Enzymatic Degradation of Îº-Carrageenan in ... - ACS Publications

Biomacromolecules 2009, 10, 1757–1767

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Enzymatic Degradation of K-Carrageenan in Aqueous Solution Pi Nyvall Colleń,† Maud Lemoine,† Richard Daniellou,‡ Jean-Paul Gue´gan,‡ Sergio Paoletti,§ and William Helbert*,† Universite´ Pierre et Marie Curie, Paris VI, CNRS, Marine Plants and Biomolecules, UMR 7139, Station Biologique, BP 74, F29680 Roscoff Cedex, France, Ecole Nationale Supe´rieure de Chimie de Rennes, CNRS, UMR 6226, CS 50837, Avenue du General Leclerc, F-35708 Rennes Cedex 7, France, and Department of Life Sciences, University of Trieste, Via Giorgieri 1, I-34127 Trieste, Italy Received February 9, 2009; Revised Manuscript Received April 27, 2009

Enzymatic degradation of standard κ-carrageenan and the low-gelling hybrid κ-/µ-carrageenan were conducted using recombinant Pseudoalteromonas carrageenoVora κ-carrageenase. The initial velocity of the enzyme was determined as a function of varying Tris or NaI concentrations and at constant 200 mM cosolutes concentration, adjusting NaI and Tris concentrations accordingly. In both cases, we observed strong inhibition of the enzyme with increasing amounts of iodide. The characterization of the κ- and κ-/µ-carrageenan ordering by optical rotation and the visualization of iodide binding on carrageenan by 127I NMR revealed that inhibition was not caused by the disordered-ordered transition of carrageenan in NaI, but by iodide binding. These results were confirmed by analysis of the degradation products by gel permeation chromatography. Degradation of carrageenan in the disordered state led to a rapid decrease in molecular mass and the production of all possible neo-κ-carrabiose oligomers. In the ordered conformation, the degradation kinetics, the decrease of average molecular weight, and the chain population distribution of degradation products varied with iodide concentration. These observations were interpreted to be the result of increasing amounts of bound iodide on carrageenan helices that, in turn, impede enzyme catalysis. Based on these results, we propose a single-helix ordered conformation state for κ-carrageenan and reject the previously advocated double-helix model.

Introduction Carrageenans are linear sulfated galactans extracted from many species of red seaweeds (Rhodophyta) and share a common backbone of D-galactose with alternating R(1f3) and β(1f4) linkages.1,2 Carrageenans are classified according to the number and the position of sulfate ester groups and by the presence or absence of a 3,6-anhydro bridge on the 1,4-linked unit.3,4 For example, the idealized structure of κ-carrageenan is a disaccharide of 1,3-linked β-D-galactopyranose-4-sulfate and 1,4-linked 3,6-anhydro-R-D-galactose. ι-Carrageenan has a very similar structure, except for the presence of an additional ester sulfate group on position 2 of the anhydro-galactose moiety (Figure 1). These two carrageenans are well-known for their ability to form iono- and thermoreversible gels and are extensively used in the food industry.5-7 Gel formation of κ-carrageenan has been the subject of many investigations and it is now fully accepted that a disorder-order transition of the macromolecular conformation is a prerequisite for gelation. However, the precise mechanism of gelation is still a matter of debate (see, for example, refs 8-10). From the first X-ray diffraction study of semicrystalline κ-carrageenan fibers that suggested that carrageenan single helices were packed side-byside11 to subsequent investigations that assumed an intertwined double-helix model,12,13 the number of strands involved the fundamental helical conformation of κ-carrageenan in the fiber is a long-standing issue. κ-Carrageenan is a polyelectrolytic polysaccharide that undergoes transitions between three basic * To whom correspondence should be addressed. Tel.: + 33 298 292 324. Fax: +33 298 292 324. E-mail: [email protected]. † Universite´ Pierre et Marie Curie. ‡ Ecole Nationale Supe´rieure de Chimie de Rennes. § University of Trieste.

Figure 1. Structure of the repetition moiety of standard κ-carrageenan. The low-gelling κ-/µ-carrageenan is a hybrid structure made of κ- and µ-carrabiose moieties. µ-Carrageenan is usually converted into κ-carrageenan by hot alkaline treatment.

conformations: disordered (dis), fundamental ordered (hel; either a single helix or a coaxial double helix, depending on the chosen model), and a side-by-side dimer (dim) of fundamental helices. The dimer represents the elementary conformational element of the gel junction, but it is also present in the initial steps of association of polysaccharide chains. As κ-carrageenan is a polyelectrolyte, the three conformations will have different values of linear charge density and different electrostatic contributions to their relative thermodynamic stabilities. The latter can be visualized in the so-called “conformational phase diagram”, as a function of important physical variables, namely, temperature (T) and ionic strength (I).14,15 Inspection of the conformational phase diagram of κ-carrageenan reveals that the (I, T) domains of existence of the hel and dim conformations are very close and largely overlap. Moreover, both conformations have the same value of optical activity because their structural differences are only due to the lateral association of the helices. Conformational and association properties of κ-carrageenan chains are strongly dependent on the nature of the salts and the ionic strength of the medium.16-20 Cations such as K+, Cs+,

10.1021/bm9001766 CCC: $40.75  2009 American Chemical Society Published on Web 05/21/2009

