THE

JOURNAL
OF BIOLOGICAL
Vol. 236, No. 4, April

Printed

CHEMISTRY
1961

in U.S.A.

Physical
I. RELATIONSHIP

Properties

BETWEEN

IODINE

J. M. BhILEYt
From

the Department

of Chemistry,

of Starch
STAIN

AND W.

University

AND

CHAIN

LENGTH*

J. WHELAN$

College of North

Wales,

Bangor,

Wales

(Received for publication, June 9, 1960)

* This work was supported

by a maintenance
grant
(to J. M. B.)
from the Department
of Scientific
and Industrial
Research
(Great
Britain).
t Present
address,
George
Washington
Medical
School,
Washington,
D. C.
1 Present
address,
The Lister
Institute,
London,
S.W.l,
England

6). Swanson realized that the presence of nonpriming

fragments
would cause the calculated chain lengths of the synthetic amyloses to be too low. The results now to be reported show that
the relationship
of color intensity to degree of polymerization
derived by Swanson was indeed incorrect, and in the sense which
she anticipated.
EXPERIMENTAL

PROCEDURE

General Methods-Phosphorylase
was prepared and its activity
measured as described by Whelan and Bailey (2). &n-Glucose
l-phosphate
was prepared by the method of McCready
and
Hassid (7) and maltohexaose
by that of Whelan, Bailey, and
Roberts (8). Inorganic phosphate was measured by the method
of Allen (9), as modified by Whelan and Bailey (2). The use
of mercuric chloride and ammonium
molybdate
as inhibitors
of enzymatic contaminants
of the phosphorylase
is described
by Bailey, Thomas, and Whelan (10). The iodine solution
used to stain the synthetic amyloses contained 0.2% iodine in
2% potassium iodide as is used in the routine determination
of
the blue value of starch fractions (11).
Synthesis of Amylose and Measurement of Iodine Stain-In
the absence of primer, the phosphorylase did not liberate phosphate from cY-n-glucose l-phosphate
when incubated
in the
presence of ammonium molybdate.
In the absence of molybdate, slow breakdown
of the Cori ester took place because of
The enzyme did not alter the intensity
phosphatase impurity.
of iodine stain of amylose when allowed to act for 19 hours at
pH 7.0 and 35. This indicated the absence of amylase and Q
enzyme, but as a precautionary
measure, mercuric chloride was
added during the synthesis of amylose to ensure suppression of
Three syntheses of amylose
the activity of such contaminants.
were performed.
Synthesis I-The
digest was made by mixing 0.1 M a-n-glucose
l-phosphate
(7 ml, adjusted to pH 7.0), maltohexaose (4.77 mg,
4.83 pmoles), 153 PM mercuric chloride (0.6 ml), 8% ammonium
molybdate (1 ml), 0.2 M acetate buffer (pH 7.0, 3.3 ml), potato
phosphorylase
(150 mg, 4.2 units), and water (to 50 ml).
The
enzyme was added last of all to the remaining digest components,
which were preheated to 35. The progress of synthesis was
followed by measuring the liberation of inorganic phosphate on
withdrawn
portions of the digest. At the same time the iodinestaining properties of the synthetic amylose were measured by
adding additional 0.5-ml samples of the digest to 0.5 ml of iodine
solution in a final volume of 25 ml. The light absorption over
the range 450 to 700 rnp was measured in l-cm cells in a Unicam
model SP 500 spectrophotometer.
The concentration of amylose

