Solution properties of chitin in alkali

The solution properties of alpha-chitin dissolved in 2.77 M NaOH are discussed. Chitin samples in the weight-average molecular weight range 0.1 x 10(6) g/mol to 1.2 x 10(6) g/mol were prepared by heterogeneous acid hydrolysis of chitin. Dilute solution properties were measured by viscometry and light scattering. From dynamic light scattering data, relative similar size distributions of the chitin samples were obtained, except for the most degraded sample, which contained aggregates. Second virial coefficients in the range 1 to 2 x 10(-3) mL.mol.g(-2) indicated that 2.77 M NaOH is a good solvent to chitin. The Mark-Houwink-Sakurada equation and the relationship between the z-average radius of gyration (Rg) and the weight-average molecular weight (Mw) were determined to be [eta] = 0.10Mw0.68 (mL.g(-1)) and Rg = 0.17Mw0.46 (nm), respectively, suggesting a random-coil structure for the chitin molecules in alkali conditions. These random-coil structures have Kuhn lengths in the range 23-26 nm.     

Solution properties of targacanthin (water-soluble part of gum tragacanth exudate from Astragalus gossypinus)

Solution properties of tragacanthin (the water-soluble part of gum tragacanth) were studied by gel permeation chromatography (GPC) combined with multi-angle light scattering and viscometry at 25 degrees C. Photon correlation spectroscopy was used to determine the hydrodynamic radius. Ultrasonic degradation was applied to obtain biopolymer fractions of different molecular weights. The dependence of intrinsic viscosity [eta] and radius of gyration (s2)z(1/2) on weight average molecular mass M(w) for this biopolymer were found to be [eta] = 9.077 x 10(-5) M(w)(0.87) (dL g(-1)) and (s2)z(1/2) in the range of M(w) from 1.8 x 10(5) to 1.6 x 10(6). The conformational parameters of tragacanthin were calculated to be 1111 nm for molar mass per unit contour length (M(L)), 26 nm for persistence length (q) and 1.87 ratio of R(g)/R(h). It was found that the Smidsr?d parameter B, the empirical stiffness parameter was 0.013, which is lower than that of several polysaccharides indicating the stiff backbone for tragacanthin. The rheological behavior of aqueous solutions of gum tragacanth and its insoluble and soluble fractions (bassorin and tragacanthin, respectively) were studied. For concentrations equal to 1%, at 25 degrees C and in the absence of salt, bassorin solution showed the highest viscosity and shear thinning behaviour. Power law and Williamson models were used to describe the rheological behaviour of bassorin and tragacanthin, respectively. Oscillatory shear experiments showed a gel like structure for the bassorin but for tragacanthin the oscillatory data were as would be expected for semi-dilute to concentrated solution of entangled, random coil polymers. NaCl changed the steady and oscillatory rheological properties of both fractions and in this way the final viscosity of bassorin was even less than tragacanthin. The calculated activation energy for bassorin and tragacanthin indicated a more rapid decrease in viscosity with temperature for tragacanthin. The plot of eta(sp,0) versus C[eta] revealed that the transition from dilute to semi-dilute regime occurs at C*[eta] = 2.82 for tragacanthin.     

Isolation and physical characterization of an exocellular polysaccharide

The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40 were investigated. Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes. Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water. Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide. Fractions were analyzed by multiangle static light scattering in aqueous 0.10 M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 +/- 0.06).10(3) and (1.62 +/- 0.07).10(3) kg/mol, respectively, were calculated so that Mw/Mn approximately 1.13. The number-averaged radius of gyration was found to be 86 +/- 2 nm. From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained. Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 +/- 4 nm was calculated. Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 +/- 3 nm, as found with GPC.     

Evaluation of radius of gyration and intrinsic viscosity molar mass dependence and stiffness of hyaluronan

Nine hyaluronan (HA) samples were fractionated by size-exclusion chromatography, and molar mass (M), radius of gyration (Rg), and intrinsic viscosity ([eta]) were measured in 0.15 M NaCl at 37 degrees C by on-line multiangle light scattering and viscometer detectors. Using such method, we investigated the Rg and [eta] molar mass dependence for HA over a very wide range of molar masses: M ranging from 4 x 10(4) to 5.5 x 10(6) g/mol. The Rg and the [eta] molar mass dependence found for HA showed a meaningful difference. The Rg = f(M) power law was substantially linear in the whole range of molar masses explored with a constant slope of 0.6. In contrast, the [eta] = f(M) power law (Mark-Houwink-Sakurada plot) showed a marked curve shape, and a linear regression over the whole range of molar masses does not make sense. Also the persistence length (stiffness) for HA was estimated. The persistence length derived by using both the Odijk's model (7.5 nm from Rg vs M data) and the Bohdanecky's plot (6.8 nm from [eta] vs M data) were quite similar. These persistence length values are congruent with a semistiff conformation of HA macromolecules.     