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NH4+, and Rb+ promote both helix formation and association. Specific binding of cations has been investigated using NMR analyses.21-23 Anions, such as I- and SCN-, also favor and stabilize helices through specific binding on κ-carrageenan, as demonstrated using 127I and 14N NMR, respectively.19,21 In contrast to the above-mentioned cations, I- and SCN- may impede, to some extent, massive helix association, depending on their ionic strength. For instance, the molar mass of κ-carrageenan does not change across conformational transitions from 0 to about 0.15 M NaI; however, molar mass clearly increases at about 0.2 M NaI and greater.8,24 These anions, which rigidify carrageenan chains by stabilizing the (secondary) helical structure, made it possible to obtain a nematic phase in concentrated carrageenan solutions.25,26 Interestingly, Chronakis and Ramzi25 were also able to model the experimental behavior of the nematic phase of carrageenan using the structural parameters of the single-helix. The determination of whether one or two strands are involved in carrageenan helices has been attempted by direct measurements of the molecular weight of ordered carrageenan prepared in I- or SCN- solutions. In addition, unfractionated and fractionated carrageenans have been analyzed by osmometry or by highly sophisticated methods of light scattering, in a standalone mode or coupled with chromatography.8,9,16,20,21,24,27-32 In light-scattering experiments, an increase in molar mass was often attributed to the adoption of well-defined multichain ordered conformation(s). However, it has been subsequently shown that this is just the result of chain association. Bongaerts and co-workers8 published a thorough study on this issue. For this reason, additional experimental evidence is needed to unambiguously determine the topology of the fundamental ordered carrageenan conformation and to complete the evidence provided by molecular weight determination methods. Unfortunately, techniques based on calorimetry14,33 or optical rotation properties34,35 are intrinsically inconclusive, because they rely on a priori assumptions of conformation and theoretical models for interpretation. Similar inconclusive studies were based on molecular modeling,30 even if coupled with sophisticated NMR techniques36 (e.g., NOESY, nuclear Overhauser effect spectroscopy). A series of investigations presented an interesting approach using acid degradation of κ-carrageenan maintained in disordered and ordered conformations and highlight variation in carrageenan reactivity in both conditions.29,37,38 The rate of degradation of κ-carrageenan in the ordered state was found to be 10-fold slower than in the disordered state.37 Differences in glycosidic bond reactivity are also associated with differences in degradation products. These products have a polymodal distribution in the disordered conformation, as expected for a random cleavage of bonds, while a bimodal distribution is observed in the case of the helical conformation. Although the ionic conditions correspond to massive chain association for both κ-8,9,24 and ι-carrageenan,39,40 the result was interpreted as supporting a double-stranded ordered conformation model for κ-carrageenan.29 Carrageenases are glycoside hydrolases found in marine bacteria involved in recycling algal biomass.41 The κ-, ι-, and λ-carrageenases specifically catalyze the hydrolysis of β(1f4) glycosidic bonds of their respective substrates. Pseudoalteromonas carrageenoVora κ-carrageenase has been purified (32 kDa) and shown to yield neo-κ-carrabiose and neo-κ-carratetraose as end products.42 This enzyme belongs to the glycoside hydrolase family GH 16, like some β-agarases, and proceeds according to a mechanism that retains the anomeric configuration.43,44 The gene

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of this carrageenase has been cloned and recombinantly overexpressed, and carrageenase structure has been solved by crystallography.45 P. carrageenoVora κ-carrageenase has also been instrumental in investigating the chemical structure of carrageenan and, more especially, in describing the composition and distribution of carrageenan hybrids.46 In terms of acid hydrolysis, the rate and mechanism of glycosidic bond cleavage by P. carrageenoVora κ-carrageenase should be directly correlated with the reactivity and the number of accessible glycosidic linkages. However, in contrast to acid degradation experiments, the κ-carrageenase employed is devoid of any sulfatase activity that can hydrolyze the sulfate ester group and thus complicate the analysis of the degradation products. We have therefore undertaken the study of the enzymatic degradation of carrageenan in various salt conditions. We aimed to compare the influence of the different conformational properties of κ-carrageenan on enzyme activity. A sample containing a fraction of µ-carrageenan (Figure 1), the biosynthetic precursor of κ-carrageenan, was also investigated for comparison. This sample, commercially identified as “lowgelling carrageenan”, will hereafter be called κ-/µ-carrageenan. Performing enzymatic degradation in carrageenan and salt conditions equivalent to those reported for acid hydrolysis, we similarly found that coil and helix conformations were not digested at the same rate and, in addition, we observed the different polymodal and bimodal distributions. However, investigating intermediate conditions by adjusting iodide concentrations, we observed a range of degradation rates and distributions of degradation products which could not be simply attributed to differences in conformation. Therefore, we propose that iodide binding is the main parameter which modulates carrageenan degradation by enzymes (and probably also by acid). Without completely dismissing the double-helix model, our kinetic results favor a single-helix conformation for orderedstate carrageenan. Nonetheless, conclusive evidence for a single helix conformation was obtained by light-scattering experiments.

Materials and Methods Preparation of K-Carrageenan. κ-Carrageenan (Cottonii X-6913) and low-gelling κ-/µ-carrageenan (Cottonii X-6042) were supplied by CP Kelco. Before use, carrageenans were purified by isopropanol precipitation as follows. Carrageenan powder (5 g) was suspended in 500 mL of cold ultrapure water (Millipore Water Purifier). The suspension was heated at 70 °C under slow stirring until the carrageenan completely dissolved. The polysaccharides in solution were precipitated by dropwise addition of 1 L of isopropanol under vigorous stirring. After centrifugation at 10500 × g, the precipitate was collected and dissolved again in 500 mL of ultrapure water. This precipitation protocol was repeated twice. Carrageenans were then dissolved in 500 mL of water prior the addition of 29.22 g NaCl. This quantity of NaCl corresponds to a salt concentration of 50 times larger than that of the ester sulfate groups in carrageenan. After overnight stirring, the solution was dialyzed (6-8000 Da MWCO Spectra/Por) against ultrapure water to remove excess salt and nonpolymeric materials in the presence of 5 mM NaOH to prevent acidification of the sample. Finally, the Nacarrageenan solution was filtered through a 0.45 µm filter (Pall Life Sciences, Acrodisc Syringe Filter, HT Tuffryn Membrane) and freezedried in the presence of 20 mM ammonium carbonate. Purification of Recombinant K-Carrageenase. P. carrageenoVora κ-carrageenase was recombinantly expressed in E. coli as described by Michel et al.47 Briefly, the enzyme was expressed in the pET20b vector (Novagen) as a His-tagged fusion protein in the periplasm of E. coli BL21 (DE3). The protein was purified by metal affinity chromatography on a column of Chemical Fast Flow Sepharose (GE Healthcare) loaded with NiSO4, followed by cation exchange chromatography

