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Iodine Binding by Amylopectin and Stability of the Amylopectin-Iodine Complex

HEATHER DAVIS, WALTER SKRZYPEK, and ARSHAD KHAN *

Chemistry Department, The Pennsylvania State University, DuBois, Pennsylvania 15801

SYNOPSIS

The iodine binding capacity (IBC) of amylopectin ( AP, from potatoes) is determined to be around 0.38% (w/w ) of the total AP in the solution. The mass of iodine bound comprises about 13.6% of the mass of AP involved with the complex, suggesting that with every four iodine atoms bound there are 23 anhydroglucose residues ( AGU) . Since our previous study indicates that four iodine atoms within the helix of 11 AGUs form a chromophore unit in the API complex, only 48% of the AGUs (11 out of 23) in the AP molecule are directly involved with the iodine. The heat of reaction for the API complex formation is determined to be around -47 kJ/mol of 1-1 units bound and is significantly lower in magnitude than that of the amylose-iodine (AI) complex [ Biopolymers, 3 1 , 5 7 ( 1991) 1. A possible mechanism has been proposed for the formation of A1 and API complexes with fixed compositions. 0 1994 John Wiley & Sons, Inc. Keywords: amylopectin, iodine binding capacity amylopectin, iodine binding energy of

iodine binding and amylopectin fine structures Amylose-iodine and amylopectin-iodine complexes of fixed composition peak shifts in amylose-iodine spectrum and different iodine species

INTRODUCTION

The major component of starch from sources like potatoes and corn is amylopectin (AP) and has amylose as a minor c~mponent.‘-~ While the blue complex of iodine with amylose is well known and extensively studied,’-5 the red complex with AP is less well known, and very little attention was given to characterize this c ~ m p l e x ~ , ~ until recently. We recently identified that four iodine atoms more or less linearly arranged within the cavity of the helix structure of 11 AGUs8 (C6H1005) form the chromophore group in the API complex. In that study we first determined unambiguously the experimental spectrum for the API complex by subtracting the H2012 spectrum (due to I2 complexes9 with water) from that of the AP-iodine solution, followed by an accurate peak resolution.” Secondly, we observed’ that the spectrum of partly hydrolyzed amylose with

~ _ _ _ _ _

* To whom all correspondence should be addressed. Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 32,2267-2274 (1994) 0 1994 John Wiley & Sons, Inc. CCC 0&37-624X/94/122267-0S

iodine was almost identical to that of the API complex suggesting that the API has small “amylose units.” In addition, the spectra calculation’ by INDO CI method (intermediate neglect of differential overlap configuration interaction) on different iodine species identified the I4 unit (1-1 distance of around 3.0 A ) to be responsible for the experimental spectrum. Unlike amylose, which is essentially a linear glucose polymer ( a 1-4 linkage) with limited branching ( a 1-6 linkage), the AP is a branched polymer (94-96% a 1-4 and 4-6% a 1-6 linkages) with an average chain length of 20-26 AGUS.~,~ Compared to the A1 complex, in which about 17 AGUs are bound to six iodine atoms,” the API complex represents a smaller “A1 helix,” having 11 AGUs and four I atoms in the chromophore?

The purpose of the present study was to examine the thermal stability of the API complex and the reactivity of the AP molecule towards iodine. For this reason we determined the iodine binding energy (BE) and the iodine binding capacity (IBC) values for AP. The BE for the API complex formation is defined as the heat of reaction (AH) per mole of I atoms or 1-1 units complexed with AP. The BE value

2267

2268 DAVIS, SKRZYPEK, AND KHAN

for the A1 complex is already known ( -110 kJ/mol of the 1-1 units bound''), and thus, by determining the BE value for the API complex, we could compare the stability of the API with that of the A1 complex. The IBC of AP is defined as follows:

Mass of bound I in API complex IBC = x 100

Total AP mass in solution

In this work the IBC value was obtained at the AP concentration that gives the maximum amount of complex for a fixed amount of iodine in the solution. This also represents a condition of no free iodine (or all iodine complexed) in the solution. The IBC value for amylose (potato) is well established (ca. 19.5%) and was determined by a number of earlier experiments involving the amperometric and potentiometric titrations. In those experiments the IBC value was obtained from the intercept of the linear portion of the iodine binding curve extrapo- lated to zero free iodine con~entration.'~-'~ This extrapolation provides a limiting condition of zero free iodine (or all iodine complexed) in the solution and can be compared to the conditions maintained in our experiment. The application of the same extrapolation method for the IBC of A P provided close to a zero value (see Ref. 17 and other references therein) and has not been reported. The failure in these earlier attempts for determining the IBC value of AP by amperometric and potentiometric titrations may be attributed to the small value of IBC for which the applied methods were inaccurate. Be- sides, these methods did not discriminate the iodine

036 - 0.24 1 0.224 J

0 6 12 18 24 30

TEMPERATURE r C )

Figure 1. The stability curve of temperature ( "C) ver- sus absorbance values (a t 550 nm) are presented here. The API complex is stable a t lower temperatures (lower than 6°C) and dissociates in increasing amounts as the temperature is increased from 6°C.

0'18 8

/ 4 -6 1.C

I'C a

0.10 1. 0.36 0.46 0.56 0.66

g/L AP

Figure 2. The IBC experimental data taken at 3,8,10, and 2OoC and their polynomial fittings are shown. The total iodine concentration in each experiment was 2.38 X gjL, and the A P concentration was varied from a small value to a large one.

complexed with AP or amylose (giving A1 complex, this might have contaminated the earlier experiments)I5 from that merely adsorbed by AP. Here we applied a spectrophotometric method which gives a major absorbance peak (in the visible range) for the API complex at around 550 nm. The 550 nm wavelength, being quite remote from the absorbance maxima of the other iodine c o m p l e ~ e s ~ . ~ ~ like H2012 or AI, is not expected to have any major interference from these species. Thus, we expect that the spectrophotometric method, as applied here, will be quite accurate for determining both the IBC and the BE values for the API complex.

EXPERIMENT

Preparation of Solutions

AP Solution

The AP concentration of 0.0762 g/L was made for the thermal stability and calibration experiments by dissolving the solid (Sigma Scientific, A 8515) in 490 mL of water and 10 mL of pH 4 buffer containing 48% of acetic acid and 24% of sodium acetate (Fisher Scientific, SO-B-85) solution. Since the solid had 94% of AP and 6% water (determined by Karl- Fischer method, Sigma Scientific), appropriate concentration corrections have been applied in re- porting the AP concentrations. Our test results with iodine and AP solutions (spectrum) indicate almost no amylose contamination. These tests were done by absorbance measurements a t around 615 nm, the

IODINE BINDING BY AMYLOPECTIN 2269

characteristic wavelength for the A1 complex. A negligibly small value of absorbance at 615 nm indicates an absence of the A1 complex. For the IBC experiments, a concentrated solution was made by dissolving 0.223 g of solid in 250 mL water and 5 mL of pH 4 solution which gives the AP concentration of 0.823 g/L (after correction).

Iodine Solution

The iodine solution of concentration 126.9 mg/L (1.0 X 10-3M I ) was made by dilution from a solution containing the same volume of concentrated iodine solution (0.10M I, S186 Fisher Scientific) and potassium iodide solution (0.30M, SP241, Fisher Scientific) and was kept a t around 6°C to prevent evaporation of iodine. This solution was used for the stability and calibration experiments, and a diluted solution (15.86 mg/L) of it was used for the IBC experiment.

Thermal Stability and IBC Experiments

The thermal stability experiments of the API complex were done by adding the same volume of AP of concentration 0.0762 g/L to iodine of concentration 126.9 mg/L and taking absorbance readings at 550 nm at 1,3,5,10,15, 20, 25, and 30°C. A spectronic 601 spectrophotometer was used for the absorbance measurements and a refrigerated temperature bath with circulator ( Model 9000, Fisher Scientific) was used for maintaining the above temperatures during the experiment. Figure 1 represents the thermal stability plot, showing almost no change in absorbance values a t temperatures lower than 6°C and a sharp decrease in absorbance as temperature was raised. This suggests that the complex reaches its maximum stability at temperatures lower than 6°C.

