The two-cross immunodiffusion technique: diffusion coefficients and precipitating titers of IgG in human serum and rabbit serum antibodies.

The two-cross technique, a new two-dimensional double-diffusion technique in gelplates, has been applied for simultaneous determination of precipitating titers and diffusion coeffients of antigen and antibody in body fluids. The advantage of this technique is that it works without using any standard solution and ensures conditions of “time-invariant sink”. The theory of the technique has been verified by experimental results on the precipitating system human serum-rabbit anti-human IgG in phosphate-buffered saline solution at pH 7.4. The results obtained using several modes of calculations from experimental parameters have been compared and found satisfactory. The accuracy and reproducibility of the results have been confirmed. It has been found that at 20°C the diffusion coefficient of human IgG in 10-times-diluted serum is (4.4 ± 0.2) × 10^?7 cm² s^?1, while the diffusion coefficient of rabbit anti-human IgG in a purified preparation is (2.9 ± 0.2) × 10^?7 cm² s^?1. The critical precipitating concentration of human IgG against rabbit anti-human IgG is invariable to concentration and amounts to 0.174 ± 0.03 mg/100 ml at pH 7.4.     

Isolation of Highly Radioresistant Bacterium,Arthrobacter radiotolerans nov. sp.

A highly γ-ray resistant bacterium, which has not been described hitherto, has been isolated from water containing mud, fur and moss at a radioactive hot spring, Misasa, Tottori Prefecture, Japan, This bacterium was Gram-positive, non-sporulating, pink to red colored and pleomorphic rod at young stage and predominantly coccoid or small short rod at old. The radiosensitivity of this bacterium was lower than that of well-known bacterium Micrococcus radio- durans. When its exponentially growing cells were irradiated in buffer solution aerated sufficiently at room temperature, shoulder dose and D₀ were calculated to be 6 × 10⁵ and 1 × 10⁶ rads, respectively. The morphological, cultural and physiological characteristics of the bacterium have been studied. These attributes suggested that the organism was a new species, and the name Arthrobacter radiotolerans has been assigned with respect to its high radioresistance.     

Interactions between purine derivatives: Electronic spectral studies. I. Electronic transitions in caffeine monomer and exciton coupling in caffeine dimer

The concentration dependence of the ultraviolet absorption spectrum of aqueous solutions of caffeine has been studied. Individual species spectra have been derived for the monomer, dimer, and tetramer of caffeine. The emission spectrum of caffeine in aqueous solution and the dichroic spectra in oriented poly(vinyl alcohol) and polyethylene films have been measured. The long-wavelength tail of the absorption spectrum of caffeine in non-polar environment has been found to incorporate at least one carbonyl(π*, n) transition. Dichroic spectral data and molecular orbital calculations have been used to assign transition moment directions to the (π*,π) transitions. The lowest energy (π*,π) transition, responsible for the near-ultraviolet absorption peak in aqueous solution of caffeine, has been used for the study of degenerate exciton interactions in the dimeric species of caffeine. Assuming that the caffeine molecules in the dimer are stacked in parallel planes, theoretical calculations of the ground-state interactions and of the degenerate exciton interactions have been combined with experimental data and a unique model for the dimer of caffeine has been derived. The transfer rate of energy between the molecules in the dimer is of the order of 10¹³_S^?1.     

Thermodynamics of gelation of sickle cell deoxyhemoglobin.

