AN.~LTTIC.

~I, BIOCHEMISTRY 45, 517-524 (1972)

Reducing Value Methods for Maltodextrins:

II. Automated Methods and Chain-Length Independence
of Alkaline Ferricyanidel

JOHN F. ROBYT, ROSALIE J. ACKERMAN, AND J. G. KENG

Departmelbtof Biochemistry al&d Biophysics, Iowa State University,
Ames, Iowa 50010

Received June 16, 1971

Hoffman introduced the use of alkaline ferricyanide as a reagent for

the determination of carbohydrates reducing groups in urine (1). The
chemistry of the procedure is less involved than the procedure with
alkaline copper (2). Ferricyanide, which is yellow, is reduced by
hemiacetals or hemiketals to ferrocyanide, which is colorless. A colori-
metric determination at 420 nm is made of the loss of yellow color,
which is an index of the amount of reducing sugar present in the sample.
Besides the simplicity of the ferricyanide reagent and the ease of
detection of the reaction, the alkaline ferricyanide procedure has an
additional advantage over the alkaline copper procedure in that the
reduction product, ferrocyanide, is not reoxidizeci by atmospheric oxy-
gen as is cuprous oxide. The reoxidation of cuprous oxide by atmos-
pheric oxygen and the complicated chemistry of the color formation
in the reaction of cuprous oxide with arsenomolybdate has caused tech-
nical problems in the accuracy and precision of the alkaline copper
procedure.
Thus, in the automated determination of micro amounts of glucose,
using the Technicon AutoAnalyzer, the simpler chemistry of Hoffman’s
procedure has made alkaline ferricyanide the reagent of choice (2).
In the quantitative determination of carbohydrate reducing groups,
it is important to know if the method is giving results that are propor-
tional to the number of reducing groups (hemiacetal groups for malto-
dextrins) present in the sample. This is particularly important in the
use of the reagent for carbohydrase assays, for carbohydrase kinetics,
1 Journal Paper No. J-6922 of the Iowa Agriculture and Home Economics Ex-
periment Station, Ames, Iowa. Project 1485. Supported by grants from the
Agricultural Research Service, U. S. Department of Agriculture, Grant 12-14-166-
9887(71), and from the Corn Refiners Association.
517
@ 1972 by Academic Press, Inc.
518 ROBYT, ACKERMAN, AND KENG

and for the determination of the molecular size of oligosaccharides and

polysaccharides by end-group analysis.
For these reasons, we have investigated the stoichiometric properties
of the alkaline ferricyanide reagent by comparing the molar reducing
values of glucose and maltodextrins (maltose through maltoheptaose) .
In addition, we describe automated procedures using the Technicon
AutoAnalyzer, which include an automated carbohydrase assay and a
continuous sampling system for carbohydrase kinetics studies.

METHODS

AutoAnalyzer Procedures
Three automated procedures, each designed for a different purpose,
have been developed. Figure 1 gives the AutoAnalyzer manifold dia-
grams for the three procedures. Procedure A is a general reducing-value
determination that has a range of l&200 pg maltose/ml. Procedure B
is an automated carbohydrase-assay system with a range of 20-360 pg
maltose/ml. Procedure C is a reducing-value sampling system designed
to follow carbohydrase kinetics continuously with a range of O-600
s/ml.
Reagents
Procedure A. Cyanide: 5.0 gm potassium cyanide, 1000 ml water, and
0.5 ml BRIJ-35 (Technicon Corp., Tarrytown, N. Y.). Ferricyanide:
0.4 gm potassium ferricyanide, 20 gm sodium carbonate, 1000 ml water,
and 0.5 ml BRIJ-35.
Procedure B. Cyanide: as in Procedure A. Ferricyanide: 0.8 gm
potassium ferricyanide, 20 gm sodium carbonate, 1000 ml water, and 0.5
ml BRIJ-35. Starch substrate: 10 mg/ml Merck soluble starch suitably
buffered for the enzyme to be assayed.
Procedure C. Cyanide and ferricyanide reagents: as described for
Procedure A. Sodium carbonate reagent contained 20 gm in 1 liter
and 0.5 ml BRIJ-35.
Dextrins
Maltodextrins, maltose through maltoheptaose, were prepared by an
a-amylase hydrolysis of amylose, which was resolved into individual
maltodextrins by charcoal column chromatography (3). The purity of
each dextrin was checked by paper chromatography.
Stoichiometry of Ferricyanide and Hemiacetal Reducing Groups
Various concentrations (0.05-0.8 m&Q of glucose and maltodextrins
were prepared. The exact concentrations were determined by an auto-
 REDUCING; VALUE; METHODS FOR MALTODEXTRI NS. II 510

PUmP reaaent ID ml /min.

tubing size
double
KCN 0.065 in 60
Sample 0.040 0.60
Air 0.056 1.20
K,Fe(CN& 0.090 2.90
Waste 0.090 2.90

calorimeter

E. +---+I-KCN a020 0.159

0.025 0.235
0.025 0.235
0.035 0.420
0.073 2.00
0.045 0.80
0.056 1.20

1.60
2.00
1.60
1.20
2.90
2.00

FIQ. 1. AutoAnalyzer ferricyanide reducing value manifolds: (A) General re-

ducing value methods. (B) Automated carbohydrase assay. (C) Continuous-time
course analysis of carbohydrate action. The 85” heating bath contained a coil
(0.16 X 1220 cm) which is equivalent to a 15 min heating period. The cell in the
calorimeter was a 15 mm flow-through cell. The wave length was 420 nm and the
aperture plate was No. 4. The incubator of Procedure B had a variable tempera-
ture control and contained various lengths of coil to give different incubation times.

mated phenolsulfuric acid procedure (4). The molar reducing values

were determined by Procedure A.

