Molecular mass and antitumor activities of sulfated derivatives of alpha-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1-->3)-alpha-D-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1-S5 and ac-PCM3-I-S1-S5, respectively. The derivatives having different weight-average molecular mass (Mw) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39-0.67 for ab-PCM3-I-S and 0.73-0.96 for ac-PCM3-I-S, respectively. The Mw and the intrinsic viscosity ([eta]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The results indicated that their Mw ranged from 2.0 to 11.3 x 10(4) for the samples ab-PCM3-I-S, and 4.7 to 40.0 x 10(4) for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native alpha-D-glucans. Therefore, a moderate range of molecular mass from 2.0 x 10(4) to 40.0 x 10(4), relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Characterization of polysaccharide-protein complexes by size-exclusion chromatography combined with three detectors

Two water-soluble samples (TM1 and TM2) were extracted from Pleurotus tuber-regium using 0.9% aqueous NaCl at 20 and 80 degrees C to obtain relatively low molecular mass fractions. The chemical structure of TM1 was analyzed to be a branched heteropolysaccharide-protein complex, and the sugar moiety was mainly beta-(1-->6)-, beta-(1-->4)-, and beta-(1-->3)-linked glucan containing galactose and mannose. TM2 was a branched polysaccharide-protein complex, and the sugar moiety was mainly beta-(1-->6)-, beta-(1-->4)-, and beta-(1-->3)-linked glucan. Preparative size-exclusion chromatography (SEC) and analytical SEC combined with three detectors were used to detect the TM1 and TM2 samples, confirming that the proteins were bonded to the polysaccharides. Furthermore, analytical SEC combined with online laser light scattering, differential refractive index detector, and UV to determine the components, weight-average molecular mass (M(w)) and chain conformation of the samples. The relatively low exponent values (nu) of S(2)(z)(1/2)=k(nu)M(w)(nu) for the samples in 0.15M aqueous NaCl at 25 degrees C suggested that TM1 and TM2 existed in compact sphere conformation in the aqueous solution. This work provided valuable information on the structure and chain conformation characterization of the polysaccharide-protein complex having relatively low M(w).     

Fractionation and characterization of a Polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography

A kind of regenerated cellulose gel (RCG) particles were treated by toluene to obtain particles with smaller mean pore size, then was mixed with the cellulose gel with pore size of 370 and 525 nm. A preparative size-exclusion chromatography (SEC) column (700 x 20 mm) packed with three gel particles was used to fractionate water-soluble polysaccharide (WEP) extracted from the sclerotia of Pleurotus tuber-regium by aqueous solution. The exclusion limit and fractionation range of the stationary phase of the preparative SEC were molecular mass 8 x 10(5) and 5 x 10(3) to 8 x 10(5), respectively. The calibration curve of the preparative SEC was represented as: log M=13.96-0.53 Ve. The WEP sample (weight-average molecular mass M(w)=2.2 x 10(4), polydispersity=2.4) was divided into three fractions with M(w) ranging from 1.4 x 10(4) to 3.4 x 10(4) by the preparative SEC column, and the fractions were characterized by gas chromatography GC, SEC combined with laser light scattering (SEC-LLS) and viscometry. The unfractionated WEP exhibited triple peaks due to different molecular mass, but each fraction exhibited single peak with the polydispersity of 1.1-1.8 in the SEC patterns. The results indicated that the preparative SEC was efficient for fractionation of polysaccharides having low molecular weight and for determination of their molecular mass.     

Molecular mass and chain conformation of carboxymethylated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Seven water-insoluble (1 --> 3)-beta-D-glucan fractions TM8-1 to TM8-7 with weight-average molecular mass M(w) ranged from 2.22 to 77.4 x 10(4) obtained from the sclerotia of Pleurotus tuber-regium were carboxymethylated to produce the water-soluble fractions CTM8-1 to CTM8-7 with M(w) ranged from 3.87 to 87.8 x 10(4). The degree of substitution (DS) of CTM8 fractions was analyzed by ir and elemental analysis (EA) to be 0.3-0.68. The M(w) and the intrinsic viscosity [eta] of the CTM8 fractions were measured by size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), MALLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependencies of [eta] and radius of gyration (z) (1/2) on M(w) for the CTM8 samples were found to be [eta] = (8.82 +/- 0.03) x 10(-3) M(w)(0.78 +/- 0.04) (cm(3) g(-1)) and (z) (1/2) = (3.09 +/- 0.05) x 10(-3) M(w)(0.75 +/- 0.06) (nm) in the M(w) range from 3.87 x 10(4) to 53.2 x 10(4). Based on current theories for wormlike chain model, the conformational parameters of the CTM8 were obtained to be 790 (nm(-1)) for M(L), 9.6 (nm) for q, which were higher than those of the native TM8 fractions, suggesting a more extended flexible chain of CTM8 in PBS. On the whole, the CTM8 fractions showed higher antitumor activity than their corresponding TM8 fractions. In view of data from molecular parameters and bioactivity, the antitumor activity of the CTM8 fractions may be correlated to its water solubility and relatively extended chain.     