This paper describes the thermodynamic behavior of gels of deoxyhemoglobin S. The solubility of the protein with respect to assembled hemoglobin fibers has been measured using a sedimentation technique. The solubility in 0.15 m-potassium phosphate buffer (pH 7.15) is found to decrease with increasing temperature, attain a minimum value of 0.16 g cm^?3 at 37 °C, and then increase at higher temperatures. The amount of polymer present at various hemoglobin concentrations and temperatures is presented as part of a phase diagram that may be useful for the calibration of other measurement techniques. The effects of varying pH and urea concentration upon the solubility have also been studied.The heat absorption accompanying gelation has been measured by scanning calorimetry. Using sedimentation data on the amount of polymer formed, molar enthalpy changes are obtained. There is a large negative heat capacity change of ? 197 cal deg. mol^?1 and ΔH = 0 near 37 °C. Calorimetric molar enthalpy changes are found to agree with those calculated from the temperature dependence of the solubility by the van't Hoff equation.Our previous two-phase, two-component thermodynamic model of gelation is extended to include the effects of solution non-ideality. A large contribution to the activity of the hemoglobin in the solution phase results from the geometric effect of excluded volume. Incorporating solution phase non-ideality permits the calculation of standard state thermodynamic quantities for the gelation process at 37 °C: ΔG^O ? ?3 k cal mol^?1, ΔH^O ~ 0, ΔS^O ~ 10 cal deg.^?1 mol^?1. The excluded volume effect is also capable of explaining observations of the minimum gelling concentrations of hemoglobin mixtures containing deoxyhemoglobin S without requiring copolymerization of the non-S hemoglobin.     

Dynamic light scattering from collagen solutions. I. Translational diffusion coefficient and aggregation effects

Solutions of native collagen extracted from rat tail tendons in neutral salt solution have been studied by dynamic light scattering. The spectra obtained are consistent with the presence in solution of both single rod-shaped collagen molecules and aggregates of molecules. No contribution to the spectrum has been detected at any scattering angle from rotational diffusion of single molecules, although a measurable broadening effect is expected at high angles. The translational diffusion coefficient D of single molecules, calculated from the broader spectral component, shows an anomalous dependence on collagen concentration with a maximum value of D_20,w = 8.6 ± 0.2 × 10^?12 m²/sec near the concentration 0.04% by weight. Above 0.05% D falls linearly with increasing concentration and takes the value D _20,w = 8.1 ± 0.2 × 10^?12 m²/sec at 0.064% collagen.     

Copper-ligand interactions and physiological free radical processes. Part 3. Influence of histidine,salicylic acid and anthranilic acid on copper-driven Fenton chemistry In Vitro

With a view to the possible use of copper(II)-^·OH inactivating ligand (OIL) complexes as regulators of inflammation, the reactivity of the copper(II)-ascorbate system with hydrogen peroxide has been investigated in the presence of three key substances: histidine (the main copper(II) low molecular mass ligand in extracellular fluid), salicylic acid (the well-known non-steroidal antiinflammatory drug, previously shown to be potentiated by copper(II) in animal models of inflammation), and anthranilic acid (an inactive substance by itself, known to be activated by copper(II) in the same models) at physiological pH (7.4) and inflammatory pH (5.5).

Such substances may affect the amount of TBARS detected in solution following copper-mediated Fenton-like reactions through three distinct mechanisms: (i) by decreasing the Cu(II)/Cu(I) redox potential, i.e. at the expense of ^·OH radical production, (ii) by scavenging ^·OH radicals in the body of the solution, and/or (iii) by acting as a true OIL, i.e. at the expense of ^·OH detection. Redox potential measurements of initial solutions have been performed in parallel to TBARS determinations to help discriminate between different ligand influences. Computer-aided speciation has been used to understand the role of copper(II) distribution on the ligand effects characterised.

Contrary to previous interpretations, histidine has been found to mainly affect ^·OH production by lowering the redox potential of the Cu(II)/Cu(I) couple. Salicylate, which has no effect on ^·OH production, has been confirmed to mainly scavenge ^·OH radicals in the body of the solution. Anthranilate, which both increases ^·OH production and decreases ^·OH detection, behaves as a potential OIL.

These results tend to confirm our previous hypothesis that copper potentiation of antiinflammatory substances is indirect, i.e. independent of any interaction between metal and drug, whereas copper activation of substances that are inactive by themselves results from specific metal-substance interactions taking place at inflammatory sites.     

