Determining the chromophore in the amylopectin–iodine complex by theoretical and experimental studies

Determining the Chromophore in the Amylopectin-Iodine Complex by Theoretical and Experimental Studies

HEATHER DAVIS and ARSHAD KHAN*

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

SYNOPSIS

A partial hydrolysis of amylose followed by the addition of iodine provides a spectrum almost identical to that of the amylopectin-iodine ( API) complex suggesting the involvement of smaller "amylose-like" units in the API complex. Our theoretical studies on different polyiodine and polyiodide species suggest that a nearly linear 1, unit stabilized within the cavity of a small "amylose-like" helix is responsible for the characteristic API spectrum. Since there are 2.75 anhydroglucose residues ( AGU) for every iodine atom in the amylose- iodine ( AI) complex and a structural similarity exists between the API and the A1 (amylose- iodine) complexes, we identify (C6H1005)ll14 to be the chromophore in the API complex. 0 1994 John Wiley & Sons, Inc. Keywords: helix in amylopectin-iodine complex I4 unit in amylopectin-iodine complex

spectra of amylopectin-iodine complex amylopectin-iodine chromophore composition amylopectin fine structures and complex formation

I NTRO DU CTI 0 N

Starch consists primarily of amylopectin ( A P ) , amylose, and other intermediate While the amylose forms a blue complex with iodine, the AP gives a red complex. Even though the amylose- iodine (AI) complex (commonly known as the starch-iodine complex) was extensively studied, 1-5

very little work was done on the API ~ o m p l e x , ~ , ~ which, like the A1 complex, is known for antimicro- bial properties? While amylose is primarily a linear glucose polymer (a 1-4 linkage) with a limited amount of branching (a 1-6 linkage), the AP is primarily a branched polymer (94-96% a 1-4 and 4- 6% a 1-6 linkages) with an average chain length of 20-26 A G U S . ~ , ~ The earlier X-ray diffraction study of model compounds by Saengerg and our direct experiment4 with the A1 complex allowed us to es- tablish the inter-iodine bond distance in the A1 complex to be around 3.0 A ".

Based on our studies" as well as studies by others," we identified that the A1 chromophore has 6 iodine atoms (inter-iodine distance 3.0) lying inside the cavity of amylose helix of about 17 AGUs. The

* To whom all correspondence should he addressed. Journal of Polymer Science: Part A Polymer Chemistry, Vol. 32,2257-2265 (1994) 0 1994 John Wiley & Sons, Inc. CCC 0887-624X/94/122257-09

helix-polyiodine interaction presumably increases the 1-1 distance from around 2.7 A" in Iz molecule to around 3.0 A " in the A1 complex. This increase in inter-iodine distance is also noticed in other iodine complexes with 0 and H containing compounds like water, alcohols, and acetone.l1*l2 In addition, we observed that almost all the spectral peaks appeared from electron transitions within the iodine units with almost no involvement of 0 and H atoms. These results on spectra were not unexpected, as iodine has weakly bound d electrons and can be readily excited to different excited states. Hence, by studying the spectra of polyiodine units with elon- gated bond distances, one can quite accurately predict the spectrum and chromophore structure in the complex.

In our theoretical studies for the spectrum of the A1 complex, we used polyiodine units with experimental inter-iodine distance and, hence, in an in- direct manner incorporated the effect of helix-polyiodine interaction. The linear I6 structure was found to be dominant a t lower temperatures with nonlinear geometries increasing in proportion as the temperature was increased. In the same article we pointed out several experimental (NMR, UV-VIS studies) and theoretical results, which suggest that the polyiodide ions, like I,, I,, I;, etc., which may form in the solution in the presence of I- ions, cannot get

2257

2258 DAVIS AND KHAN

involved with the A1 helix and, hence, I- ions in solutions merely help in dissolving iodine in water.