Enzymatic Degradation of κ-Carrageenan on a MonoS column (HR 5/5, GE Healthcare). A stock solution containing 60 µg mL-1 of κ-carrageenase with an activity of 0.06 mM equiv glucose min-1 µg-1 (0.125% κ-carrageenan in 0.2 M Tris) was stored at 4 °C. The pI of κ-carrageenase was calculated using the Compute pI/Mw tool available at http://www.expasy.org/. Enzymatic Digestion. A stock solution of 0.4% (w/v) κ-carrageenan and 0.5% κ/µ-carrageenan in 10 mM Tris buffer (2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrochloride; Sigma) pH 8.0 was prepared at room temperature under gentle stirring overnight. To ensure complete dissolution, the solution was heated at least for 1 h at 70 °C and stored at 4 °C. Salt solutions containing twice the final concentration required were prepared prior to use. The stock solution of NaI was prepared with 2 mM Na2S2O3 to prevent oxidation.21 Equal volumes of carrageenan and salt solutions were mixed after being heated separately for 10 min at 70 °C. The final concentrations of κ-carrageenan and low-gelling κ-/µ-carrageenan were 0.2 and 0.25% (w/v), respectively, in the desired salt concentration. The mixture was kept at 70 °C for 30 min before being cooled down to room temperature (22 °C). Solutions were maintained at least 12 h at room temperature prior to incubation with enzyme. The κ-carrageenase (60 µg mL-1) was diluted 50 times in the appropriate salt solution. Then, 40 µL of diluted enzyme (0.048 µg mL-1) was added to 1 mL of carrageenan solution and incubation was conducted at 25 °C. Because κ-carrageenase is inactivated in alkaline solutions48 without alteration of polysaccharide chemical structure, 12 N NaOH (10 µL per mL of sample) was added to the medium to stop the enzymatic reaction. The amount of reducing sugars produced during the enzymatic digestion was determined using a method adapted from Kidby and Davidson,49 as previously reported.46 Gel Permeation Chromatography-Multiangle Laser Light Scattering (GPC-MALLS). After filtration (0.22 µm, Millipore), the samples (200 µL) were injected on a TSK-GEL GMPW column (7.5 mm × 30 cm, Tosoh Bioscience, MW size) to determine molecular weight and on a Superdex S200 column (300 × 10 i.d.; GE Healthcare) coupled with a Peptide HR (300 × 10 i.d.; GE Healthcare) to better visualize the component distribution. For both column systems, elution was performed at 25 °C at a flow rate of 0.3 mL min-1 (Waters 626 pump) in 200 mM Tris filtered at 0.1 µm. Under these conditions, carrageenan was in the disordered state. Detection was monitored by a Waters 2414 refractive index detector (refractive index, R.I. ) 1.327), used as a mass sensitive detector, set at 890 nm and 35 °C. MALLS measurements were performed at 690 nm with a DAWN EOS system (Wyatt Technology, Santa Barbara, CA) equipped with a 30 mW Ga-As linearly polarized laser. The intensity of scattered light was measured at 12 different angles, from 35 to 143°. Chromatographic data were collected and processed using Astra software (Wyatt Technology, Santa Barbara, CA). The Zimm fit method was applied for molecular weight determinations. The calculated dn/dc was 0.115 mL g-1. Bovine serum albumin monomer (Sigma, St. Louis, MO) was used to normalize the signals recorded at various angles of detection with the signal measured at 90°. Polarimetry. Optical rotation measurements were performed using a Perkin-Elmer 341 polarimeter operating at a wavelength of 365 nm produced by a Hg lamp. Samples were kept at 25 °C in a thermostatted jacketed cell having an optical path length of 10 cm. For convenience, the raw data were converted into a “% of chain ordering”, by subtracting the angular value in the initial (disordered) condition from that at increasing iodide concentrations and then normalizing the values relative to the maximum difference between the full-order and the no-order conditions. The µ-component is considered as “helix-breaking” because it is unable to participate in the long cooperative ordered structure. Thus, for comparison purposes, the same maximum difference was used for κ-carrageenan and for κ-/µ-carrageenan. Strictly speaking, then, the calculated values should be interpreted as “% of maximum ordering by κ-carrageenan units”. 127 I NMR. NMR samples were prepared by adding 50 µL of deuterated water to 450 µL of filtered carrageenan solution containing


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various concentrations of salts in water. 127I NMR spectra were recorded on a Bruker Advance III DRX 400 spectrometer operating at 80.05 MHz and using a 5 mm multinuclei broadband BBO probe. Relaxation time was adjusted to 0.3 s and acquisition time to 82 ms (32 K of time domain for a spectrum window of 200 kHz). Experiments consisting of 2048 scans were conducted at a temperature set to 297 K. Processing of all data (spectrum size of 32 K and LB of 5 Hz) was performed on a PC with Bruker Topspin v2.0 software, and most importantly, the full width at half-maximum was determined for each sample using the “peakw” Bruker command. The average experimental line width of aqueous NaI, in the range from 10 mM to 100 mM, was 1762.4 ( 9.3 Hz.

Results Degradation and Ordering of Carrageenan: Enzyme Kinetics and Optical Activity. Experiments Performed with Increasing Electrolyte Concentrations. The degradation time course of κ-carrageenan in increasing concentrations of Tris by P. carrageenoVora κ-carrageenase was monitored by assaying the reducing power of the incubation medium. The concentration of 0.2% (w/v) carrageenan was chosen because optical rotation as well as light-scattering experiments could be performed at such concentration, and, in addition, the experimental conditions were similar to those used in the published acid hydrolysis experiments.29,37 Initial velocity was calculated on the linear section of the progress curve and corresponded to, at most, 15-20% degradation. As shown in Figure 2A, the initial velocity increased with ionic strength up to a plateau that was reached at about 150 mM Tris. For higher Tris concentrations (200-450 mM), the rate of cleavage remained constant. The curve obtained with low-gelling κ-/µ-carrageenan, which contained about 15% µ-carrabiose moieties, was very similar in shape (Figure 2B). Optical rotation of κ- and κ-/µ-carrageenan did not show any sigmoid behavior with increasing Tris concentrations (Figures 2C,D) nor was any conformational transition observed after heating the solution. In addition to the absence of carrageenan ordering, no polysaccharide aggregation was observed by light-scattering analysis (not shown). The behavior of carrageenan in Tris appeared quite similar to that reported for the chemically related tetramethylammonium salt.15,27,50,51 The initial increase in activity for concentrations below 200 mM Tris can be explained by a “salting in” effect of κ-carrageenase. At low ionic strength, protein molecules aggregate due to electrostatic interactions and, as a consequence, are less reactive. The aggregation of the enzyme at low salt concentrations was visualized experimentally by size exclusion chromatography (GE Healthcare Superose 12 column). At 100 mM NaCl, κ-carrageenase eluted as a single peak, while at 10 mM salt at least three broad peaks were observed (not shown). This “salting in” effect was independent of the salt species: it was also observed with low concentrations of NaI and other salts. The rate of κ-carrageenan degradation in aqueous NaI displayed a very different pattern. As for Tris, degradation increased with NaI concentration but activity reached its maximum at about 50 mM NaI, whereas for Tris, maximum activity was attained at about 200 mM (Figure 2A). At concentrations higher than 50 mM NaI, we observed a strong decrease in enzyme activity that was clearly dependent on the total iodide concentration, rapidly reaching a quasi-asymptote at concentrations greater than 200 mM NaI. The combination of the rapid increase and the subsequent decrease resulted in a curve with a sharp maximum. In agreement with many previous reports, our optical rotation experiments confirmed that about

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Figure 2. Effect of NaI and Tris concentration on the rate of degradation of (A) the standard κ- and (B) low-gelling κ-/µ-carrageenan incubated with P. carrageenovora carrageenase. The ordering of (C) κ- and (D) low-gelling carrageenan in NaI and Tris was analyzed by optical rotation.