Amylase Assay by Ferricyanide-Automated Procedure B

A stock solution of porcine pancreatic a-amylase (2X crystallized,
Worthington Biochemical Corp., Freehold, New Jersey) was prepared in
20 mM sodium glycerophosphate (pH 7) and 10 mM sodium chloride.
This stock solution was diluted with 20 mM soduim glycerophosphate
buffer 10 mM in sodium chloride (pH 7) until it gave approximately
200 pg of maltose in the assay syst.em. Further dilutions of this solution
520 ROBYT, ACKERMAN, AND KENG

were made to give relative amounts of enzyme of 0.1, 0.25, 0.5, and
0.75. Assays by the reading-value determination were made by using
Procedure B .
Reducing Value Determination of Time Course Reaction
of a Carbohydrase Digest
The reaction was initiated by injecting 20 ~1 (50 mg/ml) of sweet
potato P-amylase (2x crystallized, Worthington Biochemical Corp.)
into 5 ml of Merck soluble starch (5 mg/ml) in 20 mM acetate buffer
(pH 4.8) equilibrated at 25”. Immediately after mixing, the digest was
sampled by dipping the sampling line of the AutoAnalyzer manifold
into the reaction mixture (see Fig. 1C).
RESULTS AND DISCUSSION
Figure 2 gives the decrease in absorbance (420 nm) for the reduction
of ferricyanide as a function of the concentrations of reducing sugars.
The data show that, on a molar basis, glucose gives a greater amount
of reduction than the maltodextrins. The latter, maltose through malto-
heptaose, give reducing values that all lie along a single line,
indicating that the maltodextrins have an identical molar reducing
value. Thus, for maltose through maltoheptaose, alkaline ferricyanide
reducing values are proportional to the number of reducing groups,
and overoxidation does not occur as it does with dinitrosalicylate re-
agents (5).
Previously it was reported (6) that the alkaline copper reagent gave a
higher molar reducing value for glucose than for the maltodextrins.
It was recognized then that the maltodextrins are chemically distinct
from glucose in that glucose has a free C, hydroxyl in the residue
carrying the hemiacetal group, while the maltodextrins have this C,
hydroxyl substituted. This must, account for the differences between the
molar reducing values of glucose and the maltodextrins. Maltose is,
therefore, the first compound in the maltodextrin series and an ap-
propriate standard for this series.
Even though this study stopped with maltoheptaose, equimolar re-
ducing values should be expected for higher maltodextrin homologs and
polysaccharides. The chemistq of the higher homologs would not be
expected to be different from the chemist,ry of maltoheptaose; if any
differences were to be expected, they should have been manifested in
the maltose through maltoheptaose range where the differences in prop-
erties would be expected to be most dramatic. It might be possible to
argue that, when the size of the molecules become large, the reducing
end could be buried in the secondary or tertiary structure and thus
 REDUCING VALGE METHODS FOR MALTODEXTRINS. II 521

0.7

0.6

0.5

0.4

0.3

0.2

0.1

I I I, 1, I,, I , , , ,

0.1 0.2 0.3 0.4 0.5 0.6 0.7

millimole / liter

FIG. 2. LOSS of absorbance (420 nm) of ferricyanide as function of increasing

concentrations of glucose (G,) and maltodextrins, maltose through maltoheptaose
(Gz-G,). Points on upper curve : (0) maltose, (0) maltotriose, (B) malto-
tetraose, (A) maltopentaose, (A) maltohexaose, (,O) maltoheptaose.

not be available for reaction. The secondary and tertiary structure of

maltodextrins and polysaccharides in solution, however, are not static
like those of proteins or polynucleotide but are constantly undergoing
change (7). Increases in temperature or pH disrupt the secondary and
tertiary structures (8)) and consequently, it would be expected that,
with a heating period of 15 min at 85” and pH 10.5, any buried reducing
groups would be exposed and made available for reaction with ferri-
cyanide. Thus, the concept of equimolar reducing values with ferri-
cyanide or copper reagents can be extrapolated to maltodextrins and
polysaccharides larger than maltoheptaose. Maltose would then be a
valid standard for these materials.
Figure 3 shows that the automated assay of porcine pancreatic
ru-amylase by ferricyanide (Procedure B) gives a linear response that is
 ROBYT, ACKERMAN, AND KENG