Determination of association and dissociation rate constants for bilirubin--bovine serum albumin.

The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 10⁶m^?1S^?1. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10^?2s ¹. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 10⁷m^?1. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 10⁷m^?1. All experiments are carried out at 36 °C and pH 7.4.     

The flavin dimers I. The application of absorption in anti-stokes excitation region to investigate the flavin dimer formation

The dimer formation process of the flavin in aqueous solution has been studied. The difference absorption spectra with the change of concentration in Stokes and anti-Stokes excitation region of the flavomononucleotide and riboflavin were measured. The highest temperature in which the dimers still appear is discussed. It is suggested that this temperature T_d can be treated as one of the empirical parameters which describe the dimer formation process of the dyes in solutions. The aqueous solution of flavins with the concentration c?5·10^?5 M at room temperature can be treated as a flavin monomers solution. For higher concentrations the flavin monomers and dimers exist in a solution at room temperature.     

Reactions of dichloroaluminium acetylacetonate with lewis bases. 1. Ionic complex of Cl2alacac with tetrahydrofurane

The reaction of dichloroaluminium acetylacetonate with THF has been studied. The ionic complex [(acac)₂Al·2THF]⁺[AlCl₄]⁻ was found to result from the reaction. The structure of the complex has been investigated by the variable temperature ¹H NMR technique, as well as by ¹³C and ²⁷Al NMR spectroscopy. The cation complex [(acac)₂Al·2THF]⁺ is predominantly trans in dichloromethane solution (75% trans and 25% cis). In the presence of an excess of THF a fast exchange proceeds at room temperature in the cation complex between the free THF molecules and those present in the complex, which is accompanied by a stereo-chemical rearrangement of the cation complex.     

Propagation Speed in Myelinated Nerve: I. Experimental Dependence on External Na+ and on Temperature

Conduction speed (θ) in single myelinated Rana pipiens sciatic nerve fibers has been precisely measured using intracellular recording and on-line digital computer techniques. The dependence of relative speed on external Na concentration at 15°C has been found to be ln(θ₁/θ₂) = 0.524 (±0.018) ln ([Na⁺]₁/[Na⁺]₂) + 0.003. Thus θ has very close to a square root dependence on [Na⁺]₀ for these fibers. This experimental finding is not in complete agreement with a theoretical prediction based on a solution of the Hodgkin-Huxley (H.H.) equations. The effect of small temperature variations around 15°C on θ has also been measured for Rana fibers in Ringer's solution. θ has close to an exponential dependence on T and a Q₁₀ of 2.95 has been estimated.     

Induced fit as a determinative of ionophore selectivity.

The conformation of the carboxylic ionophore $\text{salinomycin}$ has been examined in a variety of solvents by circular dichroism. The solution conformation of the ionophore has been correlated with Na⁺ and K⁺ affinity and selectivity. Cation selectivity appears to be a function of both the initial conformation of the free anionic ionophore and the ability of the latter to reorient about the cation. An analogy to the induced fit model of enzyme-substrate interaction is proposed.     

A NEW METHOD FOR THE STUDY OF DIFFUSION OF BIOLOGICALLY ACTIVE MATERIAL

A simple diffusion apparatus has been described in which a layer of solution is allowed to diffuse upward into a layer of solvent. Accurate sampling is performed at various heights and the concentration of the samples is determined. The method has been illustrated with a determination of the diffusion constant of crystalline catalase, which was found to be 3.1 x 10^–7 cm.²/sec. at 4°C. The method should be especially suited to the study of biological substances endowed with specific activity and which cannot be obtained in pure solution.     