100 mM NaI was able to induce complete helix formation (Figure 2C) and that this ordered state was not associated with chain aggregation (see Table 1 and references in 9). For κ-/µ-carrageenan, a very similar apparent enzyme inhibition effect was observed at increasing iodide concentrations, although it was less pronounced since the curve showed a much gentler decrease after the maximum. As its name implies, lowgelling κ-/µ-carrageenan, which contains µ-carrabiose moieties, is well-known for its weaker gelation. The low capacity of this carrageenan to form a gel is associated with its reduced ability to form helices as shown in Figure 2D. Using the observed maximum variation range shown by κ-carrageenan (normalized to 100%), variation in optical rotation suggests that only about 60% of the hybrid macromolecule adopted a helical conformation at about 150 mM iodide. It is then apparent that the µ-component in the hybrid molecule shows a double difference compared to the κ-component: it is unable to achieve the same conformational ordering but, at the same time, it is not completely “protected” from enzymatic degradation in increasing iodide concentrations. One should notice that, upon increasing the NaI concentration from 150 to 600 mM, the amount of helices showed, at most, a further increase of about 15%, whereas the activity of the enzyme dropped by more than half. Experiments Performed at Constant Concentration of Cosolutes. Experiments presented in Figure 3 were performed with increasing amounts of NaI to which Tris was added to keep the cosolutes concentration constant at 200 mM, as required for the optimal activity of the enzyme (at such concentration, and pH ) 8, the ionic strength of the Tris solution is 94 mM). Using a range of increasing NaI concentrations from 0 mM NaI (with 200 mM Tris) to 200 mM NaI (with 0 mM Tris) had the

advantage of spanning the whole disorder-order transition, from coil to 100% helix, as illustrated by results of the optical rotation experiments also presented in Figure 3. The rate of carrageenan degradation was practically constant as a function of NaI concentration up to about 60 mM. At higher concentrations, the initial velocity decreased strongly and reached the lowest rate at 200 mM NaI. Polarimetry performed at constant 200 mM cosolutes concentration showed that carrageenan helices started to form at about 25 mM NaI and helix formation reached its maximum at the critical concentration of about 60 mM NaI (Figure 3A). Interestingly, the amount of NaI required for inducing helix formation was lower in these constant 200 mM cosolutes concentration experiments, than when no other ions were added to NaI (compared to 100 mM cf. Figure 2C). In the case of κ-/µ-carrageenan, the initial velocity versus iodide concentration curve shows a maximum between 50 and 70 mM with a gentle decrease up to 200 mM. Across the maximum, the curve displayed the same rate at 0 mM and about 120 mM and the chain reached its maximum ordering at this latter value. Maximum ordering corresponded to about 60% of that of κ-carrageenan in the same conditions, as in the abovementioned experiments at variable ionic strength. This means that the strong inhibition of the enzyme observed with κ-carrageenan as a substrate was not caused by the detrimental action of iodide ions against κ-carrageenase activity nor by the formation of an ordered conformation of the polymer. As a consequence, enzyme inhibition must be due to some modification of the properties of the carrageenan molecules, which, albeit correlated with chain conformation and with iodide concentration, affect chain accessibility.

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Figure 4. Line width at half height of the 127I NMR signals recorded on κ- and κ-/µ-carrageenan. The experiments were conducted with increasing concentrations of NaI (A) in solution and (B) at constant 200 mM cosolutes concentration (+Tris).

Figure 3. The ordering and the rate of enzymatic degradation of (A) κ-carrageenan and (B) κ-/µ-carrageenan were determined with increasing NaI concentrations, at a constant 200 mM cosolutes concentration (+Tris).

Biochemical Parameters of the κ-Carrageenase. The Michaelis constant (Km) of P. carrageenoVora carrageenase estimated in this work was about 0.22 mM (as β(1-4) linkages), which is equivalent to a carrageenan concentration of about 0.007% (w/v) κ-carrageenan. The Km value determined for the Zobellia galactinoVorans κ-carrageenase is about 0.28 mM of β(1-4) linkages or 0.009% (w/v) carrageenan concentration (Potin et al.,48 after recalculation). Although the determination of the Km using polymeric material should be interpreted with caution because the substrate changes after each bond cleavage, it gives an estimation of the saturating conditions for the enzyme. Consequently, at a concentration of 0.2% (w/v) carrageenan, the enzyme was in saturating conditions, indicating that the initial rate observed in 0 mM NaI was close to the maximum velocity (Vmax). In addition, the inhibition of the enzyme observed, for example, at 60 mM was probably underestimated because the amount of available substrate was close to saturating conditions. 127 I NMR of Carrageenan: Iodide Solutions. As reported earlier by Grasdalen and Smidsrød,21 the conformational transition of carrageenan was associated with the binding of iodide ions on the polysaccharide, which was directly monitored by the increase of the width-at-half-height (∆ν) of 127I NMR signals. In the absence of polymers, when no binding has occurred, the signal width was narrow and the measured ∆ν was of about 1800 Hz (see Materials and Methods). At 100 mM NaI in water, when helices formed, ∆ν reached a maximum value of 5400 Hz, which then decreased with increasing amounts of iodide (Figure 4A). In the case of κ-/µ-carrageenan the observed line width was always lower than that of pure κ-carrageenan, indicating a lower