The identification of the chromophore unit in the API complex was a relatively complicated problem, as the AP molecule is a highly branched polymer and no X-ray diffraction studies were reported on the API structure. In the first part of this article we unambiguously determined the experimental spectrum of the API complex (especially in the visible range) by subtracting the background due to the uncomplexed Iz as well as its complexes with 0 atoms of water (HzOI,) and some of the AGUs (in AP) in the solution followed by an accurate peak resolution. Then we determined a structural similarity between the A1 and the API complexes by hydrolyzing the amylose molecule for different time intervals followed by the addition of the iodine solution, and comparing the spectrum of the resulting solution with that of the API complex. This study indicates that the API complex has smaller “amylose-like” units and is consistent with the postulate of Higginbotham,‘ who first suggested a smaller “amylose-like” helix formation in the API complex. Since the average inter-iodine distance in the helix of amylose is well characterized (around 3.0 A O ) , we examined several average distances, especially in the neighborhood of 3.0 A O , by calculating spectra of different species, and obtained the best agreement between the theory and experiment for the inter- iodine distance of around 3.0 A ” for I4 polyiodine unit in the API complex.

EXPERIMENTAL

Preparation of Solutions

A P Solution

The AP solution was made by dissolving 0.100 g of solid (94% AP content and 6% water, Sigma Chem- ical, A 8515) in 490 mL of water and 10 mL of pH 4 (acetate buffer, Fisher Scientific) solution. This yields AP concentration of about 188 mg/L (1.2 X lop3 M in terms of C6H1005, the glucose concentration). Proper dilution of this solution was used for experimental spectra of the API complex at different temperatures.

Iodine Solution

The iodine solution of concentration 126.9 mg/L (1.0 X M I) was made by dilution from a solution containing the same volume of concentrated iodine solution (0.10 M I, S186 Fisher Scientific)

and potassium iodide solution (0.30 My SP241, Fisher Scientific) and was placed in an ice bath at around 6°C to prevent evaporation of iodine. More- over, the addition of KI increases the solubility of iodine and minimizes evaporation of it.

Amylose Solution

The amylose solid (Sigma Chemical A 0512) of 0.20 g was dissolved in 50 mL of 2.0 M NaOH and then neutralized with 50 mL of 2.0 M HC1 solution. The resulting solution was then heated to evaporate 10- 20 mL of water, and pH 4 solution was added to obtain 100 mL of total volume. This solution was diluted to amylose concentration of 195 mg/L after taking into account its p ~ r i t y . ~

a-Amylase Solution

The enzyme solution was made by dissolving 0.15 g of solid (Sigma A 2771 ) in 255 mL of water. To stabilize this solution 0.50 g of calcium chloride was added. This enzyme is known for the fast hydrolysis of inter-glucose bonds (a-D 1-4 linkage) in amylose and AP, except when the AGUs are involved in branching.

Experimental Spectra of API Solutions

The absorbance readings for the spectra of API solutions were taken at wavelengths ranging from 195 nm to 700 nm by using a spectronic 601 spectro- photometer with suprasil quartz cuvettes (Fisher Scientific). The uv range of the spectrum (195-360 nm) of the API solution required a fairly dilute solution in order to have absorbance readings within the detection range of our equipment. Each solution for the API spectrum was made by adding 5 mL of the AP solution to 5 mL of the iodine followed by its dilution by 50 mL of water. The resulting solution was then placed in a constant temperature bath (Fisher Scientific, Model 9000) for a desired temperature (3, 15, or 25OC) before measuring its absorbance values at different wavelengths. The spectra a t 3, 15, and 25”C, thus obtained, are shown in Figures 1 and 2, as the second, third, and fourth curves from the bottom.

In a second set of experiments, the enzymatic hydrolysis of AP was carried out for a fixed amount of time before adding an iodine solution into it. The purpose was to compare the spectrum of the degraded AP-iodine with that of the original AP-iodine solution (when no hydrolysis was done) and, hence, to identify the spectral peaks due to the API complex. In each of the AP hydrolysis experiments in

CHROMOPHORE IN T H E AMYLOPECTIN-IODINE COMPLEX 2259

W u z 4 m K 0 111

4 m

Z5.C API

1 S C API

J'C API

HYDROLYZED API

1 9 0 2 9 2 3 9 4 4 9 6 5 9 8 7 0 0 W A V E L E N G T H < N M >

Figure 1. The lowermost curve represents data taken at 15°C after enzymatic hydrolysis of AP by a-amylase followed by the addition of iodine. The next three curves represent spectra of API solutions at 3,15, and 25°C (uppermost). The spectra are separated from each other for the clarity of viewing.