Relative Amount of Enzyme

FIG. 3. Assay of different concentrations of porcine pancreatic cr-amylase by

an automated (Procedure B) system.

proportional to the amount of added enzyme. The system can be stand-

ardized by adding various concentrations of maltose (20-360 pg/ml)
through the substrate line while the enzyme line draws water. The
automated system permits the individual assay of a large number of
enzyme samples that might arise from column chromatographic fractiona-
tions. Procedure A may also be used for enzyme assays when the reactions
are run individually in a constant-temperature bath and stopped by
heating in a boiling water bath or by the addition of trichloroacetic acid.
Since the initial reactions of a-amylases with starch produced malto-
dextrins and since glucose is not an initial or primary product, maltose
may be used for the standardization. Porcine pancreatic a-amylase
will form glucose only from a very slow reaction with the primary
products, maltotriose and maltotetraose (9). Bacillus subtilis (10) and
 REDUCING VALUE METHODS FOR MALTODEXTRIKS. II 523

600 -

500 -

400 -

100 200 300 400 500 600

time (se4

FIG. 4. Continuous-time analysis of action of sweet potato P-amylase with starch

(Procedure C). Data were replotted from a continuous AutoAnalyzer recording
chart that had been calibrated with standard maltose solutions. (10) 0.3 pg en-
zyme/ml. (e) 0.2 pg enzyme/ml.
Aspergillus oryzue2 oc-amylase will also form glucose only in the late
stages of amylolysis when they catalyze the slow hydrolysis of the
primary products. Pullulanase (11)) which forms maltodextrins from
pullulan, amylopectin, or glycogen, can also be quantitatively assayed
by this system using maltose as a standard. In fact, any enzyme system
that yields maltodextrins as initial products can be assayed by these
procedures. Carbohydrases that form glucose as an initial product, such
as glucosidase or invertase, could be assayed quantitatively if glucose
or glucose plus fructose were used, respectively, as standards.
Figure 4 is the continuous-time course analysis (Procedure C) of the
action of ,8-amylase on soluble starch at two concentrations of enzyme.
The production and analysis of maltose was linear up to at least 10 min.
This system provides an accurate, automated determination of the initial
velocity that is necessary for kinetic studies. The system has a range of
O-600 pg/ml of apparent maltose.
SUMMARY
Three automated, alkaline ferricyanide, reducing value procedures are
described: (A) a general procedure, (B) a carbohydrase assay, and (C)
*J. F. Robyt, unpublished results
524 ROBYT, ACKERMAN, AND EENG

a continuous-sampling system for carbohydrase kinetic studies. The

stoichiometry of the ferricyanide procedure was investigated by com-
paring the molar reducing values of glucose and a series of malto-
dextrins, maltose through maltoheptaose. It was found that glucose has
a higher molar reducing value than those of the maltodextrins but
that the maltodextrins all had identical values. It was concluded that
ferricyanide procedure described is a reliable means of quantitatively
determining the number of hemiacetal groups (reducing groups) for
maltodextrins and a-1,4-polysaccharides when maltose is used as a
standard. It was further shown that the carbohydrase assay procedure
gave a linear response with enzyme concentration when the samples were
producing reducing compounds within the assay range of 20 to 360 ,ag of
maltose equivalents per milliliter and that the continuous sampling
system gave a linear response with time up to 600 pg of maltose
equivalents per milliliter.
-ACKNOWLEDGMENT
We wish to thank Professor Paul Hartman of the Department of Bacteriology,
Iowa State University, Ames, Iowa, for his critical reading of the manuscript and
for independently testing the automated procedures.

REFERENCES
1. HOFFMAN, W. S., J. Biol. Chem. 120, 51 (1927).
2. “Technicon AutoAnalyzer Methodology, Microglucose,” Technicon Corp.,
Tarrytown, New York, 1964.
3. FRENCH, D., R~BYT, J. F., WEINTRAUB, M., AND KNOCK, P., J. Chromatogr. 24,
68 (1966).
4. ROBYT, J. F., AND BEMIS, S., Anal. Biochem. 19, 56 (1967).
5. ROBYT, J. F., AND WHELAN, W. J., Anal. Biochem. 45, 510 (1972).
6. WHELAN, W. J., BAILEY, J. M., AND ROBERTS, P. J. P., J. Chem. Sot. 1953, 1293.
7. FRENCH, D., in “Symposium on Foods: Carbohydrates and Their Roles” (W.
W. Schultz, ed.), p. 40. Avi, Westport, Conn., 1969.
8. HOLLY, J., AND SZEITLI, J.,, in “Starch and Its Derivatives” (J. A. Radley, ed.),
4th ed., p. 212. Chapman & Hall, London, 1968.
9. ROBOT, J. F., AND FRENCH, D., J. Biol. Chem. 245, 3917 (1970).
10. ROBYT, J. F., AND FRENCH, D., Arch. Biochem. Biophys. 100, 451 (1963).
11. ABDULLAH, M., CATLEY, B., LEE, E. Y. C., ROBYT, J., WALLENFELS, K., AND
WHELAN, W. J., Cereal Chem. 43, 111 (1966).