amount of bound iodide for κ-/µ-carrageenan (maximum ∆ν ) 4000 Hz). For NaI concentrations higher than 100 mM (corresponding to full helical transformation), we observed a systematic sharpening of the 127I NMR signal, which is illustrated by the strong decrease in ∆ν. The contribution of free (unbound) iodide was then higher than the contribution of bound iodide. Interestingly, the slopes of these decreasing curves were also dependent on the chemical structure of carrageenan. Thus, when the amount of bound iodide was high (κ-carrageenan), the decrease in ∆ν was steeper than when bound iodide was low (κ-/µ-carrageenan). This indicates that for the same amount of iodide added to the medium, the amount of free iodide did not increase at the same rate and, in parallel, additional binding to the carrageenan chain did not occur in the same proportion. Similar observations have been previously made with SCN-, another helix-promoting anion, for which 500 mM salts were necessary to fill all carrageenan binding sites.19 Importantly, we were able to establish that only a fraction of available binding sites were occupied by iodide at the critical helix-forming concentration of NaI and that additional iodide ions are bound to ordered carrageenan with increasing iodide in the medium. This very strongly suggests that the inhibition of the enzyme is caused by an increasing amount of bound iodide on the ordered carrageenan, leaving fewer sites for the enzyme to bind to the substrate. In the case of experiments carried out as a function of iodide but at constant cosolutes concentration, the width of the 127I NMR signals was also higher (∆ν ) 6500 Hz), indicating that the ratio of bound to unbound iodide significantly increased with ionic strength. This phenomenon has already been observed by Zhang et al. 19 and was interpreted by the electrostatic screening effects of the supporting salt on the increasingly (negatively) charged polysaccharide. In addition, under the conditions of Figure 4, the amount of iodide bound by κ-/µ-carrageenan was always lower than that by κ-carrageenan. As previously observed in Figure 3A,B, when the disorder-order transition occurs, the iodide binding sites were

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Figure 5. Gel permeation chromatograms of the degradation profiles of κ-carrageenan obtained for increasing percentages of degradation. Carrageenan was incubated with P. carrageenovora carrageenase at 200 mM constant cosolutes concentration (+Tris) with (A) 0, (B) 60, and (C) 120 mM NaI. DP2, neo-κ-carrabiose; DP4, neo- κ-carratetraose; DP6, neo- κ-carrahexaose.

not filled and the degradation rate decreased with increasing concentration of iodide, the amount of helical residues remaining constant. Digestion Profiles and Molecular Weight Determination. The degradation of κ-carrageenan incubated in the disordered (0 mM NaI) and in the ordered conformation (60 and 120 mM NaI) at constant total ionic strength 200 mM (with Tris) was followed by gel permeation chromatography (GPC) coupled with a MALLS detector. Independent of the conformational conditions of the degradation experiments, the GPC-MALLS analyses of all degraded carrageenan samples were performed in conditions of conformational disorder, to avoid any (possible) effect of association on the determined molecular weight. In the case of 0 mM iodide, as the degradation of the disordered substrate proceeded (Figure 5A), the signal corresponding to the polymer fraction shifted toward higher elution volumes, indicating a decrease in molecular mass. This was correlated to the MALLS measurements (see below), which showed a rapid drop in molecular mass. In addition, the shape of the peak became broader, indicating an increase in polydispersity. In the case of the substrate in the ordered conformation, the situation seemed totally different. Indeed, for the concentration of 120 mM NaI, the degradation profiles had a bimodal distribution consisting of one high molecular mass fraction which decreased in area without significant loss of molecular mass (i.e., shift to higher retention volumes) and a second fraction composed mainly of disaccharides and traces of tetrasaccharides (Figure 5C). The degradation profile of the ordered carrageenan in 60 mM NaI appeared intermediate to that of the coil and that in 120 mM NaI (Figure 5B). Here, the production of intermediate molecular mass oligosaccharides was readily observed (compared to latter case where they were absent), and it was still possible to observe the high molecular mass fraction which did not show any shift in elution time as the cleavage proceeded.

the effect of iodide anions studied by NMR and the rate of degradation. Our results of 127I NMR presented in Figure 4A,B fully agree with those first reported by Grasdalen and Smidsrød27 and then confirmed by Norton et al.,52 Zhang et al.,19 and Zhang and Furo´.53 Although the generality of the interaction of a halide anion with κ-carrageenan (albeit quantitatively very different) prompted Norton and co-workers52 to address it as a chargecloud interaction, all other authors converged in using the term “site binding”, which does not preclude anion specificity. Following this line, we interpret the profile of the 127I line width as typical for the (weighted) average of two populations of nuclei perceiving two, very different, physical-chemical situations that deeply affect their relaxation. The accepted interpretation of this case is by use of the so-called “two-state-all-or-none” binding model, which assumes two types of states for the ligand, a “free” and “bound” one. They are endowed with an intrinsic Lw (line width) for the bound species Lw,B (presently unknown) and Lw,F for the free species. It is usually assumed that Lw,F is equal to the Lw value of iodide alone, Lw,0

Lw,exper ) Lw,B × fbound + Lw,F × ffree ) Lw,B × (Cbound/Ctotal) + Lw,0 × (1 - Cbound/Ctotal) Lw,exper ) Lw,B - Lw,F × (Cbound/Ctotal) + Lw,0 where Cbound is the concentration of iodide bound to κ-carrageenan, Ctotal is the total concentration of iodide, and fbound and ffree are, respectively, the fractions of bound and unbound (free) iodide. Lw,0 is then subtracted from Lw values determined in the presence of polysaccharide chains, Lw,exper, giving the values of Lw,corr

Lw,corr ) Lw,exper - Lw,0 ) (Lw,B - Lw,0 × (Cbound/Ctotal) ) Lw,B(relative) × (Cbound/Ctotal) (1)

Discussion Carrageenan Conformation and Iodide Binding. The experimental novelty of this work resides in the correlation of

Lw,B(relative) is not known and, hence, a quantitative determination of Cbound is presently not possible and, moreover, beyond


Figure 6. Plot of the concentration of iodide bound to κ-carrageenan, Cbound, calculated from 127I NMR data, as a function of the iodide concentration in solution, without any additional electrolytes (A, filled squares) and at constant cosolutes concentrationof 200 mM by adjusting with Tris (B, open squares). Cbound expressed in arbitrary units (a.u.); the Cbound scale is the same for both panels A and B, scaled to 1 on the highest determined Cbound value (panel A). The dashed curve in the upper part of each plot represents the best fit curve through the data points according to Langmuir equation, after attaining 100% conformational ordering. The filled circles represent optical activity data, represented as % of conformational ordering, scaled to highlight the initial sigmoid behavior of both plots. The initial Cbound data points from panel A are replotted on panel B (filled squares connected by a dotted line) to facilitate the comparison of the results in the initial range of iodide concentrations.