Correlation between antitumor activity, molecular weight, and conformation of lentinan

A (1-->3)-beta-D-glucan having (1-->6) branching (L-FV-IB) from Lentinus edodes in water was degraded into seven fractions of different molecular weights by ultrasonic irradiation, and each was further fractionated into three parts, by precipitation from water into acetone at room temperature. The weight-average molecular weight (M(w)), radius of gyration ((z)(1/2)), and intrinsic viscosity ([eta]) of lentinan and its fractions in 0.9% NaCl aqueous solution and dimethyl sulfoxide (Me(2)SO) were determined by size-exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS), LLS, and viscometry. Analysis of M(w), [eta], and (z)(1/2) in terms of known theory for worm-like chains yielded 2240 +/- 100 nm(-1), and 100 +/- 10 nm for molar mass per unit contour length (M(L)), and persistence length (q), respectively, corresponding with theoretical data for triple-helical chains. The [alpha](D) of lentinan in water-Me(2)SO mixtures indicated an order-disorder transition. The results indicated that lentinan exists as a triple helix in 0.9% NaCl aqueous solution and as a single flexible chain in Me(2)SO. Assays in vivo and in vitro against the growth of Sarcoma 180 solid tumor as well as the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method for lentinan showed that the triple-helix sample exhibited a relatively high inhibition ratio. Interestingly, the triple-helix lentinan with M(w) of 1.49 x 10(6) exhibited the highest antitumor activity in vivo, having an inhibition ratio (xi) of 49.5%, close to that of 5-fluorouracil (xi = 50.5%), whereas the bioactivity (xi = 12.3%) of its single flexible chains almost disappeared. The triple-helix conformation plays an important role in enhancing the antitumor effects of lentinan.     

Polycondensation of dicarboxylic acids and diols in water catalyzed by surfactant-combined catalysts and successive chain extension

Direct dehydration polycondensation of dicarboxylic acids and alcohols was carried out by surfactant-combined Br?nsted and Lewis acids. This procedure did not require the removal of water, because the esterification was established at the interface of the emulsion in water. Emulsion polycondensations of 1,9-nonanediol (1,9-ND) and dodecanedioic acid (DDA) (the molar ratio of dicarboxylic acid to diol = 1:1) were carried out at 80 degrees C for 48 h in the presence of 16 wt % DBSA. The corresponding polyester (M(w) = 10.1 x 10(3)) was obtained in an excellent yield (99%). Chain extension in the emulsion was carried out using hexamethylene diisocyanate as the chain extender. SEC measurements indicated the expected shift to higher molecular weight region (M(w) = 11.4 x 10(3), M(w)/M(n) = 3.4) compared with parent polyester (M(w) = 4.5 x 10(3), M(w)/M(n) = 2.2).     

Characterization of a polysaccharide-protein complex from Ganoderma tsugae mycelium by size-exclusion chromatography combined with laser light scattering

A water-soluble polysaccharide-protein complex (GM3) extracted from the mycelium of Ganoderma tsugae was characterized using size-exclusion chromatography combined with laser light scattering (SEC-LLS). Two peaks coded as fractions I and II appeared in the SEC pattern of GM3 in 0.5 M NaCl aqueous solution, corresponding to the weight-average molecular mass (M(w)) of 355 x 10(4) and 6.3 x 10(4), respectively. The relationship between the radius of gyration ((z)(1/2)) and M(w) showed that molecules of fraction I exhibited more compact coil conformation than that of fraction II in 0.5 M NaCl aqueous solution at 25 degrees C. To clarify the component of polysaccharide and protein in each fraction, the sample GM3 was treated with 0.2 M NaOH aqueous solution to degrade polysaccharide and trypsin to hydrolyze protein. The obtained products were analyzed by SEC combined with detectors such as UV, differential refractive index (DRI) and LLS. The results indicated that both the fractions I and II were protein-bound polysaccharide, but had different protein content and degree of branching, resulting in the difference of the chain conformation.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

Enhancement of superantigen activity and antitumor response of staphylococcal enterotoxin C2 by site-directed mutagenesis

Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vβ domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immuntherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.     

Location of F1 ATPase-like Genes on the Physical Map of the Bacillus subtilis 168 Chromosome

A water-soluble polysaccharide, FI, extracted from the mycelium of Granoderma tsugae, was fractionated and purified by ion-exchange chromatography, gel filtration, and affinity chromatography. Sixteen polysaccharides obtained were examined for antitumor effects on Sarcoma 180 in mice.

The three active polysaccharides obtained were as follows:

FI₀-a: A glycan-protein complex containing 9.3% protein, and having a hetero-glyco-chain of mannose and xylose.

FI₀-b-α: Molecular weight 10,000, glucan-protein complex containing 25.8% protein. The inhibition ratio was 61.8% against the solid cancer Sarcoma 180/mice; the survival ratio was more than 194% of the control group (100).