In each of the IBC experiments a certain amount (2, 4, 6, 8, 10, 12, 14, 15, 16, 17 mL) of the AP solution (0.823 g/L) was added to a fixed volume (3 mL) of iodine solution (15.86 mg/L) and water ( 15, 13, 11, 9, 7, 5, 3, 1, 0 mL) to obtain a total volume of 20 mL before absorbance readings were taken at 3, 8, 10, and 20°C. Some of these data of interest are shown in Figure 2 in which g/L of AP (added concentration) is plotted against absorbance values. The total concentration of iodine in each 20 mL solution was 2.38 X g/L.

Calibration Experiments

The calibration data (Fig. 3) are needed to convert the absorbance values of thermal stability and IBC

experiments (Figs. 1 and 2, respectively) to concentrations of AP (in g/L) complexed. Each of these experiments was done by adding a 5 mL of concentrated iodine solution (126.9 mg/L) to a relatively dilute solution of AP (0.076 g/ L) of varied amounts (1, 2, 3, 4, 5 mL) and water (4, 3, 2, 1, 0 mL) to obtain a final volume of 10 mL. This solution was then placed in the refrigerated bath for 10 min at a lower temperature (3°C) to ensure that the solution attained the bath temperature and the API complex formed to the maximum extent. The absorbance reading was then taken at 550 nm. Each of the above concentrations of AP and I were chosen in such a way so that the AP was the limiting reagent and totally involved with the complex.

We should point out that this calibration graph can be used to determine the concentration of AP complexed for any amounts of AP and I added to the solution. Since the API complex gives an absorbance maximum at 550 nm and has a fixed composition (11 AGU for every 4 I) ,8 a particular absorbance value at 550 nm, no matter whether obtained under I or AP excess condition, will give a unique concentration of the AP complexed.

RESULTS AND DISCUSSION

Determining the IBC Value

Figure 2 represents the results of the IBC experiments a t different temperatures. As expected from the stability experiments, the lower temperature curves show larger absorbance values than the higher temperature curves. Since the API complex shows maximum stability (thus the maximum amount of complex formation) a t temperatures lower than 6”C, the IBC calculations were based on ’

the 3°C curve (uppermost curve, Fig. 2) . In this figure, one can notice an initial increase in the absorbance value (a t 550 nm) with the AP concentration (for a fixed amount of I) as an increasing amount of the API complex is formed. This is also expected from the equilibrium eq. (1) in which a higher reactant concentration is expected to shift equilibrium towards an increasing amount of prod- ucts. At the AP concentration of 0.62 g/L, the 3°C curve levels off (absorbance 0.169), suggesting that almost all the iodine present (2.38 X g/L) in the solution is bound (no free iodine) by this concentration of AP giving an IBC value of 0.38% (weight I/weight AP) . This method of determining the IBC value is comparable to those reported1’-17 in the literature in which the condition of “no free

DAVIS, SKRZYPEK, AND KHAN 2270

0.35

0.31

0.27

Y 0.23 z 2 0.19

5: -2 0.15

0.1 1

0.07

0.03

0 a 16 24 32 40

glL AP COMPLEXED X 103

Figure 3. Calibration plot (3°C) in g/L of AP complexed against absorbance values taken at 550 nm for the API complex. The amount of iodine added was in large excess for which the AP concentration was completely involved in the complex formation.

iodine" was met by extrapolation. This extrapolation of the iodine binding curve to zero free iodine may, however, introduce a significant error when the IBC value (the intercept) is quite small. This may be the reason for not having any reported IBC value thus far for the API complex. Our method, on the other hand, is expected to be more accurate as it does not involve extrapolation to a small value.

