DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0–2.5 S, corresponding to a molecular weight of about 20 000–25 000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100–200 mM NaCl and is relatively independent of temperature and pH. MgCl₂ and MnCl₂ at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4·10^?11 M. There are at least 1·10⁵ DNA-binding protein molecules/HeLa cell.     

Binding Stoichiometry of the Gene 32 Protein of Phage T4 in the Complex with Single Stranded DNA Deduced from Boundary Sedimentation

Abstract

Short 145 base DNA fragments in complex with the helix destabilizing protein of bacteriophage T4, GP32, have been studied with boundary sedimentation. The sedimentation coefficient was determined as a function of concentration, protein-nucleic acid ratio, temperature and salt concentration. It can be concluded that the measured values reflect the properties of the saturated DNA-GP32 complex. A combination of the earlier obtained translational diffusion coefficient of the complex with the sedimentation coefficient yields its anhydrous molecular weight (M_w = 5.4 · 10^s D), which corresponds to a size of the binding site of 10 nucleotides per protein. This procedure is not sensitive to the presence of non-binding protein molecules and to the assumed protein concentration, and therefore, it seems more reliable than a determination from titration experiments.

Similar sedimentation measurements were performed with tRNA-complexes containing 76 nucleotides. The translational diffusion coefficient can be calculated from the measured rotational diffusion coefficient and assuming the same hydrodynamic diameter for this complex as obtained for the 145 b DNA complex. The molecular weight derived from the data then also leads to a binding site size of about 10 nucleotides. This suggests that also the short tRNA-complex forms an open, strongly solvated structure, as was proposed for the 145 b DNA-GP32 complex.     

Juvenile hormone-specific esterases in the haemolymph of the tobacco hornworm, Manduca sexta

A new sensitive method for determining juvenile hormone (JH) hydrolysis has been developed which measures the release of tritiated methanol from JH labelled in the methyl ester group. Using this assay we investigated the interaction of JH with haemolymph esterases and haemolymph JH-binding protein. Haemolymph from fifth instar larvae of Manduca sexta contains two families of esterases which can be distinguished by their reactivity with diisopropylphosphorofluoridate (DFP). One group consists of general esterases which are capable of hydrolysing free JH but not JH complexed to the binding protein and are completely inhibited by low concentrations of DFP (10⁻⁴ M). The other group (JH-specific esterases), relatively DFP resistant, has little detectable general esterase activity but can hydrolyse JH bound to the binding protein as well as free JH. The major JH-esterase has a sedimentation coefficient of 4·98 S and a diffusion coefficient of 6·4 × 10⁻⁷ cm² sec⁻¹. The molecular weight calculated from these values is 6·7 × 10⁴. The general esterases are present throughout the larval stage, but the JH-specific esterases are barely detectable until the fourth day of the fifth instar when they suddenly appear at a high concentration. Since the general esterases cannot hydrolyse bound JH, one function of the binding protein is to protect JH during transport in the early instars, thus confirming that the binding protein is a true carrier of JH. In the late fifth instar prior to metamorphosis, however, JH-specific esterases appear in the haemolymph resulting in the hydrolysis of JH complexed to the carrier protein. Thus, by lowering JH titre, the JH-esterases play an important rôle in development in M. sexta.     

Electron microscopy of a eukaryotic single-stranded DNA binding protein-DNA complex

The single-stranded DNA binding protein of Ustilago maydis decreases the contour length of φX174 DNA. When DNA complexes were prepared with subsaturating amounts of the protein, its distribution on the DNA was markedly non-random, indicating a high degree of co-operativity in its binding to single-stranded DNA. The analagous Escherichia coli, Salmonella typhimurium and bacteriophage T7 binding proteins also reduced DNA contour lengths to a similar extent, whereas the bacteriophage T4 gene 32 protein, as shown previously, increased the contour length. Despite the fact that the U. maydis protein efficiently denatures poly[d(A-T) · d(A-T)], it appears to initiate denaturation of native bacteriophage λ DNA rather inefficiently.     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein.

The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.     