the scope of this paper. Nevertheless, we used eq 1 as a purely qualitative measure of Cbound, providing the possibility of a direct comparison between the data obtained for variable ionic strength (Figure 6A) and for constant ionic strength (Figure 6B). The Cbound curves actually correspond to the plots of (Lw,corr × Ctotal) for both experiments using the same, albeit arbitrary, scale. The Cbound versus iodide concentration curves in Figure 6A,B show the same behavior. For iodide concentrations lower than 100 mM, the curve increase is sigmoid and, in both cases, it closely follows the corresponding sigmoid development of ordering as indicated by optical activity data. After reaching 100% ordering, the shape of both curves conform to the “classic” increase of the fraction of bound molecules onto a linear array of sites, most simply described by a Langmuir binding isotherm. At low iodide concentrations, in the case of constant ionic strength, the amount of iodide bound is larger than in the case of equivalent aqueous iodide, perfectly paralleling the higher fraction of ordered conformation. The combined analysis of the data of Figures 3B and 4B shows that, at the same conditions, the amount of both conformational ordering and bound iodide was lower in the case of κ-/µ-carrageenan than in the standard κ-carrageenan, both at constant cosolutes concentration and in the case of varying NaI solutions, pointing to an inability of the µ-component to provide good iodide binding site, in parallel with the already mentioned inability to undergo conformational ordering.


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Physical-Chemical Effects Modulating Degradation Rate. Another remarkable result of our work is presented in Figure 2A,B. The value of initial velocity for increasing amounts of aqueous NaI was higher than that of Tris up to about 50 mM. In addition, the rate of degradation was similar at about 75 mM Tris or NaI, condition for which carrageenan is disordered or 80% ordered, respectively (Figure 2C). This result could be regarded as quite peculiar, because the κ-carrageenase turns out to be unexpectedly more effective (or at least no less effective) on the helical conformation of κ-carrageenan than on the disordered one. Different modulating factors can be identified that allow either an increase or a decrease in initial velocity: (i) The initial increase in enzyme activity for concentrations below 200 mM Tris was already explained by a “salting in” effect of κ-carrageenase. This effect can be the result of a purely electrostatic component and a component related to lyotropic behavior. The former effect must be species-nonspecific, that is, the same for TrisH+ and Na+, and for Cl- and I- and therefore unable to explain the differences in behavior with Tris and iodide. The latter effect (which can, in principle, affect the enzyme, the polysaccharide chain19 or both) must result in a monotonic increase of the initial velocity as a function of cosolutes concentration. (ii) The disorder-order transition induced by bound iodide anions (and ionic strength) brings about a major expansion (by stiffening) of the chain which is measured by the angular dependence of the scattered light: the radius of gyration passes from 75.3 ( 5.2 nm to 88.9 ( 4.0 nm.16 This was also manifested as a sharp increase in viscosity in the range of iodide concentrations in which the transition develops.22 After helical conformation is attained and in still higher salt concentrations, chain dimensions remain practically constant.24 The ordered chain is more expanded and therefore more accessible to ligands (both iodide anions and the enzyme). (iii) The addition of NaI and Tris solutions increases the ionic strength (albeit at different extent), thereby reducing all intra- and intermolecular electrostatic interactions. Apart from the “salting in” and the lyotropic effects mentioned above, intramolecular interactions occur between fixed charges of the carrageenan chain and intermolecular interactions occur between the enzyme and the polyanion. In both cases an increase of the ionic strength is expected to reduce the rate of degradation. In fact, in the case of intramolecular interactions, the increase of the ionic strength will progressively shield repulsive interactions, thereby decreasing the electrostatic expansion and the overall chain dimensions. The result will be a decrease of the intrinsic viscosity and a reduction of ionic polymer expansion with somewhat lower accessibility. The intermolecular interactions favor the formation of the enzyme (E)-substrate (S) complex. The κ-carrageenase under study has a calculated isoelectric point (pI) of 9.5. Under neutral conditions, the enzyme then carries an overall cationic charge which forms positive patches spread on the surface of the protein.45 This condition seems to favor interaction between the cationic proteins and the polyanionic substrate. Any increase of the ionic strength is then expected to shield both intramolecular repulsive forces on carrageenan and intermolecular (E-S) attractive forces. However, the practical constancy of kinit up to high values of the concentration of Tris (see Figure 2A) suggest that such ionic effects are not the dominating ones. (iV) As it has been pointed out in the discussion of Figure 6, iodide binding on κ-carrageenan increases as a function of the total iodide concentration in solution. The shape of the binding curve indicates that iodide ions are able to induce helix formation, because the growth of Cbound and chain ordering can be perfectly superimposed up to 100% ordering. Beyond that,

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chain conformation does not change, whereas more halide anions accumulate on the helical polysaccharide. As a consequence, iodide inhibits enzyme activity by blocking the binding sites. Considering the above listed effects, one can tentatively describe the curves of Figure 2A,B in the following way. In the case of Tris solutions, the “salting-in” effect prevails up to about 100 mM. Beyond this already high value of ionic strength, the effects on intra- and intermolecular interactions are comparatively small and, as a consequence, the initial velocity of the enzyme remains relatively constant. For iodide solutions of up to about 50 mM (i.e., about 27% chain ordering), the same patterns occur as for Tris but to a greater extent. This can be ascribed to the favorable effect of solvent quality (lyotropic effect; i), conformational transition (ii), and enzyme-substrate electrostatic attractions (iii), which all make the chain much more accessible to the degrading enzyme. However, the disorder-order transition simultaneously occurs with the binding of iodide ions, which increases the enzyme inhibition caused by the “cover-and-protect-the-chain” effect (above-mentioned under iV). In terms of the Michaelis-Menten equation, this leads to a dramatic decrease of enzyme-substrate complexes [ES]. Mathematically, the positive and negative effects on the rate of degradation produce a sharp maximum at about 50 mM NaI. The negative effect rapidly develops and it counterbalances the positive effect at about 75 mM NaI, an ionic strength value for which the initial velocity is equal for both Tris and NaI. Interestingly, the comparison of the rate of degradation as a function of iodide concentration, both in solution (Figure 2A) and at constant cosolutes concentration (Figure 3A) revealed very similar behavior. At 75 mM NaI in water (which corresponded to about 50% ordering), the rate of degradation was similar to that of 75 mM Tris, without carrageenan ordering (Figure 2A). Similarly, the value of initial velocity versus increasing iodide concentration at constant cosolutes concentration (Figure 3A) stayed practically constant up to about 60 mM iodide which roughly corresponded to 100% ordering. These observations indicate that the ordered conformation, as such, is not the cause of reduced degradation. Effect of Degradation on Molecular Weight and Molecular Mass Distribution. The GPC profiles (Figure 5) of the degradation products carried on κ-carrageenan in conditions of conformational disorder are reminiscent of random depolymerization observed for acid degradation.29,37,38 For comparison purposes, weight-average molar mass (Mw) data was analyzed in relative terms, that is, using the ratio of the Mw at the given degradation percent (Mw(deg%)) over the initial value of Mw (Mw(0)). The results are plotted in Figure 7. For the case at 0 mM iodide, the shape of the curve is clearly bimodal and showed a good fit with a biexponential decay function. The initial 5% of (linkage) degradation was able to produce about 80% of decrease of Mw, whereas the remaining 95% of the degradation process accounted for 20% of the total decrease of Mw. This means that κ-carrageenase has an endolytic mode of action producing an initial number of cuts, which produces a dramatic drop of the average chain length. As long as degradation reactions continue, short oligomers are produced down to the lowest possible length. The behavior of the curves in Figure 7 for the cases of 60 and 120 mM has already been shown to be very different. To start with, it is important to stress that in both conditions κ-carrageenan attains the completely ordered conformation. The second interesting observation is that the molecular mass of the undigested fraction (i.e. high molecular fraction) did not decrease similarly as a function of the number of cleavages. The curves could also be fitted with a biexponential decay function, but it could also be fitted with only