Since our limiting absorbance value of 0.169 corresponds to the AP complexed concentration of about 17.45 X lop3 g/L (from Fig. 3), most of the AP (out of total 0.62 g/L) will remain unreacted in the solution. The amount of iodine bound (2.38 X g or 0.0187 mol I / L ) , therefore, comprises about 13.64% (w/w) of the AP (17.45 X g or 0.106 rnol C6Hlo05/L) complexed. This corresponds to 4 mol of I atoms for every 23 mol of AGUs in the complex. Since our previous theoretical and exper-

imental studies' suggest that 4 mol of I atoms with 11 AGUs form a chromophore in the API complex giving an absorbance peak at 550 nm, only 48% of the total number of AGUs ( 11 out of 23) in AP molecules (which bind iodine) are involved in the complex formation. This also means that over half of the AGUs in the API complex do not take part in iodine binding. We can explain these findings based on the finer structural features of the AP mole-

which consists of external A chains (the shortest ones) and are connected to the internal and longer B or C chains. Since the average chain lengths (CL) of AP may vary depending upon the source, we presented here the results of AP (from potatoes) debranching experiments by Robin et a1.l' The results of these experiments suggest that the fine AP structures consist of linear A chains of an average degree of polymerization ( D P ) of 15, and the B and C chains of average D P of 45 and over 60 respectively. The B and C chains comprise about 55% of the internal AP structure, and the A chains repre- sent the rest (45% ) of the structure.lg We already estimated that only around 48% (11 out of 23) of the AGUs in AP can react with iodine forming the API chromophore. Thus, it is reasonable to propose that the external A chains (45%) are involved in iodine binding and the internal B and C chains ( 55% ) remain unbound. Since the polyiodine unit of four I atoms is not stable outside of the helix and requires a minimum helix length of 11 AGUs,' we cannot expect to have an API chromophore or its characteristic spectrum with fewer than 11 AGUs in the A chains.

The earlier experiments by Bailey and Whelan 22

with the amylose chains of varied D P and iodine provide support to our API chromophore composition and assertion that a close structural relationship exists between the amylose and the A chains of AP. In their experiments a faint red color was obtained

cule 1-3.17-21

Table I. for API Complex Formation at Different Temperatures (TI: [I]initial = 5.0 X 10-4M; [AP]initi,l = 8.55 X lO-'M"

Absorbance Values (Abs), Concentrations of AP Complexed (g/L, M ) , and Equilibrium Constants ( K )

AP Complexed

T (K) Abs g/L M " K 1/T (K)-' In K

283 0.325 0.0366 3.77 x 10-5 1.71 x lo3 3.53 x 10-3 7.44 288 0.300 0.0335 3.46 x 10-5 1.46 x lo3 3.47 x 10-3 7.29 293 0.274 0.0303 3.13 x 10-5 1.23 x 103 3.41 x 1 0 - ~ 7.11 298 0.251 0.0275 2.84 x 1 0 - ~ 1.05 x lo3 3.36 x 1 0 - ~ 6.96 303 0.228 0.0247 2.55 x 10-5 8.95 X lo2 3.30 x 1 0 - ~ 6.80

a In units of 2.75 mol AGU/L.


when the average D P was around 12, and there was no red color when the D P was around 9. Even though these experiments did not involve subtraction of spectrum due to other iodine complexes ( HzOIz) in the solution or peak resolution (for which the wave lengths of maximum absorbance may be off), it provides a qualitative support to our API chromophore unit (11 AGU) below which the API red complex cannot form.

Heat of Reaction for API Complex Formation

The API complex formation can be assumed to take place in the following manner:

AP + Iodine = API complex (1)

Initial: a b 0

Equilibrium: a-c b-c C

As we discussed before, each A chain in AP is quite similar to a small amylose and the API chromophore is like a small section of the A1 chromophore. Since there are 2.75 AGUs bound to each iodine atom in the A1 complex, we can expect the same for the API complexs~'' and thus the molarity of AP is expressed in terms of 2.75 AGUs for the ease of computation of equilibrium constant values ( K ) at different temperatures. The equilibrium expression can now be written as:

[ C I

[ U - C ] [ b-C] K =

The initial concentrations, a and b , of AP and iodine respectively, were obtained from the added amounts in stability experiments (Fig. 1 ) . The concentration of AP complexed, c , was obtained by first expressing each of the absorbance values a t 10, 15, 20, 25 and 30°C (Fig. 1 ) to AP complexed concentration in g/ L (using the calibration plot of Fig. 3 ) followed by a conversion in moles of 2.75 AGUs and correction for the number of AGUs bound (48% ) . As we already pointed out, within the AP molecules complexed only 48% of the AGUs ( A chains) are directly involved in iodine binding for which this correction was needed. Since each mole of the 2.75 AGUs binds 1 mole of I atoms, the equilibrium iodine concentration, i.e., the concentration of iodine not involved in the complex, was determined by subtracting the molarity involved in the complex from the initial concentration. In a similar manner the equilibrium concentration of AP was determined. In Table I, the equilibrium constant values at different temperatures are presented.

As expected, these equilibrium constant values are significantly smaller than those of the A1 complex.'' For example, at 298 K, the equilibrium constant of A1 is about 27 times larger than that of the API complex suggesting only a small amount of the API complex formation and hence a greater thermal instability of the API complex. Figure 4 shows a Vant Hoff's plot in which 1 / T (K-') is plotted against In K. From the slope value ( - M/8.314) of the straight line plot, the heat of reaction (AH) was calculated to be -23.5 kJ/mol of I atoms or -47.0 kJ/mol of 1-1 units. This BE value for the API complex is significantly lower than that of the A1 complex (-110 k J ) .ll A significantly lower formation constant ( K ) and the IBC values compared to those of the A1 complex are consistent with the low BE value for the API complex. Our proposed A1 and API chromophore structures are presented in Figures 5 (a ) and 5 (b) , respectively. The structure in Figure 5(b) is shown as a small segment of the A1 chromophore [Fig. 5(a)] . Even though the shorter helix (in API complex) is known to give a less stable complex than a longer onez3 (in A1 complex) , it is not known whether this remarkable dif- ference in the BE values are merely due to the differences in the helix CL or the structural variations in helices, from the shorter to the longer ones.

Possible Mechanism of the Al and API Complex Formation

Before concluding, it will be appropriate to put for- ward a possible mechanism of the A1 and the API complex formation. The amylose molecule in the aqueous solution probably remains as a long chain

3.30 3.35 3.40 3.45 3.50 3.55

in x 103

Figure 4. Vant Hoff's plot of 1/T (1 /K) vs. In K is presented here. The slope of the straight line is equal to - AH/8.314 J, where AH is the heat of reaction for 1 mol of I atoms bonded to 2.75 mol of AGUs in the API complex.


0

I 0 I

I \

\ I I

0

(a) (b)

Figure 5. unit along the helix axis. The inter-iodine distance in each case is around 3.0 A.

Proposed A1 and API chromophore structures: ( a ) has a I6 and ( b ) has a I4

of extended helical segments connected by random nonhelical segmentsz4 [Fig. (6a) 1. The polyiodine unit, as we already mentioned, is not stable without the helix structure around.’ During its formation, an iodine molecule presumably enters into the cavity of the extended helix [Fig. 6 ( a ) ] , followed by its polarization by the oxygen atoms, l1 which in turn, helps the other iodine molecules to align within the cavity. The interaction of a polyiodine unit with the extended helix may form a “tighter” helix [Fig. 6 ( b ) 1, thereby providing a greater stability to the polyiodine complex with the amylose or AP chains. Since the AP molecule has only shorter A chains which are capable of binding iodine, only the shorter polyiodine, 14, may be stabilized.

