THE

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

CHEMISTRY 1961

Printed

in U.S.A.

Physical
I. RELATIONSHIP

Properties
IODINE
AND W.

of Starch
STAIN AND CHAIN LENGTH*

BETWEEN J. M. BhILEYt

J. WHELAN$ Wales, Bangor,

From

the Department

of Chemistry,

University

College of North

Wales

(Received for publication, June 9, 1960)

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,
* 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

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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

970
TABLE Iodine-staining properties

Physical
I
in range

Properties

of Starch.

Vol. 236, No. 4

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.
of amylose Time of incubation min
DP

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 mr

min.
30 52 70 100 122 184 238 310 372 423 501 571 613 672 36 58 70 90 107 135 169 195 218 230 240 256 269 278 308 325 366 415 568 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

Iodine color

P.V.

B.V.

Xmas !J

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

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

Red-purple Purple Blue-purple Blue Blue-green

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

0 0

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

490 490

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

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

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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.
RESULTS

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

1.4 -

PEAK

VALUE

(P.V.)

,LUE

( B.V.) Y - 640 =620 5 ; kCl j

PEAK

WAVELENGTH

( x max.)

- so - 580 - 560 - 540 - 520 - 500

20

, 40

I 60

I 80

I 100

I 120

1 140

I 150

1 IBO

I 200

I 220

CHAIN

LENGTH

OF

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

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.

April

1961

J. M. Bailey

and W. J. Whelan

971

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

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.

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AVERAGE

CHAIN

LENGTH

FIG. length

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

average

chain

972

Physical

Properties

of Starch.

Vol. 236, No. 4

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.
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

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

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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. 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,

Peat,

April

1961
(1940). WHELAN, MACEY, C. T., J.,

J. M. Bailey and W. J. Whelan

W. J., Biochem. A., AND PEAT, J. Chem. Sot., S., 4640 166,

973

9. ALLEN, R. J. L., Biochem. J., 34,858 10. BAILEY, J. M., THOMAS, G. J., AND J., 49, lvi (1951). 11. BOURNE, E. J., HAWORTH, W. N., J. Chem. Sot., 924 (1948). 12. COWIE, J. M. G., AND GREENWOOD, (1957). 13. BOURNE, E. J., AND WHELAN, W. 578 (1950).

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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. Sot., 66, 142 (1943). 17. GILBERT, G. A., AND MARRIOT, J. V. R., Trans. Faraday Sot., 44, 84 (1948). 18. GRAY, R. G., M.S. thesis, George Washington University, 1959.

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