969

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The formation of color by the interaction of starch and iodine

is one of the most useful and characteristic reactions of the polysaccharide.
The relatively advanced state of our knowledge
of starch structure is due in great measure to the ability of
iodine to detect small amounts of starch (as little as 1 pg per
ml) and to reveal changes in its degree of polymerization
caused
by enzymatic and chemical treatment (1). The blue color of
the stain is due to the amylose component of starch.
The
other component, amylopectin,
gives a red-purple
color which
is much less intense than the amylose stain. When hydrolyzed
in random fashion by acid or by a-amylase, both polysaccharides
gradually lose the capacity to stain with iodine.
The blue
amylose color becomes purple, then red, brown, and finally
disappears.
It is of interest to know precisely the relationship
between the chain length of the amylose and the color and intensity of the iodine stain.
The products of partial acidic or amylolytic
hydrolysis of
amylose reveal the color spectrum of chains of varying length,
but are too heterogenous to be suitable for deriving the desired
relationship.
Amyloses of known degree of polymerization
and
degree of homogeneity
can be prepared, however, by phosphorylase-catalyzed
synthesis from a maltodextrin
primer, with
a-n-glucose
l-phosphate
as the chain-lengthening
donor substrate (2). The degree of polymerization
of the amylose is
calculated from the amount of inorganic phosphate released (and
hence the moles of n-glucose incorporated
into the polymer)
during synthesis from a known amount of a pure maltodextrin
The glucose units are added in random fashion to the
primer.
end of each priming molecule so that the resulting polymer has
a Poisson-type (4) distribution
of chain lengths which may be
calculated accurately.
The correctness of values of degree of
polymerization
and chain-length
distribution
calculated in this
way has been confirmed analytically
(3, 4). Amyloses synthesized by a similar method were examined for iodine-staining
properties by Swanson (5), but the primer used was a partial acid
hydrolysate
of Schardinger
P-dextrin
(cyclohepta-amylose).
Since it was a mixture of unknown composition, it was not possible to determine what proportion
of the /3-dextrin fragments
Only maltodextrin
molewas represented by priming molecules.
cules having four or more D-glucose units are efficient primers (2,

970

Physical

Properties

TABLE

DP 6 to 81
Maltohexaose
primer was incubated with phosphorylase
and
a-n-glucose l-phosphate.
At the times indicated, portions were
withdrawn
for phosphate
determination
and iodine-staining.
DP was calculated from phosphate liberation.
P.V. and B.V.
were calculated from optical density of stain at X,sX and at 680
mp, respectively.
Iodine-staining

Time of
incubation

properties

of amylose

Iodine color

DP

in range

P.V.

Xmas

B.V.

min

!J

8.6
12
14.5
17
20
23
25
28
31
33
35
36
38
40
42
43
45
47
48
50
52
53
55
58
61
68
75
81

None
Faint red
Red

Purple
Blue-purple
Blue
Blue-green

VALUE

0
0

0.27
0.52
0.71
0.88
0.98
1.03
1.11
1.17
1.19
1.19
1.24
1.31
1.18
1.30
1.19
1.26
1.25
1.32
1.28
1.35
1.31
1.31
1.34
1.32
1.29
1.31
1.33

Red-purple

PEAK

1.4 -

490
490

0.028
0.042
0.075
0.12
0.16
0.21
0.31
0.37
0.35
0.40
0.48
0.45
0.54
0.53
0.58
0.61
0.69
0.71
0.78
0.78
0.80
0.86
0.86
0.98
1.06
1.12

505
515
520
525
533
537
546
550
554
555
557
562
565
568
571
575
575
575
575
580
584
590
600
695
610

(P.V.)

,LUE

( B.V.)
Y

PEAK

WAVELENGTH

( x max.)

- 640

=620

- so
- 580
- 560

kCl
j

- 540
- 520
- 500
20

,
40

I
60

CHAIN

I
80

I
100

LENGTH

I
120

1
140

OF

I
150

1
IBO

I
200

I
220

AMYLOSE

1. Relationship
of chain length and iodine-staining
properties of synthetic amyloses. Additional
details are given in
Tables I and II in the text.
FIG.

was calculated from the initial amount of maltohexaose and the

The results are given in Table I
inorganic phosphate released.
and Fig. 1.
Syntheses d and S-The
digest components in the second and

Vol. 236, No. 4

TABLE
II.
Iodine-staining
properties
of amylose
in range DP 6 to 668
All the amyloses stained blue-green except the first, which
stained purple.
Those of DP 308 to 568 were prepared in a separate experiment. For additional details see Table I.
Time of
incubation

DP

P.V.