Colleń et al.

Figure 7. Decrease in molecular mass of κ-carrageenan incubated with P. carrageenaovora carrageenase as a function of degradation percentage. The experiments were conducted at a constant cosolutes concentration (+Tris) of 200 mM with 0 (filled circles), 60 (open circles), and 120 mM (open squares) NaI.

for one exponential component using reasonable parameters, as indicated by qualitative inspection of the curves. As the percent of conformational order was 100% for both cases, we assume that these undigested fractions correspond to longer segments (or blocks) of iodide-bound carrageenan in 120 mM than in 60 mM NaI. We could obtain the complete digestion in 60 mM (Figure 5B) with prolonged incubation, but not with 120 mM NaI. Because of the reversibility of iodide binding, the enzyme can probably compete with the anion at 60 mM NaI and, as soon as the carrageenan chain becomes shorter, iodide binding becomes weaker, allowing more cleavage. Evidence that the cooperative units of helical segments that bind iodide are quite long is in agreement with previous observations.29 In the case of 120 mM NaI, although iodide binding is reversible, the equilibrium is so strongly displaced toward the halide-bound form as to impede any efficient binding of the enzyme. Comparison of Acid and Enzymatic Degradation of Carrageenan. Enzymatic degradations presented in this work were achieved in 2 g L-1 carrageenan and with iodide concentrations similar to those applied in acid hydrolysis experiments performed by Hjerde et al.29,37 ([10 mM LiCl + 100 mM HCl ] and [200 mM LiI + 100 mM HCl], respectively). The degradations presented herein, done with 0 and 120 mM NaI at constant ionic strength (200 mM), seemed to proceed in a way very similar to acid hydrolysis. When carrageenan was in the disordered state, we observed fast degradation and production of all possible degradation products. In 120 mM NaI concentration, the rate of digestion slowed down and a bimodal distribution of carrageenan products was obtained. In the case of acid hydrolysis, the bimodal distribution of the degradation products and the lower rate of degradation were interpreted by Hjerde et al.29,37 as due to a double-helix conformation. Their assumption was that the observed resistance to degradation in the ordered state was to be entirely ascribed to the intertwining arrangement of the double helix, iodide binding being granted only a “modulation” role in degradation. It was also assumed that the double-stranded carrageenan helix could support some random cleavage of glycosidic bonds without dissociating, based on the double helix behavior of DNA. As soon as some short fragments were liberated in the medium, adopting a disordered state, they could be disrupted 10 times more rapidly than the less reactive components involved in the double helix. According to this model, the bimodal distribution recorded was composed of a high molecular mass fraction corresponding to the ordered, double



helical fraction and a low molecular mass fraction, representative of components which lost their helical conformation. However, an internal inconsistency of the model designates the double-helix formation as the cause of the observed decrease of acid degradation rate in κ-carrageenan, since the observed rate constant of acid hydrolysis performed on coil or ι-carrageenan helix was the same, which is not the case with κ-carrageenan.37 Because ι-carrageenan helices cannot bind iodide, it seems presumptuous to conclude that in κ-carrageenan it is iodide binding, and not the coil-helix transition, that triggers acid hydrolysis. The enzymatic degradation of carrageenan in the ordered state cannot be interpreted as straightforwardly by the double-helix model. Indeed, when carrageenan is in the ordered conformation, the rate of its degradation is directly correlated with the amount of iodide bound and indirectly to the amount of helices formed: Figure 3A,B shows that when passing from 60 to 200 mM NaI, the initial velocity decreased while the amount of bound iodide increased in a complementary fashion (as shown by NMR experiments), although the amount of helices remained constant (as indicated by optical activity). As a result, the ordered conformation has the capacity to bind increasing amounts of iodide which, when bound, drastically reduce the accessibility of glycosidic bonds, and as a consequence, inhibit catalytic efficiency (of both enzyme and acid). Similarly, acid hydrolysis of κ-carrageenan in 100 mM LiI was eight times faster than in 200 mM LiI although κ-carrageenan had adopted the fully ordered conformation in both concentrations.37 This very strong difference of reactivity of acid toward glycosidic bonds in 100 and 200 mM LiI was explained, on one hand, by an increase of charge density of the carrageenan double-helix, which entrapped increasing amounts of iodide and, on the other hand, by the increasing ionic strength of the medium, which was thought to slow down acid catalysis. Our enzymatic experiments were performed at constant cosolutes concentration, suggesting that only the helix charge density argument is valid, which, however, can be equally used with iodide bound on carrageenan single or double helices. However, one cannot escape underlining that, on purely electrostatic grounds, the unfavorable increase of electrostatic free energy on iodide anions binding to the double-helix polymer would be much larger than that expected in the single-helix case.