Cylindrical poly(oligo-DNA)

Oligo-desoxyribonuleic acids (ODNs) having a sequence of 5'-TCC ATG ACG TTC-3' were modified at the 5' end by introduction of an amine group via a C6-amino linker. After subsequent reaction of the amine group with N-methacryloyloxysuccinimide, polymerizable ODNs were obtained. Free radical homopolymerization results in the formation of comb polymers, which possess an ODN side-chain at each repeating unit of the main chain. Mainly due to steric repulsion, the main chain has to adopt a semi-flexible wormlike shape instead of the otherwise preferred coiled structure. This leads to the formation of cylindrical poly(oligo-DNA) molecules. Characterization by static and dynamic light scattering of the poly(oligo-DNA) in aqueous solution gave a radius of gyration Rg,app = 67.8 nm, a hydrodynamic radius Rh,app = 44.6 nm, and a characteristic ratio of rho = Rg/Rh = 1.52, indicating the cylindrical shape in solution. In addition, the cylindrical poly(oligo-DNA) molecules were adsorbed onto mica and visualized by atomic force microscopy.     

Pressure cell assisted solution characterization of polysaccharides. 2. Locust bean gum and tara gum

Following the work carried out on guar gum in our first paper of a series, the "pressure cell" solubilization method was applied to two other less highly substituted galactomannans: locust bean gum (LBG) and tara gum. True molecular solution of the polymers was achieved using appropriate temperature, time, and pressure regimes. The technique of capillary viscometry was used to determine the intrinsic viscosity [eta] of the "pressure cell" treated and untreated samples. Molecular weight (M(w)) and radius of gyration (R(g)) were determined by light scattering. The data obtained for LBG and tara gum were compared statistically with reliable data found for guar gum in the literature. The variation in [eta] with M(w) followed the Mark-Houwink-Sakurada relationship, giving the exponent alpha = 0.74 +/- 0.01 for galactomannans consistent with random coil behavior. The characteristic ratio, C(infinity), and the chain persistence length, L(p), were both calculated for LBG and tara gum using the Burchard-Stockmayer-Fixman (BSF) method which is appropriate for flexible to semiflexible chains. A general value of 9 < C(infinity) < 16 and 3 < L(p) < 5 nm can now be estimated with statistical confidence for all galactomannans. According to our statistical analysis, the chain persistence length was found to be insensitive to the degree of galactose substitution.     

Associating hyaluronan derivatives: a novel horizon in viscosupplementation of osteoarthritic joints

The association of two high-molecular-weight hyaluronan (HA) derivatives, namely a beta-cyclodextrin (HA-beta-CD) and an N-acylurea (EDC-HA), dissolved in aqueous NaCl, was studied. The weight-average of the molecular weights (Mw) of HA-beta-CD and of EDC-HA was 185.3 and 86.8 kDa, respectively. However, the Mw value determined for the equimolar mixture of the two biopolymers equaled 556.0 kDa. Similarly, the radius of gyration (dimension) Rg = 80.6 nm of the above equimolar mixture was significantly greater than the values found for the single macromolecules, i.e., 40.2 nm for HA-beta-CD and 23.8 nm for EDC-HA. These data indicate that the two kinds of substituents, borne by the HA polymeric chains, form host-guest inclusion complexes resulting in polymacromolecular associates/aggregates.     

Characterization of pectin, flash-extracted from orange albedo by microwave heating, under pressure

Pectin was acid extracted from orange albedo by microwave heating under pressure. Extraction times ranged from 2.5 to 8 min. Solubilized pectin was characterized for molar mass (M), rms radius of gyration (Rg) and intrinsic viscosity [eta] by HPSEC with online light scattering and viscosity detection. M, Rg and [eta] all decreased with increasing extraction time. Nevertheless, at heating times of 2.5 and 3.0 min, M, Rg and [eta] were significantly higher than a commercial citrus pectin when the albedo:solvent ratio was 1:25 (w/v). At the heating time of 2.5 min Mw was 3.6 x 10(5), Rgz was 38 nm and [eta]w was 10.8 dL/g. Chromatography revealed that solubilized pectin distributions were bimodal in nature and that the low-molar-mass fraction increased at the expense of the high-molar-mass fraction with increasing extraction time. Scaling law exponents revealed that the high-molar-mass fraction was extremely compact in shape, whereas the low-molar-mass fraction was more asymmetric in shape. Possibly these results indicated that at short extraction times, pectin was solubilized as compact aggregated network structures that were broken down to their more asymmetric components with increased heating times.     