FA-1-b-α: Molcular weight 16,000, a complex of glycan: protein = 42:58 w/w, consisting of glucose as a main component, and associated with arabinose, mannose, xylose, and galactose. This had a tumor inhibition ratio of 56% and a survival ratio of more than 182%.

Comparison of active glycan with the fruiting body and mycelium: Among water-soluble polysaccharides of fruiting body, FI₀-a and FA-1, with antitumor activity, were both glucogalactan-protein complexes of molecular weight 10,000, but that of mycelium was a homoglucan protein complex in FI₀-b-α and heteroglucan protein in both FA-l-a and FA-l-b-α. The heteroglucan had a low tumor inhibition ratio, but caused a high survival ratio in mice.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

Refolding of recombinant human interferon ��-2a from Escherichia coli by urea gradient size exclusion chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon ??-2a (rhIFN??-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhIFN??-2a would pass along the 8.0?C3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 × 10⁸ IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhIFN??-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhIFN??-2a refolding in terms of specific activity and mass recovery.     

Comparative analysis of glycoprotein and glycolipid composition of virulent and avirulent strain membranes ofMycoplasma hyopneumoniae

The glycoproteins and glycolipids from membranes of virulent strain Z and avirulent strain M ofMycoplasma hyopneumoniae have been compared. The proteins and the glycoproteins were identified by SDS-polyacrylamide gel electrophoresis and concanavalin A-biotin labeling, respectively. The membrane preparation contained approximately 34 protein bands with molecular weights between 20 KD and 100 KD. The concanavalin A-biotin system reacted with a glycoprotein of a molecular weight of approximately 28,000 from avirulent strain M and did not react with the correspondent band from virulent strain Z. The membrane glycolipids of both strains consisted of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the percentages of 160, 180, and 181 fatty acids comprised more than 80% of the total fatty acids of membrane glycolipids. The 180 fatty acid of MGDG in avirulent strain M was twofold higher than that of virulent strain Z.     

Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform

Optimization was carried out for the recovery of microbiol poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90-min treatment time, and a 1:1 (v/v) chloroform-to-aqueous-phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, M(n) of recovered PHB was about 300,000 and the weight average molecular weight M(w) was about 1,020,000, compared to the original M(n) of 530,000 and M(w) of 1,272,000. The moderate decrease in both M(n) and M(w) might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in M(w) probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. (c) 1994 John Wiley & Sons, Inc.     

Distribution of elements binding to molecules with different molecular weights in aqueous extract of Antarctic krill by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000-20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000-20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.     

Fractionation and characterization of a conjugate between a polymeric drug-carrier and the antitumor drug camptothecin

A conjugate between the antitumor drug camptothecin and the polymeric drug-carrier poly[N-(2-hydroxypropyl)methacrylamide] was synthesized and fractionated. The conjugate samples, both fractionated and unfractionated, were characterized with a multi-detector SEC system using three on-line detectors: a multi-angle light scattering photometer, a viscometer, and a refractometer. The used mobile phase (DMF + 0.01 M LiBr + 0.05 M CH(3)COOH) derives from previous experience with similar conjugates. Narrow molar mass distribution fractions of the conjugate obtained by means of a semipreparative LC system were used to derive the coefficients of the Mark-Houwink-Sakurada relationship and to check the universal calibration of the SEC system. This study has demonstrated that the conjugate elutes according to the hydrodynamic volume. Thus, a conventional SEC method that uses only an on-line refractometer detector, commercially available narrow standards, and the universal calibration is adequate for the characterization of the molar mass distribution. Also the size and the conformation of the conjugate were studied by means of the gyration radius-molar mass power law.     

In vitro degradation behavior of microspheres based on cross-linked dextran

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time. The amount and nature of the formed degradation products were established for microspheres with different cross-link densities by FT-IR (Fourier transformed infrared spectroscopy), NMR, mass spectrometry, SEC analysis, and XPS (X-ray photoelectron microscopy). The dex-HEMA microspheres DS 12 (degree of HEMA substitution; the number of HEMA groups per 100 glucose units) incubated at pH 7.4 and 37 degrees C showed a continuous mass loss, leaving after 6 months a residue of about 10% (w/w) of water-insoluble products. NMR, mass spectrometry, and SEC showed that the water-soluble degradation products consisted of dextran, low molecular weight pHEMA (M(n) approximately 15 kg/mol), and small amounts of unreacted HEMA and HEMA-DMAP (intermediate reaction product of the Baylis-Hillman reaction of HEMA with DMAP (4-dimethyl aminopyridine)). Microscopy revealed that the water-insoluble residue consisted of particles with shape and size similar to that of nondegraded microspheres. However, these particles had lost their mechanical strength as evidenced from micromanipulation experiments. FT-IR and XPS (X-ray photoelectron microscopy) revealed that these particles consisted of pHEMA, of which a small fraction was soluble in methanol (M(n) ranging between 27 and 82 kg/mol). The insoluble material likely consisted of lightly cross-linked pHEMA. In conclusion, in vitro degradation of dex-HEMA microspheres results in the formation of water-soluble degradation products (mainly dextran), leaving a small water-insoluble residue mainly consisting of pHEMA.