the visible as well as the uv range, 5 mL of the AP solution was allowed to react with 10 mL of the a- amylase solution (enzyme) for about 20 minutes at 25°C followed by the addition of 5 mL of iodine and 40 mL of water. The resulting solution was then placed in a constant temperature bath for a desired temperature before its absorbance reading was taken at different wavelengths. These spectra a t different temperatures were identical; thus, one representa- tive hydrolysis spectrum at 15OC is presented as the bottom curve in Figures 1 and 2.

W 0 Z 4 m K 0 111

4 m

4 0 0 4 6 0 5 2 0 5 8 0 6 4 0 7 0 0

W A V E L E N G T H < N M >

Figure 2. For clarity, the visible range of the spectra of Figure 1 are shown here with proper magnification. Both the experimental data points and the spline fittings are shown. The lowermost curve, as in Figure 1, is obtained after the enzymatic hydrolysis of AP, and the next three represent the spectra of API solutions a t 3, 15, and 25°C.

In separate experiments we also established that the enzymatic hydrolysis reaction of AP or amylose is stopped after the addition of an iodine solution. We carried out each of these experiments by taking the absorbance readings (at characteristic wavelengths of API or A1 complex) of the iodine containing enzyme-AP or enzyme-amylose solution at different time intervals (after the addition of iodine) and observed almost no change in absorbance values. It should be pointed out that the hydrolysis reaction would have decreased the concentration of AP or amylose and, hence, the API or A1 complex lowering the intensity of its spectrum with time.

Figure 1 represents the spectra of API solutions ranging from 195-700 nm. Figure 2 represents the visible range of the Figure 1 spectra magnified for better viewing. While the uv range of the spectra ( 195-360 nm, Fig. 1 ) does not show any significant change with temperature or enzymatic hydrolysis, the visible range (Fig. 2 ) displays a remarkable decrease in absorbance as the temperature is raised or the hydrolysis of AP is performed. The 3°C visible spectrum (the second curve from the bottom of Fig- ure 2 ) shows a number of structures, each of which becomes broader with reduced intensity as the temperature is increased from 3°C to 25OC (uppermost curve). At 25"C, the visible API spectrum quite closely resembles that of the hydrolysis (bottom curve) reaction with a significantly reduced intensity, and, hence, suggests only a small API complex concentration at this temperature. The absorbance

w 0 Z 4 m K 0 111

6 m

J 4 0 5 4 6 4 5 2 3 5 8 2 6 4 1 7 0 0

WAVELENGTH CNM>

Figure 3. The first, second, and third curves (from the bottom) are obtained by the enzymatic hydrolysis of amylose for 2,4, and 6 min followed by the addition of iodine, and the uppermost (fourth) curve is due to the API complex obtained after subtracting its background. A remarkable similarity between the third curve (6 min hydrolysis) and that of the API complex (fourth curve) suggests a structural similarity between the partly hydrolyzed A1 and the API chromophores.

2260 DAVIS AND KHAN

I 5w M30 WAMLO\IGRI (NM)

?igure 4. The resolution of the visible region of the !xperimental API spectrum at 3OC is shown with com- Ionent peaks (broken lines) together with an overall fit

(solid line) to the experimental points. The component peaks appear at 412,458,550, and 640 nm having relative intensities of 0.3, 1, 12, and 2, respectively. It should be pointed out that the peak intensity at 550 nm is about f the intensity at the 350 nm peak.

curve of the hydrolysis reaction (60% AP degraded) provides the background, the major part of which is due to the shoulder band of the 350 nm peak (Fig. 1) given by H201z (WI) complexes or similar Iz complexes with other oxygen containing molecules present in the solution" (some of the AGUs may also be involved). Since we have a large excess of water in AP solutions, the WI complex will predom- inate. This hydrolyzed spectrum of Figure 2 (bottom curve) is subtracted from the 3°C curve (second from the bottom, Fig. 2) to obtain the uppermost curve of Figure 3 and, hence, represents the visible spectrum due to the API complex. The resolution of this API spectrum is done by using the PeakFit program13 and is shown in Figure 4 with the component peaks (Gaussian functions) at around 412, 458, 550, and 640 nm, having relative intensities of 0.3,1,12, and 2, respectively. The component peaks are shown with broken lines, and the over-all fit to the experimental points are shown with a solid curve.