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Model of Iodide-Bound Carrageenan Degradation. We have shown that the main parameter affecting the rate of enzymatic degradation is the amount of iodide bound in ordered carrageenan. Nevertheless, the accessibility of the catalyst (acid or enzyme) to the glycosidic linkages is probably also modulated by carrageenan conformational ordering. In a double-helix conformation, the geometrical constraints as well as the energy required to dissociate the intertwined strands would reduce the efficiency of any catalyst. Based on this assumption, the bimodal distribution was previously taken as evidence of a double helix. However, we observed variation in the length of degradation products for ordered carrageenan prepared in different iodide concentrations (Figure 5). Notably, we observed that when more iodide was bound (i.e., at 120 mM vs 60 mM iodide), fewer intermediate products were produced. This suggests that the distribution of the degradation fragments was modulated by the amount of bound iodide only, because the helical conformation was fixed at 100%. Assuming that enzymatic degradation proceeds randomly as shown for carrageenan coils (Figure 5), cleavage inside the carrageenan chain should lead to shorter macromolecules that are less able to adopt a long, highly cooperative, helical conformation29,54 and, as a consequence, to be stabilized by bound iodide. Then the shortened fragments should offer more available and more reactive glycosidic linkages for the catalyst: they will be degraded more quickly than the iodide-bound carrageenan chain. This assumption is consistent with variation in degradation rates, which decreases with increasing bound iodide, and with the distribution of carrageenan degradation products. The conclusion of the analysis of the results so far reported is that the alleged kinetic argument of Hjerde et al.29,37 in favor of the double-helical model does not hold: chain stabilization that produces the peculiar distribution of degradation products is not due to conformation as such but, rather, to the occupation of binding sites by iodide anions. In fact, when site occupation is low (i.e., at low iodide concentrations), the helical conformation does not show lower accessibility to either protons (acid degradation) or to κ-carrageenase (enzymatic degradation) compared to the disordered conformation.

Table 1. Molecular Mass of κ-Carrageenan Determined for Various Iodide Concentrationsa iodide, mM

ionic strength, mM

ordering, %

Mw g mol-1

note

(A) This Work 0 60 120

200 200 200

0 100 100

265000 294400 354000

avg: 279700 ( 11500 (rel. std. ) 4.1%) ) (avg) + 27%

(B) Slootmaekers et al.22 0 40f150 200

20f150 40f150 200

0 4f100 100

335125 ( 14565 326375 ( 30390 391000

avg: 330750 ( 23460 (rel. std. ) 7.1%) ) (avg) + 19%

(C) Bongaerts et al.8,9 0 150 200

0f150 150 200

0 100 100

154000 ( 13000 162000 206350 ( 1060

avg: 157000 ( 10000 (rel. std. ) 6.4%) ) (avg) + 31%

(D) Grasdalen and Smidsrφd21 0 150 200

150 150 300

0 100 100

720000 610000 not determined

avg: 665′000 ( 55000 (rel. std. ) 8.3%)

(E) Hjerde et al.27 0 110 200

110 110 300b

0 100 100

110000 not determined 260000

avg: 110000 ) (avg) + 140%

a To compare our data with previous investigations, average molecular weight and standard deviation (STD) were calculated. b Degradation conditions: GPC-LALLS in 200 mM (after dialysis) but without thermal pretreatment.8

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Figure 8. Schematic representation of the different conformational and iodide binding situations encountered with increasing NaI concentrations. Without iodide, κ-carrageenan is in a disordered (dis) conformation. The increasing amount of iodide induces helical (hel) conformation of the polysaccharide, associated with iodide binding. The amount of bound iodide increases with anion concentration in the medium and impedes the enzymatic degradation of κ-carrageenan. For high iodide concentrations, carrageenan dimerization (dim) starts to occur and the carrageenan increases in molecular mass.

Given that these conceptually inconclusive considerations are based on kinetic arguments alone, we turned to the MALLS data on molecular mass. The values of the weight-average molar mass, Mw, determined at constant ionic strength 200 mM are reported in Table 1A. The MALLS results at 0 and 60 mM show that the weight-average molar mass of the polymer does not change when going from 0 to 100% ordering, respectively, with a remarkably low relative standard deviation value of 4.1%. Bongaerts et al.9 reported 10 different sets of determinations showing that κ-carrageenan can pass from 100% disordered conformation to 100% ordered without any change of molar mass. For comparison, we extracted values from the studies of Slootmaekers et al.,24 Bongaerts et al.,9 and Grasdalen and Smidsrød,21 that are reported in Table 1B-D. In these studies, the observed Mw constancy ranged from 6.4 to 8.3%. The disordered-ordered conformation transition of κ-carrageenan induces no change in molar mass and it is thus an intramolecular process. Therefore, the single-helix model is compatible with previous molar mass determinations as well as our enzymatic degradation kinetics, whereas the intertwined double-helix model can be dismissed. Upon increasing the iodide concentration to 120 mM at a total ionic strength of 200 mM, a 27% increase of Mw was observed (Table 1A). In the same conditions, Slootmaekers et al.24 and Bongaerts et al.9 reported an increase of 19 and 31%, respectively (Table 1B,C). This demonstrates that iodide is not a “universal” stabilizer of the single-helix conformation of κ-carrageenan. On increasing both iodide and ionic strength, progressive chain association starts developing. The molecular mass is particularly sensitive to association, but it cannot give per se an indication of the fraction of repeating units involved in such “dimeric” side-by-side contacts (dim, as defined in the Introduction).

Conclusion We have shown that the enzymatic degradation of κ-carrageenan by P. carrageenoVora carrageenase (and also by acid hydrolysis) is negatively affected by the amount of bound iodide on ordered carrageenan chains rather than by the ordered conformation of the polysaccharide as such. These kinetic observations, combined with our (and other previous) molecular mass determinations, help distinguish

Colleń et al.

several conformational and iodide-binding situations. At first, as schematically represented in Figure 8, when little or no iodide is present in solution, carrageenan adopts a disorder (dis) conformation (Figure 8I), which can be easily degraded. Increasing iodide concentration induces a helical conformation (hel; Figure 8II), which (i) does not inhibit enzyme activity, (ii) binds iodide anions, and (iii) has the same molecular mass as the disordered conformation. These properties are only compatible with a single-helix conformation. Iodide binding on the single helix increases with increasing NaI concentration (Figure 8III) and proportionally impedes the efficient binding of the enzyme. Nevertheless, at this stage the molecular mass of carrageenan remains constant. Finally, high iodide concentrations make carrageenan even more recalcitrant to enzyme degradation because of two converging negative effects on the amount of free ordered polysaccharide repeating units. Namely, more hel segments are occupied by a still increasing amount of bound iodide anions (by more effect of the mass-action law), whereas other hel segments start being involved in chain dimerization (Figure 8IV), also a consequence of the high ionic strength of the solution, as shown by an increase of molecular mass. Acknowledgment. This work was supported by the French National Center for Scientific Research (CNRS ATIPE program) and Pierre and Marie Curie University (Paris VI).

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