B.V.

Xmax

1.27
1.20
1.29
1.28
1.35
1.30
1.25
1.33
1.38
1.38
1.42
1.42
1.39
1.44

0.46
0.85
1.04
1.09
1.10
1.10
1.12
1.19
1.20
1.21
1.27
1.27
1.26
1.29

550
595
600
605
610
615
620
620
620
625
625
630
630
630
635
640
645
645
645

min.
30
52
70
100
122
184
238
310
372
423
501
571
613
672

mr

36
58
70
90
107
135
169
195
218
230
240
256
269
278
308
325
366
415
568

third experiments were the same as

amounts of cu-n-glucose l-phosphate
primer (Synthesis 2, 0.955 mg, 0.965
mg, 0.193 pmole).
The digests were
measurements were made as before.
Table II and Fig. 1.

in the first except for the

(5 ml) and maltohexaose
pmole; Synthesis 3, 0.191
again incubated at 35 and
The results are given in

RESULTS

In the present experiments, amyloses were synthesized from

pure maltohexaose
primer.
The products were not isolated,
but were examined for iodine-staining
properties in the presence
of the buffer salts, oc-n-glucose l-phosphate,
etc., also present
in the digest. These substances did not interfere with the stain.
The iodine concentration
used was that employed in the standard conditions established and routinely used for determining
and characterizing
starch fractions (11). The conclusions and
quantitative
relationships
reported here refer, therefore, to
fractions stained under these standard conditions.
The upper
This limit was
limit of DPr examined was 568 glucose units.
imposed by the tendency of amyloses of high DP to retrograde
To some extent the point at which retrogradafrom solution.
tion occurs is determined by the concentration
of the amylose.
For this reason it was necessary to perform three experiments,
the first to cover the range DP 6 to 81 (Table I), the others the
range DP 6 to 278 (Table II) and the range 6 to 568 (Table II).
In this last experiment,
the tendency of the high molecular
weight amyloses to retrograde made the values for B.V. and
P.V. (see below) unreliable, and only the values for the color
and wave length of peak light absorption (X,,,) are given, since
these properties are not affected by retrogradation.
A lower
molar concentration
of amylose was produced in the second and
* The abbreviations
used are: DP, degree of polymerization;
B.V., blue value ; P.V., peak value.

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6.25
11.25
15.75
20
25
30
32
38
44
48
51.5
55
60.5
64.5
69.5
73
79
84
89.5
96.5
101.5
107.5
114
122.5
137
168
205
253

of Starch.

April

1961

J. M. Bailey

971

and W. J. Whelan

proportion of the chains in the distribution

have a greater length
than the average.
Since the phosphorylase-catalyzed
synthesis of amylose has
been shown (2) to involve the completely random addition of
glucose units to all available priming ends (multichain synthesis),
the resulting polymer which is built up will be one in which the
various molecules will be present in a Poisson distribution
(4).
The form of this distribution
may be calculated from the formula:
e-u. 2)z
P, = ~
x!
where P, is the fraction of the priming molecules which have had
x glucose units added, when v is the average number of glucose
units added per priming molecule.
Thus, for example, when the
average DP of the synthetic polymer was 12 units, (the point
at which an iodine stain first became visible), 20% of the molecules in the distribution
had DP 18 units or more. The proportion of the polysaccharide which was iodine-staining,
and hence
had passed the minimal DP, was determined for different integral
average values of DP by plotting the curve of percentage of
maximal P.V. against average DP as shown in Fig. 2. A series
of distributions
of DP for different average values of DP was
now calculated by the Poisson formula given above. Since
intensity of iodine stain is dependent upon the weight of polysaccharide rather than its molar concentration,
these distributions
were corrected by the different weight factors for the polymers
in the distribution.
A family of curves was now obtained by plotting average DP
against percentage of chains (by weight) in the distribution
which were greater than x units, where 2 ranged from 10 to 27
units.
These were compared with the experimentally
determined curve. The curve of best fit was that given by the 1%
unit polymer.
Thus at average DP 17 units, 50% of the polysaccharide was iodine-staining,
and at this average DP, 50%
by weight of the distribution
consisted of chains of 18 units or
greater.
It is concluded, therefore, that under the standard
conditions, 18 glucose units is the minimum necessary for formation of an iodine complex having a visible stain.
Determination
of Minimal
DP for Optimal Iodine-stainingThe form of the B.V./DP curve shown in Fig. 1, with the relatively sharp inflexion between 60 and 80 glucose units and the
subsequent linear but slow increase in B.V. with DP, suggests
that there may be some optimal DP reached around this point.