As we already pointed out, the assignment of the uv spectral peaks to the API complex was somewhat less straightforward, as neither the hydrolysis reaction nor the increased temperature made any significant change in the uv spectrum for which a background subtraction was not possible. We, therefore, analyzed the spectra of both the hydrolyzed and unhydrolyzed AP-iodine solution in the

uv range and compared their peak intensities. The peak res~lut ion '~ from 150-400 nm of the 3°C API curve (before the hydrolysis reaction) yields maxima at around 197, 213, 228, 289, and 350 nm with relative intensities of 9, 4, 8, 1, and 0.5, respectively, and is presented in Figure 5 (a ) . The resolution of the hydrolyzed curve, presented in Figure 5 (b) , gave almost the same component peak positions and their intensities except a t 228 nm, the intensity of which is decreased to around 7. Most of these component peaks were obtained by using Voigt spectroscopic functions, except the last two (289 and 350 nm), which were obtained by using Gaussian functions. Different types of spectroscopic functions were cho- sen in order to obtain the best possible fit with the experimental data. The peak intensities in the visible range, as pointed out earlier, are significantly lower than those in the uv range and at 550 nm, for ex- ample, it is about f of that of the 350 nm peak. We should point out that these absorbance values, obtained by using the spectronic 601 spectrophotom- eter, are quite accurate. The maximum absorbance value of 2.95 that we measured at 225 nm had an error of f0.059, and the smallest value of 0.030 at 700 nm had an error of f0.003. The ratio of the peak intensity a t 289 and at 350 nm is around 2 : 1, the characteristic ratio for the WI complexes1' in the solution. These complexes are also known to absorb strongly at around 200 nm, for which we assigned 197, 289, and 350 nm peaks to WI complexes. In addition, the iodine molecule in the gas phase ab- sorbs at around 215, 246, and 270 nm14 and their absorption peaks in the aqueous solution may be shifted close to the experimental peaks at 213, 228, and 289 nm. The broad absorbance peak at around 289 nm may, therefore, be due to WI complexes as well as uncomplexed iodine molecules overlapping quite close to each other. Since the ratio of peak intensities a t 289 and at 350 nm is around 2 : 1, we can postulate that the peak intensity a t 289 nm is primarily due to the WI complexes with water.

The above analysis allows us to explain why the temperature increase or enzymatic hydrolysis does not show any significant effect on the uv range of the API spectrum. If there are absorbance peaks (in uv range) due to the API complex overlapping with those of the WI complex, a decrease in absorbance due to hydrolysis or heating (which decreases the API complex concentration) is more likely compen- sated by an increased concentration of I2 or WI complex resulting from the dissociated API complex. As we mentioned later, the theoretical calculations do suggest a weak API peak at around 212 nm and

CHROMOPHORE IN THE AMYLOPECTIN-IODINE COMPLEX 2261

a strong peak at around 231 nm. A small decrease in absorbance at 228 nm (close to theoretical 231 nm peak) due to hydrolysis provides some support to our theoretical result. As already pointed out, only a small decrease, rather than a large one, at this wavelength is due to an increased iodine concentration, which presumably compensates for much of the decrease in absorbance.

150 203 250 3M) 350 4M) W A M LENGTH (NM)

(b)

Figure 5. Figures 5 (a ) and 5(b) represent the peak resolution of the uv region of the API spectrum before and after the hydrolysis of AP, respectively. The component peaks are shown with the broken lines and the symbols represent the experimental points. In 5 ( a ) , the component peaks were obtained at around 197,213, 228, 289, and 350 nm with relative intensities of around 9, 4, 8, 1, and 0.5, respectively. The peak positions and their relative intensities in 5 (b) were about the same as those in 5 (a ) , except, at around 228 nm, the intensity was decreased to around 7.