Solution properties of gellan gum: change in chain stiffness between single- and double-stranded chains

Nine samples of gellan gum in the sodium form, ranging in weight-average molar mass from 3.47 x 10(4) to 1.15 x 10(5) at 40 degrees C, were investigated by static and dynamic light scattering and viscometry in 25 mM aqueous NaCl both at 40 and at 25 degrees C. The ratios of the molar mass at 25 degrees C (in the ordered state) to that at 40 degrees C (in the disordered state) were in the range of 1.99 to 2.07, supporting the scheme of the conformational transition of gellan gum between a disassociated single chain and an associated chain composed of two molecules. Focusing on the effects of polydispersity, the intrinsic viscosities, radii of gyration, and hydrodynamic radii were analyzed on the basis of unperturbed wormlike chain models. The persistence lengths were evaluated as 9.4 nm at 40 degrees C and 98 nm at 25 degrees C.     

Neutron scattering study of the (gamma-B) catalytic domains of complement proteases activated C1r and C1s

The catalytic domains of activated C1r and C1s, comprising the C-terminal region of the A chain (gamma), disulphide-linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For activated C1s (gamma-B), a molar mass of 45,000 +/- 5000 g/mol, and a relatively large radius of gyration (Rg) of 28 +/- 1 A were determined, excluding a single globular domain. The corresponding values for activated C1r (gamma-B)2 (90,000 g/mol, Rg = 34 +/- 1 A) are consistent with a dimer involving the loose packing of two (gamma-B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data.     

Complex coacervation of whey proteins and gum arabic

Mixtures of gum arabic and whey protein (whey protein isolate, WP) form an electrostatic complex in a specific pH range. Three phase boundaries (pH(c), pHphi(1), pHphi(2)) have been determined using an original titration method, newly applied to complex coacervation. It consists of monitoring the turbidity and light scattering intensity under slow acidification in situ with glucono-delta-lactone. Furthermore, the particle size could also be measured in parallel by dynamic light scattering. When the pH is lowered, whey proteins and gum arabic first form soluble complexes. This boundary is designated as pH(c). When the interaction is stronger (at lower pH), phase separation takes place (at pHphi(1)). Finally, at pHphi(2) complexation was suppressed by the charge reduction of the gum arabic. The major constituent of the whey protein preparation used was beta-lactoglobulin (beta-lg), and it was shown that beta-lg was indeed the main complex-forming protein. Moreover, an increase of the ionic strength shifted the pH boundaries to lower pH values, which was summarized in a state diagram. The experimental pH(c) values were compared to a newly developed theory for polyelectrolyte adsorption on heterogeneous surfaces. Finally, the influence of the total biopolymer concentration (0-20% w/w) was represented in a phase diagram. For concentrations below 12%, the results are consistent with the theory on complex coacervation developed by Overbeek and Voorn. However, for concentrations above 12%, phase diagrams surprisingly revealed a "metastable" region delimited by a percolation line. Overall, a strong similarity is seen between the behavior of this system and a colloidal gas-liquid phase separation.     

Shape, symmetry, hydration and secondary structure of the legumin from Vicia faba in solution

The following physical parameters of the legumin from Vicia faba were determined by means of small-angle X-ray scattering, quasi-elastic light scattering and circular dichroism: molar mass, M = 3.5 X 10(5) g/mol; radius of gyration, Rg = 4.45 nm; maximum dimension, L = 13 nm; translational diffusion coefficient, D0(20),w = 3.38 X 10(-7) cm2 X s-1; alpha-helix content about 15%; content of beta-sheets 10%; dihedral point group symmetry of the molecule 32.     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

Intramolecular complex formation of poly(N-isopropylacrylamide) with human serum albumin