AVERAGE

FIG.
length

CHAIN

LENGTH

2. Relationship
of peak value
(P.V.)
to
of amyloses
during
early stages of synthesis.

average

chain

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third experiments by reducing the amount of primer added to

the synthesis.
Four properties of the iodine stain were recorded, (a) the
color, (b) B.V., (c) P.V., and (d) X,,,.
B.V. is defined as the
optical density to light of wave length 680 rnp of a mixture of 1
mg of polysaccharide,
2 mg of iodine, and 20 mg of potassium
iodide per 100 ml of solution contained in a 4-cm cell (11). The
ratio of optical density to polysaccharide
concentration
obeys
Beers law, and B.V. may therefore be calculated from measurements made with concentrations
of polysaccharide
other than
that specified.
P.V. refers to the optical density at X,,, under
conditions specified for B.V. determination.
Fig. 1 shows the
variation with DP of R.V., P.V., and X,,, of the iodine-stained
synthetic amyloses.
Relationship between DP and A,,,-The
colors of the iodine
complexes are noted in Table I. Almost all the visual changes
in color take place below DP 45 units.
After passing the achroic
point at average DP 12 (see below), the iodine color changes
through brown, red, purple, and finally becomes blue at about
DP 45, when X,,, is at 570 mp. Thereafter X,,,, continues to
rise, but no visible change in the quality of the color takes place.
The threshold value of X,,, at DP 12 is 490 rnp, almost the same
as for rabbit liver glycogen, the sample of glycogen tested having
Between DP 12
an average unit-chain DP of 13 glucose units.
and DP 50, X,,, increases in linear fashion; thereafter the rate
The data
of increase drops and attains another steady value.
in Table II and projection of the curve in Fig. 1 show that X,,,
of 645 rnF is not attained before DP 350 to 400. This value of
x rnlLXis that found for natural potato amylose.
achroic
Relationship between DP and B.V.-From
the apparent
point at DP 12, the B.V. rises regularly and rapidly with chain
length in the range of DP from 12 to 70 (Fig. 1). The rate of
increase then falls abruptly and attains a new steady value.
Projection
of this part of the curve shows that B.V. 1.4, the
minimal value found for natural potato amylose, is not reached
The DP
until the polymer has a length of 400 glucose units.
of potato amylose is of the order of several thousand (12). The
iodine-staining
qualities of an amylose of DP several hundred
units thus differ little from those of one with several thousand
units.
In this range of DP, therefore, B.V. and X,,, are very
insensitive indexes of molecular size. This is of importance in
relation to attempts to interpret
the mechanism of enzymatic
degradation
of amylose, in which iodine-staining
properties of
the products have been used as a guide to molecular size (see (13)
for a more full discussion).
Relationship between DP and P.V.-A
striking feature of the
relationship between P.V. and DP (Fig. 1) is the rapid increase
in P.V. from the achroic point of average DP 12 to close to its
Since the
maximal value at an average DP of only 30 units.
P.V. is a measure of the extinction coefficient of the polysaccharide-iodine
complex, we have interpreted
this to mean that
in the range of average DP 12 units to average DP 30 units, an
increasing proportion
of the total polysaccharide
present becomes iodine-staining
(i.e. the chains pass a minimal length
necessary for formation of a visible complex with iodine at the
standard iodine concentration).
for Iodine-stainingDetermination
of Minimal
DP Necessary
To determine the minimal DP for iodine-staining,
it is not sufficient to note merely the lowest average DP at which an iodine
stain is first visible, since at this average DP, a considerable