Structural Similarity between Al and API Chromophores

The structural similarity was established by hydrolyzing amylose for certain periods of time before adding the iodine solution and comparing the spectrum of the resulting solution with that of the API complex. In each experiment 5 mL of the enzyme solution was allowed to hydrolyze a solution of 5 mL of amylose (195 mg/L) mixed with 5 mL of water for 2, 4, and 6 min at 25°C followed by the addition of 5 mL of iodine solution (126.9 mg/L). The resulting solution was then placed in a constant temperature bath so that the solution attained 15OC before measuring its absorbance values a t different wavelengths. Since the A1 complex shows maximum thermal stability4 at 15°C or lower, the cooling was necessary before taking the absorbance readings. The bottom curve in Figure 3 represents the result of 2 min of hydrolysis reaction followed by those of 4 and 6 min. The shifting of the peak positions of the A1 solution from those of the A1 complex” (similar to the 2 min curve, bottom curve in Fig. 3 ) to API complex (6 min curve, third from the bottom) is quite obvious in this figure. These hydrolysis re- actions (from 0-6 min) are expected to give an increasing number of smaller amylose fragments in the solution to form complexes with iodine. Since the 6 min curve represents a spectrum of 90% degraded A1 chromophore ( 17 AGUs and 6 I atoms) lo

and is remarkably similar to that of the API complex (uppermost curve, Fig. 3 ) , we can suggest that the API chromophore will involve a shorter “amylose helix” (fewer than 17 AGUs) and enclose fewer than 6 iodine atoms. The percentage of the amylose (chromophore unit) degraded was calculated from the absorbance values of the amylose-iodine solution at 615 nm before and after the hydrolysis reaction. Because of the structural similarity between the A1 and the API chromophores, we can expect an inter- iodine distance of around 3 A O (average distance for the A1 ~ o m p l e x ~ ’ ~ ) in the API complex. This hy- pothesis about the API chromophore structure gains support from the following theoretical studies.

THEORETICAL SPECTRA

As mentioned earlier, our previous theoretical and experimental studies suggested the existence of a polyiodine 16 unit, rather than one of the polyiodide ions like I,, I;, I;, etc. inside the amylose helix of the A1 complex and is responsible for the charac-

2262 DAVIS AND KHAN

teristic A1 absorbance spectrum.4v'0 Since the present experimental studies indicate a remarkable structural similarity between the A1 and the API complexes, we feel that the mechanism of the API complex formation will be similar to that of the A1 complex and may involve polyiodine units enclosed by a helix. The theoretical results given below further support this expectation.

The spectra calculations were done by applying the INDO CI (intermediate neglect of differential overlap configuration interaction) method in which the basis functions included s, p, and d orbitals on each iodine and had around 200 configurations obtained by single excitations from the occupied ground state to different excited states. The reli- ability of this method in predicting the correct geometry and spectra as well as more detailed discus- sions of the basis functions can be found else- where.l0-l2.l5 All these calculations were done by using the ZINDO series of programs developed by Zerner and co-workers.16

We already reported theoretical spectra lo for several linear polyiodine and polyiodide species with the 1-1 distances of 3.0 A ". Among them, the linear I, unit [Fig. 6 ( a ) 3 , giving the absorbance peaks at 212, 231 (strong), and 598 nm, showed the closest resemblance to the API experimental spectrum. Even though a number of experimental peaks were not predicted by this structure, the striking spectral similarity encouraged us to undertake the following calculations on different geometries of I, for a better agreement with the experiment and, hence, to correctly identify the predominant polyiodine structure in the complex.

