase on 0-(2-hydroxyethyl)an^lose with isolated hydroxyethyl substituents
GÂ glucoaiaylase
PPA porcine pancreatic a-amylase
t enzymic cleavage point indicated as a, b, c, and d 158
PPA*-^ 0 +OO0
opc^—IGA" PPA"''
•••OOCK>Op"; b.e ÙC a b he e PPA
D HE 0 + HE
P0
. PPA •••OOO^DOO*^ a.d bHE d PPA"*" PPA" 0 + op0 - ' oopp
I HE PPÂ
HE ^ H^ obo0 » ' 's. h PPA"' fc ^GA.
HE /
-.•CC^OOO-:, t t t PPA •' a b * Figure 51. Binding specificities of the glucose binding subsites in porcine pancreatic a-amylase. The catalytic site of the enzyme is located between subsites II and III, and the numbering of the subsites is started from the reducing end. The substituents which are permitted are indicated above each individual subsite 160 i
Substituents permitted
2-0- 3-0- ^ ^ 3-0- „ 6-0- NONE 6-0«
IV III li II
ENZYME 161
necessary in the binding and catalytic reaction. Previous investigation in our laboratory (40) shows that maltobionic acid lactone is a competitive inhibitor. These facts taken together suggest that the enzyme forces the glucose residue at subsite III into a half-chair conformation resembling that of a reaction intermediate (41). Models show that such a conformation is easily induced by torsion at the C2 -C3 bond, holding the C3 and C6 hydroxyl groups fixed, and exerting force on the C2 hydroxyl group.
5. The binding requirements at subsites I and V are not critical.
The C2 hydroxyl group of the glucose residue is important for binding at subsite II, and the C6 hydroxyl group is necessary for binding at subsite
IV. 162
VI. BIBLIOGRAPHY
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3. Robyt, J. F. and French, D. Multiple attack hypothesis of a-amylase action: Action of porcine pancreatic, human salivary and Aspergillus oryzae a-amylases. Arch. Biochem. Biophys., 122, 8 (1967).
4. Robyt, J. F. and French, D. The action pattern of porcine pancreatic ct-amyl&se in relationship to the substrate binding site of the enzyme. J. Biol. Chem., U5, 3917 (1970).
5. Robyt, J. F. and French, D. ÎSiltipls attack and polarity of action of porcine pancreatic o-amylase. Arch. Biochem. Biophys., 138, 622 (1970).
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7. Kainuma, K. and French, D. Action of pancreatic alpha-amylase and sweet potato beta-amylase on 6^- and 6^-a-glucosylmalto-oligosaccharldes. FEBS Letters, 6, 182 (1970).
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20. Meyer, R. H., Fischer, E. H. and Bernfeld, P. L'isolement de I'a- apylase de pancreas. Experlentla, 362 (1946).
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VII. ACKNOWLEDGMENTS
I wish to express my deep appreciation to my major professor. Dr.
Dexter French, for the opportunity of working In his laboratory. His understanding, encouragement, and Inspiring guidance made the completion of Çhls work possible. A special thank you to Dr. French for his extra help during the final preparation of this manuscript and the warm friend ship of Dr. and Mrs. French througjhout the author's stay at Iowa State
University.
Thanks are also du@ to Drs. J. F« Robyt, C» L. Tipton, J. H. Sloneker,
S. Klkumoto, and T. J. Barton for their technical help and valuable dis cussions.
Sincere gratitude is extended to all the members of Dr. French's and
Dr. Robyt's research groups, especially Mr. J. W. Bolcsak, Mr. S. A. Brown,
Mr. J. Zikopoulos and Mrs. Wendy B. Llnder for their encouragement and friendship. I wish to express appreciation to Mrs. Ardythe Ho viand, my typist, whose very able and wiiiing asaisuanee lightened the wsrk losd in preparing this manuscript, and to R. L. Mellgren with whom I had a good time taking course work and studying for exams.