Periploca sepium Bunge as a model plant for rubber biosynthesis study

Periploca sepium Bunge (Chinese silk vine) is a woody climbing vine belonging to the family Asclepiadaceae. It originally comes from Northwest China. Periploca resembles the Para-rubber tree, Hevea brasiliensis, regarding a similar body plan to produce a milky exudate containing rubber latex. The Periploca plant was assessed as a rubber-producing plant by rubber structure elucidation and its molecular weight distribution. A rubber fraction purified from the milky exudate was subjected to 1H NMR analysis, and a characteristic signal derived from cis-polyisoprene was observed. In addition, when the molecular weight distribution of rubber components in the exudate was measured (using size-exclusion chromatography), the number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity (Mw/Mn) were estimated to be Mn = 1.3 x 10(5), Mw = 4.1 x 10(5), and Mw/Mn = 3.1, respectively. Furthermore, the presence of polyisoprene, with Mn = 4.0 x 10(4), Mw = 7.6 x 10(4), and Mw/Mn = 2.5, was also confirmed in plantlets obtained from shoots as a result of tissue culture.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

The thermal depolymerization of porcine submaxillary mucin

The time dependence of the molecular weight, radius of gyration, and hydrodynamic size distribution for porcine submaxillary mucin (PSM) in solution have been studied using static and dynamic light scattering. The weight average molecular weight (Mw) of PSM in 6 M guanidine HCl, pH 7, is initially 3 X 10(6) and decreases with time in three phases: rapidly from 3-2 X 10(6), less rapidly from 2-0.9 X 10(6), and slowly below 0.9 X 10(6). The rates of decrease are much greater at pH 2. The energy of activation associated with each phase is 20 kcal/mol, which is similar to that reported for peptide bond cleavage at an aspartic acid residue. Addition of mercaptoethanol to PSM in 6 M guanidine HCl leads to a rapid decrease in Mw to 0.9 X 10(6), followed by a very slow further decrease. These results suggest that native PSM consists of subunits (Mw = 0.9 X 10(6] that are linked by disulfide bonds to form dimers (Mw = 2 X 10(6] and then higher aggregates. This cross-linking appears to occur at unglycosylated regions of the protein core, which are believed to be richer in aspartic acid than the rest of the molecule.     

Determination of the molecular weight of microbial polysaccharides using high performance exclusion chromatography

An automatic method of determining the molecular weight parameters (Mw, Mn) of microbial polysaccharides such as dextran, pullulan was developed based on the use of high performance size-exclusion chromatography on the two types of columns: Zorbax PSM 60 + 300 + 1000 and SynChropack GPC 100 + 500 + 1000. The Mw and Mn values were determined for a number of domestic and foreign dextran preparations. Changes in the molecular weight of pullulan and hydroxyethylstarch resulted from acid and enzymatic hydrolysis were estimated.     

Preparation and characterisation of copper(II) hyaluronate

Amorphous copper complexes of the general composition Cu(C14H20O11N)2 x xH2O have been prepared with high- and low-molecular-weight hyaluronic acid (HA). Optimal conditions for preparation are obtained at pH values from 5.0 to 5.5, with a molar ratio of HA versus Cu2+ of 1:1, and at a mass concentration of 5 and 10 mg/mL for high- (Mw = 1.8 x 10(6) Da) and low-molecular-weight sodium hyaluronate (Mw = 2 x 10(5) Da), respectively. The coordination polyhedron of the copper ion has been elucidated by EXAFS and XANES spectroscopy. Copper atoms are octahedrally coordinated in both cases with four equatorial Cu-O bond lengths of 1.95 A, and two axial Cu-O bonds of 2.46 A. Visible spectra of acidic aqueous solution suggest that substitution of axial oxygens by NH groups occurs at pH 6.5 or higher. If the pH value of the copper(II) hyaluronate solution increases above 6.5, the coordination of copper(II) changes. It is very likely that the N atom coming from the acetamido group enters into the coordination sphere of the copper(II) ion.     