We first tried different 1-1-1 angles in I4 and some of these structures are shown in Figures 6 ( a ) - ( d ) . Figures 6 (b) , (c ) , and ( d ) represent cis, trans, and step-like geometries (planar) in which different I- 1-1 angles were used for theoretical spectra calcu- lation. In each of the geometries tested, the strongest peak was predicted to be at 231 nm. The existence of the other peaks, as well as their intensities, varied with the values of the 1-1-1 angle. When the angles were set a t 179") the cis-geometry predicted the absorbance maxima at 212,411,455,598, and 639 nm with relative intensities of 4, 0.4, 0.8, 12, and 0.2. When the angle was 178.5", the relative intensities changed to 4,0.4,1,12, and 0.4, and, when the angle was 178" or 175") the 411 nm line disappeared and a new peak (weak) developed at 198 nm. For the cis 178" structure, the relative intensities at 198, 212, 411, 455, 597, and 638 nm were 1, 4, 0, 4, 12, and 1.2, and, for 175" structure, the relative intensities

were 8,4 ,0 , 24, 12, and 8. As mentioned before, the resolution of the visible range of experimental API spectrum (at 3°C) gave absorbance maxima at 412, 458,550, and 640 nm with relative intensities of 0.3, 1,12, and 2, and are explained quite well by a nearly linear I4 structure (slightly bent symmetrically) , yieldingpeak positions at 411,455,598, and 639 nm. The trans and step geometries [Figs. 6 (c ) and (d) ] predicted the same peak positions with the strongest one at 231 nm and relatively weaker ones at 212, 412, and 597 nm. For the trans geometry, for ex- ample, when the bond angle was 175") the relative intensities at 212,412, and 597 were 24, 24, and 12, and for the step-geometry at 175" angle the intensities were 3, 3, and 12; moreover, they did not give all the experimental peak positions or intensities.

In order to see whether a change in 1-1 distance in I4 from 3.0 A" explains the experimental spectrum, we examined calculated spectra for a shorter 1-1 distance (2.9 A " ) as well as a longer (3.1 A" ) one and found that the 3.0 A' provided the best result. These test results indicate that the shifting of some of the peak positions together with the ap- pearance or disappearance of others take place when the inter-iodine distances change. The peak intensities, however, change remarkably with the varia- tion of the angle and have already been discussed. When the inter-iodine distance in I4 was set at 2.90 A" and symmetrically bent [like Fig. 6 ( b ) ] to an angle of 179" or 178", the spectral peaks in the visible range were obtained at 420, 554 (strong), and 601 (weak) nm; the inter-iodine distance of 3.10 A ", on the other hand, gave peak positions at 452 (weak), 495,650 (strong) , and 684 (weak) nm. None of these I, structures could, however, explain all the experimental peaks.

Even though most of the I, structures discussed here, with varied shapes [Figs. 6 ( a ) - ( d ) ] , angles, and inter-iodine distances, could not predict all the experimental peak positions and intensities to justify their predominant involvement in the API complex formation, it seems likely, however, that a small number of these species may exist in the complex (because of thermal agitation) and can account for the significant broadening of the experimental spectrum. Based on all these calculations, we can conclude that a t 3°C a slightly nonlinear symmetrically bent I4 structure [like 6(b) ] with an inter-iodine distance of around 3.0 A" is predominantly involved in the API complex, giving absorbance maxima at 212, 231 (very strong) , 411,455, 598, and 639 nm. Since these peak positions and their intensities in the visible range match quite well with those of the

CHROMOPHORE IN THE AMYLOPECTIN-IODINE COMPLEX 2263

I /I

I \

1

I

I /I

I /I

I

I

I \

I

I

I

I

Figure 6. Different I4 units, linear (a ) as well as some of the nonlinear geometries (b-d) , used in theoretical calculations are shown.

experiments a t 412, 458, 550, and 640 nm, we can conclude that the peak positions and their intensities in the uv range are also correctly predicted by this polyiodine structure. Hence, we can say that the uv experimental peaks at 213 and 228 nm, being fairly close to the theoretical peaks at 212 and 231 nm, might have resulted from the close overlapping of the peaks due to both the API complex and I2 not involved in the complex formation. As already pointed out, a decrease in the 228 nm peak intensity after the hydrolysis reaction may also suggest its possible involvement with the API complex. Since the peak intensity a t 213 nm remains almost un- changed even after hydrolysis or heating, we can suggest that the intensity due to the API complex may be quite small a t this wavelength (as theory predicts) compared to that due to 12, for which a small decrease in the intensity is readily compen- sated by an increased iodine concentration resulting from the hydrolysis. We should also point out that a small amount of discrepancy exists between the theoretical and the experimental peak positions and their intensities and presumably results from the solute-solvent interactions, which were ignored in these calculations. The spectra due to polyiodide ions (like I,, I;, I;, etc.) have already been reported" and do not match with the experimental spectrum of the API complex and, hence, suggest that these species are not involved in this complex formation, as was also expected from the structural similarity between API and A1 chromophores.