972

Physical

Properties

DISCUSSION

The most recent theory as to the nature of the complex which

starch forms with iodine has developed from preliminary observations by Hanes (14) on the decrease in the iodine color of starch
during enzymatic degradation.
On the basis of these results,
Hanes postulated that a coil of 6 glucose units was necessary
for the display of iodine-staining
properties, and that the amylose
chain was coiled in solution, giving a long spiral.
Each turn
of this spiral contained 6 glucose units, and the iodine molecules
lay in the interior.
The suggestion was adopted and extended
by Freudenberg et al. (15), who showed by means of models that
the amylose molecule could exist in the form of a helical spiral
with each turn of the helix containing 6 glucose units, and that
the interior of the helix had a hydrocarbon
lining due to the
inward-pointing
hydrogen atoms of the glucose units.
The
stain given with iodine was attributed to this lining, by analogy
with the purple color which iodine displays in hydrocarbon
solvents.
Further confirmation
of the helical structure was
given by the work of Bates, French, and Rundle (16). By using
a potentiometric
technique, they were able to show that natural
amyloses bind iodine strongly, and in amounts up to 21 y. by
weight, corresponding
to 6 glucose units for each iodine atom.
X-ray diffraction
studies were in agreement with the helical
structure, with the iodine molecules arranged along the spiral
with their axes along its major axis.
Gilbert and Marriot
(17) examined the complex formed between amylose and iodine in dilute potassium iodide solution,
the conditions used here for determination
of B.V.
They found
that the blue color given by amylose was well developed when
ions of the type 31z.21- were present.
They considered that
such ions were probably present in a linear resonating form, and
that the red complexes which were formed by shorter chains were
due to their inability to stabilize an ion of this size. On the
basis of these results, it would seem that the large increase in
B.V. which occurs in the region DP 18 to 72, represents the
* By the true curve is meant the relationship
obtained with monomolecular
amyloses.

which would be

Vol. 236, No. 4

building up of such a linear stable complex through various

resonating ions, until an ion of optimal stability is reached.
One of the most important
factors determining
iodine stain
in the low molecular weight region is iodine concentration.
For
example, the B.V. of a maltodextrin
fraction of average DP 24
was increased 50% in intensity by a lo-fold increase in iodine
concentration
over the standard
concentration
used in our
experiments.
When the dextrin was hydrolyzed
by acid to
about half its original size, the B.V. fell to a small fraction of its
original value, but now increased a-fold by a similar increase in
iodine concentration
(18). Similarly,
structural
factors are
important.
The cyclic Schardinger
dextrins containing
only
5 to 8 glucose units will stain with iodine vapor when they are
in the crystalline form. In addition, the iodine stain of a sample
of liver glycogen was increased 20-fold by the lo-fold increase
in iodine concentration,
whereas that of a sample of heart glycogen from the same animal was only increased 6-fold (18). It
seems that adsorption
phenomena, in addition to the specific
method of complexing discussed above, are responsible for these
effects. In experiments in which branch linkages were introduced into the synthetic amylose molecules by the action of Q
enzyme, it is known that the ability to form iodine complexes is
very much decreased.
The results of these experiments have
been of considerable assistance in interpreting
the structures of
some of the naturally occurring branched polymers, and will be
reported elsewhere.
SUMMARY

1. Synthetic amyloses of average degree of polymerization

in
the range 6 to 568 glucose units were prepared by an enzymatic
method.
The iodine-staining
properties have been examined
in terms of color, intensity of stain (blue value), and wave
length of peak light absorption.
2. Under the standard conditions used, the chains become
iodine-staining
when they are 18 units long. Thereafter
the
blue value increases rapidly and linearly with chain length
until the chains are about 72 units in length.
From this point
the rate of increase falls to about 5% of the initial value.
The
blue value of naturally occurring amyloses is not reached until
the chains exceed 400 units.
3. The results are discussed in terms of possible structures
for the starch-iodine
complex.
Acknowledgment--We
are grateful to Professor Stanley
F.R.S., for his advice and encouragement.