Characterization and production of a new extracellular polymer from Pseudomonas sp. GSP-910

Summary A new strain, Pseudomonas sp. GSP-910 has been isolated from soil and has been found to produce large quantities of an extracellular, highly viscous polysaccharide in a simple salt medium. Good polymer production (6.16 g·l^-1) occurs on a sucrose-containing medium (2%) at high phosphate concentration (80 mM·l^-1) and 0.5 g·l^-1 of nitrogen source NH₄Cl. The relative proportions of sugars in the polymer are: glucuronic acid 8.8%, glucose 28.07%, galactose 56.8%, and it is partially acetylated (6.32%). The isolated polymer exhibits higher viscosity at dilute concentrations than xanthan gum and it is stable at different temperatures, over a wide range of pH and in the presence of monovalent salt. In the presence of divalent cation (CaCl₂ 0.5%), 910-gum in aqueous solution (1%) solidifies to a resilient gel.     

Infrared and raman spectroscopy of carbohydrates. 3. Raman spectra of the polymorphic forms of amylose

The Raman spectra of V_a-, Vh_h-, and B-amylose have been recorded, and are interpreted in terms of the proposed mechanism for conversion from the V- into the B-form. Lines occurring at 1263 and 946cm^-1 with V-amylose shift to 1254 and 936 cm^-1 on conversion into the B-form; at the same time intensity changes are observed for the lines at 2940 and 1334 cm^-1. These effects are consistent with the mechanism proposed for V→B conversion, involving an extension of the helix and changes in the intramolecular hydrogen-bonding. In addition, the spectra of amylose dissolved in aqueous salt solution and in methyl sulfoxide have been recorded. The results indicate that amylose does not adopt the V-conformation in methyl sulfoxide solution.     

Synchrotron-excited time-resolved fluorescence spectroscopy of adenosine,protonated adenosine and 6N, 6N-dimethyladenosine in aqueous solution at room temperature

The fluorescence behavior of adenosine in neutral solution has been studied by time-resolved spectroscopy using synchrotron excitation and timecorrelated single photon counting, and by decay time measurements. Three emissions have been identified and correlated with three excitation spectra. The assignment of these transitions has been made by comparison with similar measurements on ⁶N, ⁶N-dimethyladenosine (6 DMA), and on adenosine in acid solution (ADO H⁺). It is proposed that two of the transitions of adenosine which correlate with 6DMA originate from coplanar and orthogonal rotational conformers of the amino group. The other transition, correlating with ADO H⁺ may originate either from the ³H-imino tautomer, or from a differently solvated rotational conformer.A partial presentation of this work has been made at the Second Congress of the European Society for Photobiology Padova, Italy, 6–10 September 1987     

THE KINETICS OF PENETRATION : XII. HYDROGEN SULFIDE

The rate of entrance of H₂S into cells of Valonia macrophysa has been studied and it has been shown that at any given time up to 5 minutes the rate of entrance of total sulfide (H₂S + S^-) into the sap is proportional to the concentration of molecular H₂S in the external solution. This is in marked contrast with the entrance of ammonia, where Osterhout has shown that the rate of entrance of total ammonia (NH₃ + NR₄ ⁺) does not increase in a linear way with the increase in the external concentration of NH₃, but falls off. The strong base guanidine also acts thus. It has been shown that the rate of entrance of H₂S is best explained by assuming that it enters by diffusion of molecular H₂S through the non-aqueous protoplasmic surface. It has been pointed out that the simple diffusion requires that the rate of entrance might be expected to be monomolecular. Possible causes of the failure of H₂S to follow this relationship have been discussed.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

A problem encountered in a study of the effects of lanthanide ions on enzyme-catalyzed reactions.

Investigations have been made of the time-dependent loss of the ability of dilute lanthanide solutions to inhibit creatine kinase. Using [¹⁵²Eu]Cl₃, it has been demonstrated that the loss is due to the reduction in concentration that occurs because of the adsorption of lanthanide ions onto walls of containers. Further, it has been shown that the adsorption varies with pH and the composition of the container. The results also indicate that dilute solutions of EuCl₃ do not undergo concentration changes when stored in Pyrex glass vessels either under slightly acidic conditions or in buffered solution at pH 8.0.