It is interesting to point out that the observed API spectrum is not due to an isolated I4 unit in the solution. We established that fact by first optimizing its geometry by INDO SCF UHF (self consistent

field unrestricted Hartree-Fock) method and then calculating its spectrum by the INDO CI method. The optimized structure has a linear geometry having the central 1-1 length of 3.4 A " and each of the other two of 2.9 A O with a fairly small binding energy (5 Kcal/mol) relative to separated I2 molecules. The predicted spectral peaks for this structure appear at around 197, 200 (most intense), 349 (very weak), and 537 nm and do not explain most of the experimental peak positions. A small binding energy value together with the poorly matched spectrum casts doubt about the stability of such a structure in the solution, especially when the solvent molecules can readily break such a weak bond between two I2 molecules. In addition, we already know that the water molecules in the solvent can form only the binary complexes l1 with the iodine molecule ( H2012) and, hence, the stabilization of the polyiodine unit, 14, will require a helix structure, which may stabilize the polyiodine unit and can prevent the solvent molecules from disrupting its bond. This finding supports the postulate of Higginbotham' regarding a helix formation in the API complex. The actual variations of the inter-iodine distances in the API helix structure can be obtained theoretically by optimizing the geometry of polyiodine in the presence of the helix structure, which involves quite a large number of atoms and electrons and, hence, becomes a very time-consuming computation (computer time). As in A1 chromophore study, we tried to mimic the helix-polyiodine interaction effect by as- suming an average inter-iodine distance close to that observed experimentally in the A1 helix ( 3 A O ) and made slight changes in inter-iodine lengths as well as angles for the best theoretical fit to the experimental spectrum. Since our previous studies on iodine complexes suggest that the spectra are almost solely due to the iodine units with the complex, our comparison of the calculated spectra of polyiodine units with those of experimental data are expected to provide a reliable chromophore unit for the API complex. A good agreement between our calculated and experimental API spectrum suggests that nearly linear I4 units with an average inter- iodine distance of around 3.0 A " are primarily responsible for the characteristic API absorbance peaks. We should point out that almost all the spectral peaks are due to electronic transitions to the same excited state, the LUMO (lowest unoccupied molecular orbital) from different filled MOs, except the 212 nm peak, in which the SLUM0 (second lowest unoccupied molecular orbital) is involved. Since the first few excited states are reliably pre-

2264 DAVIS AND KHAN

dicted by this INDO CI method,l2 its predicted spectrum can be considered to be quite reliable.

API CHROMOPHORE COMPOSITION

Because of the structural similarity between the API and the A1 complex, we can assume that there are 2.75 AGUs bound to every iodine atom in the API complex? Since four iodine atoms with inter-iodine distances of 3.0 A O are involved in the complex, the total number of AGUs involved, and, hence, the chromophore in the complex can be represented by (C6H1005)1114 ( 11 AGUs and 4 I) . This result suggests that if the linear AP chain has fewer than 11 AGUs, the I4 unit cannot be stabilized and, hence, there will be no API complex formation with its red color. Indeed, the previous experimental results by Bailey and Whelan17 on different chain lengths (CL) of amylose and their complexes with iodine indicate that the degree of polymerization (DP) of 12 gives a faint red color, which disappears a t a smaller DP value of around 9. Even though there were no reported results at DP of 10 or 11 to directly compare with our results, these DP values l7 are close enough to suggest that the minimum CL for the API complex is around 11 AGUs. We believe that the faint red color a t amylose DP of 12 is due to the “API type” complex formation. We should caution that in these earlier experiments, no background correc- tion (due to WI complexes) or peak resolution was done and, hence, the reported wavelengths of maximum absorbance may not be that accurate, especially when the absorbance values are low.