Peat,

REFERENCES
S., in R. T. WILLIAMS
(Editor),
Biochemical society
symposia, No. 11, University Press, Cambridge, England,
1953, p. 1.
2. WHELAN,
W. J., AND BAILEY,
J. M., Biochem. J., 68,560 (1954).
3. PARRISH,
F. W., AND WHELAN,
W. J., Nature
(London),
183,
991 (1959).
4. MOULD,
D. L., AND SYNGE,
R. L. M., Biochem.
J., 68, 571
(1954).
5. SWANSON,
M. A., J. Biol. Chem., 172,825 (1948).
6. FRENCH,
D.,
AND WILD,
G. M., J. Am. Chem.
Sot., 76, 4990
(1953).
7. MCCREADY,
R. M., AND HASSID,
W. Z., J. Am. Chem. Sot.,
66, 560 (1944).
8. WHELAN,
W. J., BAILEY,
J. M., AND ROBERTS,
P. J. P., J.
Chem. Sot., 1293 (1953).
1. PEAT,

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If the second part of the B.V./DP

curve (i.e. that beyond 80
units) is produced backwards, it intersects the portion of the
curve with steep slope (i.e. that below 60 units) at DP 72. (Note
also that it cuts the axis of zero B.V. at DP 18.) The significance
of the inflexion at DP 72 was analyzed by plotting Poisson distributions for polymers with average values of DP in this range.
The following conclusions were reached: (a) B.V. is a linear
function of DP in the region 18 to 72 units.
(b) At DP 72 there
is a sharp inflexion.
The B.V. then continues to increase in a
regular manner, but at a rate which is only 5% of the initial
rate. The shape of the experimentally
determined
curve, i.e.
the divergence from linearity which begins at about average DP
48, is due to the following effects. Up to DP 48 the B.V. increases linearly with DP until some of the chains in the distribution begin to pass 72 units.
The experimental
curve then falls
below the true curve2 and does not meet it again until all the
chains in the distribution
have passed DP 72. For example, in
a synthetic amylose having average DP 77, about 25% of the
chains are still less than 72 units in length.
All the chains in
the distribution
will not exceed 72 units until the average DP
is about 100 units.
This was the point at which the experimental curve then achieved the second phase of linearity.

of Starch.

April

1961

9. ALLEN, R. J. L., Biochem.

J., 34,858
10. BAILEY,
J. M., THOMAS,
G. J., AND
J., 49, lvi (1951).
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W. N.,
J. Chem. Sot., 924 (1948).
12. COWIE,
J. M. G., AND GREENWOOD,
(1957).
13. BOURNE,
E. J., AND WHELAN,
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578 (1950).

J. M. Bailey and W. J. Whelan

(1940).
WHELAN,
MACEY,
C. T.,
J.,

W. J., Biochem.
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14. HANES, C. S., New Phytologist,

36, 101 (1937).
15. FREUDENBERG,
K., SCHAAF,
E., DUMPERT,
G., AND PLOETZ,
T., Naturwissenschuften,
27, 850 (1939).
16. BATES, F. L., FRENCH,
D., AND RUNDLE,
R. E., J. Am. Chem.
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17. GILBERT,
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thesis,
George
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1959.

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ARTICLE:
Physical Properties of Starch: I.
RELATIONSHIP BETWEEN IODINE
STAIN AND CHAIN LENGTH
J. M. Bailey and W. J. Whelan
J. Biol. Chem. 1961, 236:969-973.

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