Influence of the concentrations of d-xylose and yeast extract on ethanol production by Pachysolen tannophilus

To determine the most favorable conditions for the production of ethanol by Pachysolen tannophilus, this yeast was grown in batch cultures with various initial concentrations of two of the constituents of the culture medium: d-xylose (s_o), ranging from 1 g·l⁻¹ to 200 g·l⁻¹, and yeast extract (l_o), ranging from 0 g·l⁻¹ to 8 g·l⁻¹. The most favorable conditions proved to be initial concentrations of S_o=25 g·l⁻¹ and l_o=4 g·l⁻¹, which gave a maximum specific growth rate of 0.26 h⁻¹, biomass productivity of 0.023 g·l⁻¹·h⁻¹, overall biomass yield of 0.094 g·g⁻¹, specific xylose-uptake rate (q_s) of 0.3 g·g⁻¹·h⁻¹ (for t=50 h), specific ethanol-production rate (q_E) of 0.065 g·g⁻¹·h⁻¹ and overall ethanol yield of 0.34 g·g⁻¹; q_s values decreased after the exponential growth phase while q_E remained practically constant.     

Characterization of hemoglobin from Phoronis architecta (phoronida)

• 1.1. Phoronis architecta hemoglobin is composed of four distinct hemoglobin subunits with minimum MW's of 16–17,000 or 17–19,000 daltons. All four hemoglobins are monomeric when oxygenated. Two of the monomers combine to form dimers when bound with carbon monoxide.
• 2.2. In cellulo, Phoronis architecta hemoglobin has a half-saturation (P₅₀) value of 1.3 ± 0.1 mm Hg, shows cooperative oxygen binding (Hill coefficient = 2.7 ± 0.3), and no Bohr effect from pH 6.6 to 7.9. In vitro, the hemoglobin has a P₅₀ of 0.76 ± 0.21 mm Hg but shows no cooperativity (0.90 ± 0.15 (SD)).
• 3.3. The oxygen dissociation constant (K_off) from hemoglobin is 2.7 ± 0.2 sec⁻¹, and the computed oxygen association constant (K_on) is 2.5 × 10⁶ M⁻¹ · sec⁻¹ (1.9–3.6 × 10⁶ M⁻¹ · sec⁻¹).

     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

The DNA-binding protein of Pf1 filamentous bacteriophage: amino-acid sequence and structure of the gene

The amino-acid sequence of the single-stranded DNA-binding protein of bacteriophage Pf1 and the nucleotide sequence of the corresponding gene have been determined. The protein has 144 amino acids and a molecular weight of 15 400; the gene consists of 435 nucleotides. The amino-acid sequence was determined by Edman degradation, carboxypeptidase A, B, and P digestion of intact protein and of peptides derived by chymotrypsin, Staphylococcus aureus V8 protease, and trypsin digestion. The nucleotide sequence was determined by the dideoxy method after random cloning of fragments of Pf1 DNA into M13. No sequence homology could be established between the amino-acid sequence of the DNA-binding protein of Pseudomonas aeruginosa-specific bacteriophage Pf1 and bacteriophage fd of Escherichia coli.     

Effects of dehydration and low temperatures on the oxidation of high-potential cytochrome c by photosynthetic reaction centers in Ectothiorhodospira shaposhnikovii

Effects of temperature and dehydration on the efficiency of electron transfer from membrane-bound high-potential cytochromes c_h to the reaction-center bacteriochlorophyll (P-890) in Ectothiorhodospira shaposhnikovii have been studied. A kinetic analysis of the cytochrome oxidation suggests that there are at least two conformational states of the c_h-P-890 complex, of which only one allows photoinduced electron transfer from cytochrome to P-890⁺. Lowering the temperature of dehydration leads to a change in the proportion of the populations in the two conformations. The observed 2-fold deceleration of cytochrome oxidation can be related only to the diminution of the amount of photoactive cytochromes per reaction center. The rate constant for the transfer of an electron from cytochrome c_h to bacteriochlorophyll is 2.8 · 10⁵ s⁻¹ and is independent of temperature and dehydration (as estimated within the accuracy of the experiments). The effects produced by low temperature and dehydration are completely reversible. The thermodynamic parameters of the transition of the cytochrome from the nontransfer to electron-transfer conformation were estimated. For room temperature (+ 20°C) in chromatophore preparations, ΔG = −5.4 kJ · M⁻¹, ΔH = 60 kJ · M⁻¹, ΔS = 0.22 kJ · M⁻¹ · K⁻¹. For Triton X-100 subchromatophore preparations, the absolute values of the above parameters are significantly lower: ΔG = −2.8 kJ · M⁻¹, ΔH = 18 kJ · M⁻¹, and ΔS = 0.075 kJ · M⁻¹ · K⁻¹. To a larger extent, the above parameters are diminished for chromatophore preparations in an 80% glycerol solution: ΔG = −1.7 kJ · M⁻¹, ΔH = 6 kJ · M⁻¹, ΔS = 0.025 kJ · M⁻¹ · K⁻¹. The data suggest the hydrophobic character of the forces that maintain the P-890-c_h complex in the electron-transfer conformation. The results obtained suggest that electron tunneling within the complex cannot occur until a specific conformational configuration of the complex is formed. The efficiency of cytochrome c_h oxidation is determined by the temperature, the degree of dehydration and the environmental conditions, whereas the transfer of an electron itself in the electron-transfer configuration is essentially independent of temperature and hydration.     

Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding

Magnesium binding to cation-depleted blue bacteriorhodopsin (b-bR) was studied spectrophotometrically as well as by following stopped-flow kinetics. There exist three kinetically different steps in the binding process, yielding purple bacteriorhodopsin (p-bR). Since only the firtst step is dependent on the concentration of the reactants, the reaction scheme

can be proposed as the simplest model, with MgbR being the first intermediate and ΣI denoting a set of successive intermediates. According to this model k₁, k₋₁ and k₂ are calculated to be 2.8 × 10⁴ M⁻¹ · s⁻¹, 5.0 × 10 s⁻¹ and 1 × 10⁻² s⁻¹, respectively.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

Algal biomass production,N uptake and N2 fixation in a synthetic medium

An experiment was conducted in the growth chamber to quantify the biomass production, N removal and N₂ fixation from a synthetic medium by Chlamydomonas reinhardtii and Anabaena flos-aquae. Nitrogen was supplied at a concentration of 100 mg liter⁻¹ of NO₃⁻−¹⁵N and NH₄⁺−¹⁵ (3·5 atom %), respectively. After 21 days Chlamydomonas reinhardtii removed an average of 83·8 and 78·7 mg N liter⁻¹ as NO₃⁻ and NH₄⁺, respectively. Averages of 0·89 and 0·71 g liter⁻¹ (first batch), 1·63 and 0·95 g liter⁻ (second batch) algal biomass were collected from NO₃⁻ and NH₄⁺ media, respectively. Uptake rates of 0·11 mg ¹⁵N g⁻¹ algae day⁻¹ from NO₃⁻ medium and 0·10 mg ¹⁵N g⁻¹ algae day⁻¹ from NH₄⁺ medium were calculated. Algal cells grown in NO₃⁻ and NH₄⁺ medium contained 71 and 65 g N kg⁻¹ (first batch), 39 and 58 g N kg⁻¹ (second batch), respectively. Anabaena flos-aquae produced averages of 0·58 and 0·46 g liter⁻¹ (first batch), 0·55 and 0·48 g liter⁻¹ (second batch) after 14 days of growth from NO₃⁻ and NH₄⁺ media, respectively. Blue-green algal biomass contained higher N (81–98 g kg⁻¹) than green algae. Isotope dilution method for determining N₂ fixation indicated that 55% and 77% of total N of blue-green algae grown in NO₃⁻ and NH₄⁺ media, respectively, was derived from the atmosphere.     

Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures

The influence of initial glucose concentrations on the production of biomass and lutein by Chlorella protothecoides CS-41 was investigated in batch cultures using both shake flasks and fermentors. The maximum biomass concentration increased from 4·9 to 31·2 g litre⁻¹ dry cells with an increase in initial glucose concentration from 10 to 80 g litre⁻¹. An even higher initial glucose concentration (100 g litre⁻¹) resulted in a lower biomass concentration, a lower specific growth rate, a lower growth yield coefficient and a considerably longer lag phase, which might be due to substrate inhibition. The initial glucose level also had a significant effect on the production of lutein. In a 3·7-litre fermentor an increase in lutein production from 19·39 to 76·56 mg litre⁻¹ was obtained with an increase in initial glucose concentration from 10 to 40 g litre⁻¹, within which range, lutein yield coefficient was constant (Y_Itn=1·90±0·02 mg g⁻¹). A simple substrate inhibition model was developed, which fitted the experimental data better than the classical Haldane model. A group of time-dependent kinetic models for algal cultivation in fermentors were also constructed, which were in good agreement with the experimental results and could be employed to predict the production of biomass and lutein, and the consumption of glucose in fermentor cultures.     