Complexation of human serum albumin (HSA) with poly(N-isopropylacrylamide) (PNIPA) ranging in molecular weight (M(PNIPA)) from 2.1 x 10(4) to 1.72 x 10(6) was studied in an aqueous system (pH 3) containing NaCl as a supporting salt. Dynamic light scattering, static light scattering, electrophoretic light scattering, and dialyzing techniques were used as the experimental tool in a suitable combination. The measurements were performed mainly at 25 degrees C and at 0.01 M NaCl as a function of mixing ratio (r(m), molar ratio of PNIPA to HSA). The results of DLS and ELS evidently demonstrated the formation of a water-soluble complex through mixing of HSA and PNIPA. A detailed analysis of SLS data with the aid of dialysis data revealed that the resulting complex is an "intramolecular" complex consisting of a PNIPA chain with several of bound HSA molecules. Both hydrodynamic radius (R(h)) and radius gyration (R(g)) of intramolecular complexes decreased as r(m) was increased. This result correlated well to the fact that the number (n) of bound proteins per polymer decreases with increasing r(m). The size and the molar mass of the complex became large depending on M(PNIPA), but the increase of M(PNIPA) led to a decrease in n at r(m) < 1. The increase in NaCl concentration from 0.01 to 0.3 M brought about the increase in the size and the molar mass of an intramolecular HSA-PNIPA complex prepared at r(m) = 1.1. This was found to be due to an increase of n. A similar trend was observed when temperature rose from 25 to 32 degrees C (close to lower critical solution temperature of PNIPA). However, the effect of temperature on the increase of was strong in comparison with that of ionic strength. On the basis of these results obtained, the complexation mechanism was discussed in detail.     

Effect of organic anions on the photoreaction of photoactive yellow protein

In order to clarify changes in the structure and surface properties of photoactive yellow protein (PYP) upon light absorption, the spectroscopic properties and solution structure of its photo-intermediate (PYP(M)) were examined in the presence of various anions. At identical ionic strengths, citrate slowed the decay rate of PYP(M) more than acetate. Although the absorption spectrum in the dark was not affected by organic anions, citrate induced a 5-nm blue shift of the absorption maximum for PYP(M). Solution X-ray scattering experiments indicated that the radius of gyration (Rg) and apparent molecular weight in the dark were constant in all buffer systems. However, the Rg of PYP(M) in citrate buffer at high concentration was 16.2 (+/-0.2) A, while the Rg of PYP(M) in acetate buffer was 15.6 (+/-0.2) A. The apparent molecular weight increased 7% upon PYP(M) formation in citrate buffer at high concentration compared to other conditions. These results suggest that citrate molecules specifically bind to PYP(M). A cluster of basic amino acid residues with a hydrogen bond donor would be exposed upon PYP(M) formation and responsible for the specific binding of citrate.     

Structure of a highly substituted beta-xylan of the gum exudate of the palm Livistona chinensis (Chinese fan)

Structural features of the acidic, highly substituted glycanoxylan (LCP; 87% yield) from the gum exudate of the palm, Livistona chinensis, family Arecaceae, were determined. It had [alpha]D -30 degrees, Mw 1.9x10(5) and a polydispersity ratio Mw/Mn of approximately 1.0. Acid hydrolysis gave rise to Rha, Fuc, Ara, Xyl, and Gal, in a 1:6:46:44:3 molar ratio, and 12% of uronic acid was present. LCP had a highly branched structure with side-chains containing nonreducing end-units (% values are approximate) of Araf (15%), Fucp (4%), Xylp (7%), GlcpA, and 4-Me-GlcpA, and internal 2-O- (5%) and 3-O-substituted Araf (8%), and 2-O-substituted Xylp (14%) units. The (1-->4)-linked beta-Xylp main-chain units of LCP were substituted at O-3 (4%), O-2 (17%), and O-2,3 (16%). Partial acid hydrolysis gave 4-Me-alpha-GlcpA-(1-->2)-[beta-Xylp-(1-->4)](0-2)-Xyl, identified by showing that the uronic acids were single-unit side-chain substituents on O-2. Milder hydrolysis conditions removed from O-3 other side-chains containing Fucp and Araf nonreducing end-units and internal Arap, and 2-O- and 3-O-substituted Araf units. Carboxyl-reduced LCP contained 4-O-methylglucose and glucose in a 3.2:1 molar ratio, arising from GlcpA and 4-OMe-GlcpA nonreducing end-units, respectively. The gum contained small amounts of free alpha-Fucp-(1-->2)-Ara, which corresponds to structures in the polysaccharide. Free myo- and D- or L-chiro-inositol were present in a 9:1 ratio.     