Correlation between the antitumor activity of a polysaccharide schizophyllan and its triple-helical conformation in dilute aqueous solution

Eight samples of a polysaccharide schizophyllan ranging in weight-average molecular weight Mw (in water) from 5 x 10(3) to 1.3 x 10(5) were prepared and their antitumor activity (expressed in terms of the tumor inhibition ratio) against Sarcoma 180 ascites, intrinsic viscosities [eta], and gel-filtration chromatograms in aqueous solution were determined. The tumor inhibition ratio was essentially unity for samples with Mw higher than 9 x 10(4), but reduced to zero or even to a negative value when Mw was lower than 10(4). The [eta] data combined with the chromatographic data showed that above Mw approximately 9 x 10(4) the predominant species of schizophyllan in aqueous solution is the previously found rigid triple helix, whereas below Mw approximately 9 x 10(4) both triple helices and single chains coexist in the solution and the fraction of triple helices decreases monotonically to zero as Mw is decreased to 5 x 10(3). From these findings it was concluded that the antitumor potency of schizophyllan in water is related to the amount of triple helices relative to that of single chains.     

Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown

Leaks were induced in human erythrocytes by brief (tau = 1-40 microseconds) discharges of high electric fields (3-20 kV/cm). Leak permeabilities were characterized by measuring (a) net and tracer fluxes of K+ and nonelectrolytes under protection of the cells against colloid-osmotic lysis, or (b) rates of colloid osmotic lysis in various salt solutions. The induced permeabilities are essentially stable for hours at 0-2 degrees C. Leak permeability P increases exponentially with the breakdown voltage ED according to a function of the general type P = bED. The basis b varies with the pulse length. A log-linear presentation reveals a biphasic linear relationship with a break at which the slope (= log b) decreases markedly. Elevated ionic strengths of the suspension medium during the electric discharge enhance leak formation. Leak permeability exhibits an apparent activation energy of 29 +/- 5 kJ/mol, indicative of diffusion through aqueous pathways. Somewhat differing equivalent pore radii emerge from measurements with different probes: 0.6-0.8 nm from tracer fluxes of polyols (Mr = 3600, ED = 4-7 kV/cm) and 0.8-1.9 nm from osmotic protection studies with polyethylene glycols (Mr = 200-3300, ED = 6-10 kV/cm). These numbers and the non-monoexponential increase of leak permeability with the field strength suggest a dual mechanism for the increase of leak permeability: an increase of the number of pores at low breakdown voltage and an additional increase of pore size at higher voltage. Estimated numbers of pores range from 1 to 10 per cell, which suggests dynamic fluctuating structural defects to be involved. The leaks discriminate small monovalent inorganic ions in the sequence of free solution mobility. Organic anions are discriminated according to size and charge. Common properties of these electrically induced defects and of chemically induced leaks (diamide, periodate, t-butylhydroperoxide) in the erythrocyte membrane suggest close similarities in the molecular organization.     

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.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA.

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

Chain conformation of water-insoluble hyperbranched polysaccharide from fungus

Water-insoluble polysaccharide (TM3a), extracted from sclerotia of Pleurotus tuber-regium, was identified as a hyperbranched beta-d-glucan from the results of one- and two-dimensional NMR and GC-MS analysis. The degree of branching of TM3a is 65.5%. TM3a was fractionated by using a non-solvent addition method into 14 fractions, and its solution properties in 0.25 M LiCl/dimethylsulfoxide (DMSO) solution were studied systematically by using static laser light scattering, dynamic light scattering, and viscometry at 25 degrees C. The dependences among the values of intrinsic viscosity ([eta]), radius of gyration (z 1/2), and hydradynamic radius (Rh) on weight-average molecular weight (Mw) were found as the following: [eta] = 0.46Mw0.30+/-0.01, z 1/2 = 4.79 x 10-2Mw0.43+/-0.04, and Rh = 5.01 x 10-2Mw0.41+/-0.02 in the Mw range from 1.94 x 105 to 2.06 x 107 for TM3a in a 0.25 M LiCl/DMSO solution at 25 degrees C. The current theory of polymer solution was applied to explain the relationship among the fractal dimension, ratio of geometric to hydrodynamic radius (rho = z 1/2/Rh), and MwA2/[eta] of TM3a. The results indicated that TM3a existed as a compact chain conformation with a sphere-like structure in LiCl/DMSO solution. Furthermore, by using transmission electron microscopy, we observed directly the spherical molecules with an average diameter of 23.0 +/- 1.8 nm.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Characterization of epidermal growth factor receptor in rat buccal mucosal cells