It is quite puzzling to point out that a particular

stoichiometry exists for each of the A1 (17 AGUs, 6 I ) and the API (11 AGUs, 4 I ) chromophores, despite the fact that the amylose CL may be con- siderably longer than 17 AGUs and the AP molecule may have A chains longer than 11 AGUs. If an I’ species is to be stabilized by an A1 type of helix, the minimum CL of 22 AGUs (2.75 AGUs per I atom4,’’) will be needed. However, our previous studies with the A1 solution did not show any evidence for such a complex formation ’’ involving the polyiodine unit of more than 6 I atoms. Similarly, in our studies with the API solution,’ we did not see any evidence for the complex formation with the polyiodine units of 6 or 8 I atoms. One possible reason may be the fact that most of the extended helix segments in the amylose molecule have CL ranging from 17-21


AGUs per helix followed by random nonhelical structures [Fig. (6a) 1. Since the helix CL shorter than 22 AGUs cannot bind Is, the complex with 22 AGUs and 8 I cannot form in any significant amount. It is also known that the longer helices are saturated prior to the shorter ones with iodine,23 and thus, a predominant complex formation will involve six I atoms rather than four I atoms within the helix of 17-21 AGUs. Even though the A1 chromophore requires six I atoms and 17 AGUs in the helix, any additional AGUs in the helix (up to 21 AGUs) will not affect the A1 spectrum in any major way, as the spectrum is almost entirely due to the polyiodine unit.8711 The longer helix, being more stable than a shorter one, may however, provide a stronger shield against the thermal dissociation of the polyiodine. Similar arguments can be made to the A chains of the AP molecule. The linear A chains have an average CL value of around 15 AGUs l9 and will predominantly bind I4 units. The longer A chains (longer than 16 AGUs) may not be available for the iodine binding if pairs of them are involved in the double helix formation. In addition, it is also possible that the longer A chains (longer than 16) may have extended helix segments of CL 11-16 AGUs and thus cannot bind 6 I atoms for an A1 type of complex formation.

Peak Shifts and Iodine Species

The foregoing analysis may also allow us to explain the observed peak shifts (visible range) from 615 nm to around 550 nm in the A1 spectrum with the extent of amylose hydrolysis? Even though the A1 complex has a definite stoichiometry ( 17 AGUs and 6 I ) , as the enzymatic hydrolysis takes place, an increasing number of the shorter amylose chains are formed with the depletion of the longer ones. While the shorter ones, with the helix CL of 11-16 AGUs, can accommodate only the I, unit giving the "API type" spectrum, the longer ones ( AGUs 17-21 ) can predominantly stabilize the I6 unit giving the A1 spectrum. Thus, with the time of amylose hydrolysis, as the concentration of the A1 complex decreases and API increases, the net spectrum due to the overlap of A1 and API spectra8,11 will also change. We examined the results of such an overlap by taking our reported spectra of A1 and API complexes.8711 When the A1 peak intensity at 615 nm was two times larger than that of the API peak (540 nm, after peak resolution it shifted to 550 nm) , the combined spectrum had a major peak at 590 nm. However, when these intensities were the same, the major peak shifted to 570 nm. Thus, the observed peak shifts

(a)

( b )

Figure 6. ( a ) Two extended helix (or loose helix) segments of amylose joined by a random nonhelical structure. Such a structure may exist in the aqueous solution in the absence of iodine. ( b ) Two tighter helix segments are shown, each of which may result from the interaction of a polyiodine unit with an extended helix. For clarity, the polyiodine unit, 16, is not shown within the helix segment of Figure 6(b) .

in our hydrolysis experiments can be explained by the overlap of A1 and API spectra of varied intensities caused by different concentrations of the A1 and API complexes in the solution.

In the same way we can also explain the peak shifts in Bailey and Whelan's (BW) experiment" with different DPs (degree of polymerization) of amylose and their complexes with iodine. In their experiments a t the average DP of around 12, the major peak was observed at 490 nm, which gradually shifted to longer wavelengths with the increase in the DP value. At the average DP of 12, we expect. only a very few amylose chains significantly longer than 12 AGUs, and hence, there may be an insig- nificant amount of the A1 complex in the solution. Thus, a t this DP, we expect primarily the API spectrum overlapping with that of the HzOIz ( WI) . We examined numerically the result of such an overlap by using the API and WI spectra. The WI spectrum was obtained from that of the hydrolyzed API (60% AP degraded') after the peak resolution for the sep- aration of the API complex contribution. When the WI peak intensity a t 360 nm was five times larger than that of the API complex at 540 nm, the major peak of the net spectrum was at 540 nm. However, when the WI intensity was 15 times larger, the major


peak in the combined spectrum shifted to 480 nm. Thus, the peak at 490 nm in the BW experiment was due to the overlap of WI and API spectra. As the DP was increased, the A1 complex concentration was increased and the API concentration decreased as longer chains were predominantly bound by the longer polyiodine units.23 This presumably caused the peak shifts towards the longer wavelengths in BW’s experiments.22