Light scattering studies of bovine skin proteodermatan sulfate

The proteodermatan sulfate (PDS) of bovine skin is a low molecular weight proteoglycan with a molecular structure consisting of a protein chain and a sulfated polysaccharide chain covalently linked at the 4-serine of the protein. Static and dynamic laser light scattering methods have been used to determine the weight-average molecular weight, Mw, zeta-average radius of gyration, Rg zeta, and zeta-average translational diffusion coefficient, Dto, zeta, of bovine skin PDS. We have also characterized the two components of PDS, i.e., the protein core and the dermatan sulfate (DS) chain. (The latter contained an N-terminal-linked penta- or tetrapeptide.) Interpretation of the PDS data is complicated by the block copolymer nature of its structure. When appropriate corrections are made, our results indicate that Mw for PDS monomer is 62,000 when dissolved in 4M guanidine hydrochloride (GdnHCl), and increases to 610,000 in 0.15M NaCl. Mw for the core protein in 4M GdnHCl is 39,000, and this also increases substantially to 650,000 in 0.15M NaCl. In contrast, Mw for the DS chain is 24,000 in 0.15M NaCl, indicating that there is minimal self-association of DS in 0.15M NaCl. Thus we conclude that the self-association of PDS involves the protein core. Comparison of Rg zeta and Rh, the average hydrodynamic radius, suggests that trace amounts of aggregation persist for the PDS and its core protein even in 4M GdnHCl. This conclusion is supported by evaluation of the second moments of the dynamic light scattering correlation function. Comparisons of the observed Dto, zeta for PDS with predicted values using hydrodynamic theory are consistent with a "lollipop" conformation for the molecule.     

Low viscosity hydrogel of guar gum: preparation and physicochemical characterization

Guar gum was cross-linked with glutaraldehyde and characterized by GPC, rheology, WADX, SEM and TGA. This guar gum is a galactomannan polysaccharide, that contains small amount of arabinose, glucose and uronic acid, besides galactose and mannose. The polymer has high molar mass, with Mw, Mn and Mv values of 2.0x10(6), 1.2x10(6) and 1.9x10(6)g/mol, respectively. The reticulation follows a slow process and lead to a viscosity increase of 40 times compared with the original gum solution. The final viscosity was similar to that of Hylan G-F 20, a hyaluronate derivative, commercially used in viscosupplementation treatment. The gel contains 95.6% of water and the amount of residual glutaraldehyde is much lower than the LD-50. Porous structure was detected by SEM and thermal stability was improved by the cross-linking. The low viscosity, the small amount of remained glutaraldehyde, and the thermal stability indicates that the guar hydrogel has potential to be applied as biomaterial with specific rheological requirements.     

Chain conformation of sulfated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Six water-insoluble (1-->3)-beta-D-glucan fractions TM8-1 to TM8-6 with weight-average molecular mass Mw ranging from 5.76 to 77.4x10(4) obtained from the sclerotia of Pleurotus tuber-regium were sulfated to produce the water-soluble fractions S-TM8-1 to S-TM8-6 with Mw from 6.0 to 64.8x10(4). The degree of substitution (DS) of S-TM8 fractions was analyzed by elemental analysis (EA) to be 1.14-1.74. The 13C NMR results indicated that the C-6 was fully substituted, and C-2, C-4 were partially substituted by the sulfo-groups. The Mw and the intrinsic viscosity [eta] of the S-TM8 fractions were measured, respectively, by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependences of [eta] and radius of gyration z(1/2) on Mw for the S-TM8 samples were found to be [eta]=1.89x10(-2) Mw(0.70) (cm3/g) and z(1/2)=1.12x10(-4) Mw(0.81) (nm) in the Mw range tested. Based on current theories for a wormlike chain model, the molar mass per unit contour length ML and persistence length q of the S-TM8 were calculated to be 990 nm(-1) and 8.5 nm, respectively. The relatively higher q value suggested a more expanded flexible chain of S-TM8 in PBS. The water-solubility and relatively expanded chain conformation of the STM8 fractions were considered to be significant to their antiviral activity.     

Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering

Aggregation of jack bean urease (JBU) is involved in many alterations of its biological properties, notably the ureolytic and entomotoxic activities. In order to investigate this phenomenon, protein aggregates were characterized by dynamic (DLS) and static light scattering (SLS) spectroscopies through determination of apparent hydrodynamic radii, the average molecular masses, radii of gyration and second virial coefficients. No effect of disulfide reducing agents on protein association was observed contrasting with previous reports implicating their function in the prevention of JBU aggregation. The influence of freeze-thawing cycles on protein aggregation was also investigated. Our results showed that after freeze-thawing cycles the native form of JBU with apparent hydrodynamic radius of 7 nm and radius of gyration of 12 nm is replaced by high-order oligomers and this aggregation is not reverted neither by dithiothreitol (DTT) treatment nor by high concentration of salts. Altogether the data help to understand the complex behavior of JBU in solution and may correlate with the diversity of biological properties of this enzyme.