1. Receptor binding for epidermal growth factor (EGF) in rat buccal mucosa was characterized. Binding of [125I]EGF to rat buccal mucosa was time, temperature, cell number and [125I]EGF concentration dependent. 2. The [125I]EGF binding was reversible and specific. Unlabeled EGF competed for binding to buccal mucosal cells with an IC50 of 1.25 nM, whereas insulin failed to compete. 3. Scatchard analysis of the binding data revealed a curvilinear plot with dissociation constants of 3.39 nM and 2.14 microM, and binding capacities of 1.23 x 10(4) and 3.38 x 10(5) receptors per cell for high and low affinity sites, respectively. 4. Crosslinking of [125I]EGF to buccal mucosa followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major protein with Mw 170,000 which shares similar molecular weight with other known EGF receptors from different tissues and species. 5. The study is the first report to provide biochemical parameters of the specific EGF receptors in rat buccal mucosa.     

Activation of lipid peroxidation in the adrenal cortex by metal ions

The processes of lipid peroxidation have been studied in bovine adrenal cortex in vitro. The lipid peroxidation rate in this tissue is shown to be dependent on the content of metal ions. EDTA, deferroxamine and penicyllamine inhibit spontaneous lipid peroxidation by 25, 50 and 42%, respectively. The ability to activate the process permits arranging metal ions in the following sequence: Fe2+ greater than Fe3+ greater than Cu2+ greater than Mg2+ greater than Mn2+. The maximum activation of lipid peroxidation is observed at Fe2+ and Fe3+ concentrations within the range of 5 x 10(-6) x 10(-4) M.     

Interaction of divalent cations with beta-galactosidase (Escherichia coli).

Although the addition of various divalent metals to beta-galactosidase resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was shown to be due to Mg2+ impurities. Calcium did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The dissociation constants for Mg2+ and Mn2+ were 2.8 X 10(-7) and 1.1 X 10(-8) M, respectively, and in each case one ion bound per monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 X 10(-5) M. Data were obtained which showed that Mg2+, Mn2+, and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a twofold drop in the Vmax for the hydrolysis and transgalactosylis reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl beta-D-galactopyranoside (PNPG), or o-nitrophenyl beta-D-galactopyranoside (ONPG); Km values were not effected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the three divalent metal ions which bound to beta-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.     

NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II)

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.     

Physicochemical properties and antitumor activities of water-soluble native and sulfated hyperbranched mushroom polysaccharides