Self-diffusion of water-soluble fullerene derivatives in mouse erythrocytes

Self-diffusion of water-soluble fullerene derivative (WSFD) C₆₀[S(CH₂)₃SO₃Na]₅H in mouse red blood cells (RBC) was characterized by ¹H pulsed field gradient NMR technique. It was found that a fraction of fullerene molecules (~13% of the fullerene derivative added in aqueous RBC suspension) shows a self-diffusion coefficient of (5.5 ± 0.8)·10⁻¹² m²/s, which is matching the coefficient of the lateral diffusion of lipids in the erythrocyte membrane (D_L = (5.4 ± 0.8)·10⁻¹² m²/s). This experimental finding evidences the absorption of the fullerene derivative by RBC. Fullerene derivative molecules are also absorbed by RBC ghosts and phosphatidylcholine liposomes as manifested in self-diffusion coefficients of (7.9 ± 1.2)·10⁻¹² m²/s and (7.7 ± 1.2)·10⁻¹² m²/s, which are also close to the lateral diffusion coefficients of (6.5 ± 1.0)·10⁻¹² m²/s and (8.5 ± 1.3)·10⁻¹² m²/s, respectively. The obtained results suggest that fullerene derivative molecules are, probably, fixed on the RBC surface. The average residence time of the fullerene derivative molecule on RBC was estimated as 440 ± 70 ms. Thus, the pulsed field gradient NMR was shown to be a versatile technique for investigation of the interactions of the fullerene derivatives with blood cells providing essential information, which can be projected on their behavior in-vivo after intravenous administration while screening as potential drug candidates.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

Influence of planting geometry on photosynthetically active radiation interception and dry matter production relationships in pearl millet

A field experiment was conducted at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Center, Patancheru, India to study photosynthetically active radiation (PAR) interception and dry matter production relationships in pearl millet (Pennisetum americanum (L.) Leeke). Two pearl millet genotypes, BJ 104 (G₁) and ICH 226 (G₂) were sown at three planting geometries obtained by using combinations of row and plant spacings (S₁: 37·5 cm × 26·6 cm; S₂: 75·0 cm × 13·3 cm; S₃: 150·0 cm × 6·6 cm) such that plant population was constant at 100 000 ha⁻¹ in all treatments. Cumulative intercepted PAR was maximum (330 MJ m⁻²) in G₂S₂ and minimum (268 MJ m⁻²) in G₁S₃. Conversion efficiency values ranged from 1·87 g MJ⁻¹ in G₁S₂ to 2·32 g MJ⁻¹ in G₂S₃. Final above-ground dry matter followed the pattern of cumulative intercepted PAR and maximum dry matter (7·22 Mg ha⁻¹) was produced by G₂S₂ while G₁S₃ produced minimum dry matter (4·97 Mg ha⁻¹).     

Afaltoxin B1 transport in rat blood plasma. Binding to albumin in vivo and in vitro and spectrofluorimetric studies into the nature of the interaction

Binding of [³H]aflatoxin B₁ to rat plasma was investigated in vivo and vn vitro. Column chromatographic and polyacrylamide gel electrophoretic analysis clearly demonstrated that aflatoxin B₁ bound primarily plasma albumin. Very little binding activity was shown by other plasma proteins. Spectrofluorimetric studies were undertaken to gain some insight into the nature of the aflatoxin-albumin interaction. Quenching of the lone tryptophan fluorescence intensity upon aflatoxin binding was due, at least in part, to a ligand-induced conformational change in the albumin molecule. Aflatoxin B₁ binds an apolar site with an association constant of 30 mM⁻¹ at pH 7.4 and 20°C. Neither charcoal treatment of rat albumin nor the presence of 0.15 M NaCl had many significant effect on the interaction. The association constant was pH-dependent, increasing about 1.7-fold as the pH increased from 6.1 to 8.4. This pH dependence is ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (ΔH = −29.3 kJ·mol⁻), with a ΔS value of −13.8 J·mol⁻¹·K⁻¹.