AP FINE STRUCTURES AND API COMPLEX FORMATION

In order to identify the type of AP fine structure involved in iodine binding, it is necessary to discuss the AP structural features. Several models have been proposed so far for the AP structure.” Among them the cluster model has been widely a~cepted.l~-’~ Ac- cording to this model, the exterior A chains in the AP cluster link to other chains only by their reducing residues; B chains link to another B or C chains and may carry one or more A or B chains as branches; and the C chain carries the sole reducing group in the molecule and is often regarded as a B chain. The concept of A, B, and C chains was first introduced by Peat et al.2zsz3 and has been extensively used in describing the fine structures of AP. Even though

Robin et a1.” defined A and B chains in a different sense, their experimental results on the debranching of AP indicate clusters of highly ordered chains with an average DP of 15 AGUs (comparable to A chains). These clusters are linked to each other by much longer chains (average DP of 45 AGUs), which correspond to B chains. A further refinement of the cluster model was proposed by Hizukuriz4 in which the B chains are classified as B1, B2, B3, etc., depending upon the number of clusters (1 ,2,3, etc.) they contain. While the average CL of the A chains range from 12-16 AGUs, the fractions B1, B2, and B3 range from 20-24,42-48, and 69-75, respectively, in four different APs examined. From these structural features of AP, we can postulate that the API complex formation (requiring 11 AGUs and 4 I atoms) will involve primarily the A chains. If the B chains are involved, one will expect formation of a number of longer helix structures with CL of 17 or more AGUs per helix. Each such helix can enclose 6 I atoms inside and form an “A1 type” of blue complex.” Since the longer helices are saturated with iodine before the shorter ones,25 we can expect a significant amount of the “A1 type” complex formation involving the B chains. Since in our experiments with the API solution we did not see any absorbance peaks for the A1 complex, we can say that the A chains with DP values of less than 17 and more than 10 are involved in the API complex formation. We should point out that the average CL of around 15 AGUs suggests that some of the chains may be considerably longer than the average value. Since we did not see any evidence of iodine binding by long chains of AP (with CL of 17 AGUs or more), we can postulate that pairs of longer A chains in AP may form double helicesz6 and may not be available for iodine binding.

It is intriguing to discover that a definite API chromophore composition of 11 AGUs and 4 I atoms explains all the spectral features of the API complex in spite of the fact that the A chains in the AP molecule has varied chain lengths.3~‘8-20~z7-z9 A s we already pointed out in the introduction, the spectra of iodine complexes are solely due to the iodine species involved, hence, the I6 and I4 are, respectively, the polyiodine units responsible for the characteristic spectrum of the A1 and API complexes. Since these longer polyiodine species are not stable in the solution, a helix structure is needed to contain these species and to stabilize them. While in the A1 complex a minimum of 17 AGUs are needed, in the API complex 11 AGUs are needed to contain all the iodine atoms in I6 and 14, respectively. Thus, the CL

CHROMOPHORE IN T H E AMYLOPECTIN-IODINE COMPLEX 2265

with D P of 11-16 AGUs will give the API complex in which at least 11 AGUs need to form a helix. Any extra AGUs (beyond 11) in a longer helix will still contain the I4 unit giving the same API spectrum. From our previous studies lo we know that the polyiodide ions like I,, 1, cannot be involved in the complex formation; hence, in between D P of 11-16, we do not expect to see any other iodine species complexed other than 14. However, as the D P increases to 17 AGUs or more, I6 can be enclosed, giving its characteristic A1 spectrum. This may be the reason for an increased iodine staining power for the debranched AP molecules30 in which some of the longer B chains are available for the A1 type of complex formation.

All the computations were performed by using an IBM ES/3090-6003 computer a t the facilities of the Pennsyl- vania State University Center for Academic Computing. We also acknowledge the assistance of W. Skrzypek, M. Pasqualle, J. Agosti, D. Shaffer, C. Owens during the com- pletion of this work, and editorial assistance of S. Delaney.

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Received April 2, 1993 Accepted February 28, 1994

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Determining the chromophore in the amylopectin–iodine complex by theoretical and experimental studies