A water-soluble hyperbranched beta-glucan, coded as TM3b, extracted from sclerotia of an edible fungus (Pleurotus tuber-regium) was fractioned into eight fractions coded as F1-F8 by a nonsolvent addition method. Five fractions were treated with chlorosulfonic acid at 35 degrees C to synthesize successfully sulfated derivatives coded as S-F2, S-F3, S-F4, S-F5, and S-F8 with degree of substitution of 0.28-0.54. The 13C NMR results of these sulfated beta-glucans indicated that while the C-6 position was fully substituted, C-2, C-3, and C-4 were only partially substituted by the sulfate groups. The weight-average molecular weights (Mw) and intrinsic viscosities ([eta]) of the native and sulfated TM3b fractions were determined using multi-angle laser light scattering and viscometry in 0.15M aq NaCl at 25 degrees C, respectively. The dependences of [eta] on Mw for TM3b and sulfated TM3b were found to be [eta]=0.18Mw(0.28+/-0.03) (Mw range from 3.30 x 10(4) to 3.90 x 10(7)) and [eta]=2.24 x 10(-2)Mw(0.52+/-0.06) (Mw range from 3.24 x 10(4) to 3.15 x 10(5)) in 0.15M aq NaCl at 25 degrees C, respectively. It revealed that both the native TM3b and its sulfated derivatives exist in a spherical chain conformation in 0.15M aq NaCl. Furthermore, the native and sulfated TM3b fractions showed potent antitumor activities in vivo and in vitro. The sulfated derivatives exhibited relatively higher in vitro antitumor activity against human hepatic cancer cell line HepG2 than the native TM3b. Water solubility and introduction of sulfate groups were the main factors in enhancing the antitumor activities.     

Ferriprotoporphyrin IX mediated oxygen activation/insertion reactions: the anerobic reduction of the aniline-Fe3+-microperoxidase-8 complex by NADH

A spectrophotometric study of the reduction of the Fe3+ microperoxidase-8-aniline (Fe3+-MP-8-An) complex has been carried out. Addition of NADH to a solution of Fe3+-MP-8-An under strictly anerobic conditions results in the formation of a species with lambda max = 414 nm (Fe3+-MP-8-An lambda max 407 nm). The kinetics of formation of this species show an induction period (tau) which follows saturation kinetics with respect to [aniline] with Km(app) = 2.2 x 10(-3) mol dm-3, i.e., close to that obtained in the preceding paper from O2 consumption kinetics mediated by MP-8. Addition of an anerobic solution of the NADH reduced MP-8-An complex, to a saturated O2 solution at pH 12 in the presence of 0.5 mM NADH and aniline 10 mM results in the virtual elimination of the induction phase, which has previously characterized O2 consumption kinetics in ferriprotoporphyrin IX oxygen activation systems. The Arrhenius activation energy for the reduction of the Fe3+-MP-8-An complex is close to that observed for the first reductive step in the cyt P-450 O2 activation cycle. Anerobic reduction of Fe3+-MP-8 by sodium dithionite in 20% MeOH/Aq at pH 8 followed by anerobic titration of the Fe2+-MP-8 (lambda max 420.5 nm) with aniline at pH 12 gives rise to a species lambda max 415 with KD for the process = 4.4 x 10(-3) mol dm-3 (+/- 1.2 x 10(-3) mol dm-3).     

Poly(styrene/alpha-tert-butoxy-omega-vinylbenzylpolyglycidol) microspheres for immunodiagnostics. Principle of a novel latex test based on combined electrophoretic mobility and particle aggregation measurements

The principle of a novel latex agglutination test based on combined results of electrophoretic mobility and particle aggregation measurements is described. Poly(styrene/alpha-tert-butoxy-omega-vinylbenzylpolyglycidol) (P(S/PGL)) microspheres were synthesized by a one step soap-free emulsion copolymerization of styrene and alpha-tert-butoxy-omega-vinylbenzylpolyglycidol macromonomer with number average molecular weight Mn = 2700 (polydispersity [Mw]/[Mn] = 1.10). Particles with monomodal size distribution (number average diameter Dn = 220 nm) and surface fraction of polyglycidol equal to f = 0.42 mol % were obtained. Human serum albumin (HSA) was covalently bound onto the surface of P(S/PGL) microspheres activated with 1,3,5-trichlorotriazine. In a model immunodiagnostic assay for anti-HSA, in which P(S/PGL) particles with covalently bound HSA have been used, the electrophoretic mobility and aggregation of microspheres were measured simultaneously. This approach allowed detection of anti-HSA in the serum in the range of anti-HSA concentrations from 0.1 to 150 microg/mL. The highest changes in electrophoretic mobility were registered for microspheres with surface concentration of immobilized HSA equal to Gamma = 9.2 x 10(-4) g/m2.