CONCLUDING COMMENTS

Each A1 and API complex has a fixed chromophore composition of 17 and 11 AGUs bound with polyiodine units of six and four I atoms, respectively. In the API complex the A chains are involved with the helix CL of 11-16 AGUs and in the A1 complex the helix segments of 17-21 AGUs are predominantly involved. The small BE value for the API complex may be solely due to the shorter helix which may be more sensitive to the thermal agitation than the longer ones (in A1 complex) or there may be a less “tight helix” [in between Figs. 6 ( a ) and 6 ( b ) 3 in the API complex which cannot stabilize the polyiodine unit that well for thermal stability. The con- firmation of our hypothesis regarding the mechanism of the A1 or API complex formation and the remarkable differences in the BE values will require further studies on amylose and AP structures and their interactions with iodine.

The authors acknowledge the assistance of J. Agosti, M. Minick, R. Itle, A. Buckvich, C. Owens, and G. Ferraccio during the progress of this research and the editorial assistance of s. Delaney. We also wish to thank D. B. Thompson and R. Yuan for giving us some of the relevant references.

REFERENCES AND NOTES

1. W. Banks and C. T. Greenwood, Starch and Its Com- pounds, Edinburgh University Press, Edinburgh, UK, 1975.

2. B. Solomon, Adv. Biochem. Eng., 10,131-77 (1978). 3. L. F. Hood, Food Carbohydrates, Symposium Papers,

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1982 pp. 217-236. J. Fonslick and A. Khan, J. Polym. Sci. Part A: Polym. Chem., 27,4161 (1989). G. Ziegast and B. Pfannemuller, Int. J. Biol. Mac- romol., 4 , 419 (1982). R. S. Higginbotham, J. Textile Inst., 40, T795-808 (1949). A. R. Archibald and I. D. Fleming, et al., J. Chem.

H. Davis and A. Khan, J. Polym. Sci. Part A: Polym. Chem., 32,2257 (1994). J. Fonslick, A. Khan, andB. Weiner, J. Phys. Chem., 93,3836 (1989). PeakFit, Peak Analysis Software, Jandel Scientific, 1991. M. Minick, K. Fotta, and A. Khan, Biopolymers, 31, 57-63 (1991). S. Lansky, M. Kooi, and T. J. Schoch, J. Am. Chem. soc., 71,4066 (1949). B. L. Larson, K. A. Gilles, and R. Jenness, Anal. Chem., 25,802 ( 1953). D. M. W. Anderson and C. T. Greenwood, J. Chem. Soc., 3016 (1955). T. Kobayashi and S. Yoshida, Bull. Agr. Chem. Soc. Jpn., 24,538-540 (1960). G. K. Atkins and C. T. Greenwood, Carbohyd. Res., 3,81,152 (1966). C. T. Greenwood, in The Carbohydrate Chemistry, W. Pigman and D. Horton, Eds., Academic, New York, 1970, Vol I1 B, p. 471. J. M. Williams, in Starch and Its Derivatives, J. A. Radley, Ed., 4th ed., Chapman and Hall, London, 1968. J. P. Robin, C. Mercier, R. Charbonniere, and A. Guilbot, Cereal Chem., 51 , 389 (1974). E. B. Sanders, D. B. Thompson, and C. D. Boyer, Cereal Chem., 67,594 (1990). S. Hizukuri and Y. Maehara, ACS Symp. Ser. 458 , 212 (1991). J. M. Bailey and W. J. Whelan, J. Biol. Chem., 2 3 6 , 969 ( 1961 ).

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24. J. Szejtli, ACS Symp. Ser., 458, 1-10 (1991).

Received August 13, 1993 Accepted March 14, 1994