Low velocity gradient flow birefringence and viscosity changes in hyaluronate solutions as a function of pH.

The flow birefringence and extinction angle over a velocity gradient range of approximately 5–100 sec^?1, and the zero shear-viscosity have been obtained from human umbilical cord hyaluronic acid at concentrations of 0.25, 0.125 and 0.0625%, and pHs 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 and constant ionic strength 0.1. The data indicate a large change in optical anisotropy as a function of pH, with most of the transition in the pH range 7.0–7.5, i.e., across the physiological range. The sign of the anisotropy changes between pH 8.0 and 8.5. These results, together with changes in the extinction angle and intrinsic viscosity as a function of pH, suggest a pH-dependent structural change in the system. Due to the abruptness of the transition, as evidenced by the intrinsic viscosity and flow birefringence, it is probable that the structural transition is cooperative. If the data are interpreted in terms of the Rouse-Zimm Gaussian subchain theory, a modification of the model in terms of the Haller-Cerf concept of internal viscosity is required. Thus, the demonstrated properties of hyaluronate solutions indicate a system with memory of stress. Due to the presence of large concentration effects discernible in the extinction angle measurements, hyaluronic acid probably exists as a network in solution. The results are discussed with respect to the mechanoelectrical transducing properties of hyaluronates and stress-dependent changes in ORD already reported.     

pH control of the third virial coefficient of hyaluronate solutions calculated from van der Waals forces by means of the Lifshitz-McLachlan susceptibility theory

Positive third virial coefficients and osmotic coefficients have been calculated for human umbilical cord hyaluronic acid solutions at pHs 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 and constant ionic strength 0.1. The calculations are based on experimental axial flow birefringence and radial linear dichroism data previously reported and the Lifshitz-McLachlan field theory of van der Waals forces. The second virial coefficients are negative, according to both this analysis and light scattering evidence, and reflect the tendency of hyaluronic acid to associate. This negativity denies the assumption of force additivity required by virial expansion theory.The results are in reasonable agreement with those of light scattering studies, and indicate the extreme nonideality of hyaluronate solutions with a high degree of pH control of osmotic pressure. The data are explained within the context of statistical mechanical and field theories of van der Waals forces, and the osmotic pressure of a solution is related to its optical properties. The numerical method used offers a way of exploring the applicability of modern interparticle force theory to biological systems.     

Studies of the birefringence and birefringence dispersion of polypeptides and proteins

An apparatus has been constructed which permits the polarimetric observation of streaming solutions of macromolecules. The apparatus is a streaming birefringence device allowing the usual measurements of birefringence parallel to the cylinder axis but which in addition transmits light in the radial direction. Installation of the apparatus between the polarizer and analyzer of a Rudolph polarimeter makes possible the measurement of changes in optical rotation, dichroism and birefringence. The present work is concerned with the latter effect. The systems studied were α-helical polyglutamic acid, paramyosin, and collagen (ichthyocol). The combined measurements of radial and axial birefringence completely determines the refractive index ellipsoid of the streaming fluid. This result in turn permits the testing of the Peterlin-Stuart distribution function for streaming in a Couette device, apart from a proportionality constant. The comparison between theory and experiment is very satisfactory provided the system is reasonably homogeneous with regard to molecular length and is sufficiently dilute. On the other hand, it is concluded that the Peterlin-Stuart optical factor seriously overestimates the “form” birefringence in agreement with recent results and conclusions of Taylor and co-workers. The apparatus permits the study of the dispersion of the birefringence in the radial direction. The dispersion of collagen follows a one-term Sellmeier formula and is dominated by absorption bands in the neighborhood of 2000 A. On the other hand, the dispersion of the α-helical systems is complex and requires a multiterm Sellmeier formula. This contrast between the two kinds of polypeptidc helices is similar to results obtained with other optical techniques and is attributed to the splitting of absorption bands in the α-helix.     

Isolation of human fibrinogen by axial and radial flow chromatography from Nitschmann fraction I

An axial column (3×2.6 cm) and a radial flow column (3.5×5 cm) packed with DEAE Sepharose Fast Flow media had been evaluated for the separation of human fibrinogen. Nitschmann fraction I dissolved in buffered saline (0.015 M NaCl buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Under radial flow conditions, sample flow up to 15 ml min^–1 (i.e., 18 bed volumes h^–1) was achieved. The operating pressures were below 0.2 MPa, even though the elution velocity was 30 ml min^–1 (i.e., 36 bed volumes h^–1).     

Conformational changes in coupling factor 1 may control the rate of electron flow in spinach chloroplasts.

The relationship between the rate of electron flow, internal H⁺ concentration and the magnitude of the H⁺ concentration gradient (ΔpH) in chloroplasts illuminated at various light intensities has been examined. At an external pH of 7.0, the internal H⁺ concentration is a linear function of the rate of electron flow except at saturating light intensity. In contrast, at pH 8.1, this relationship between electron flow and internal H⁺ concentration holds only at values of ΔpH below about 2.8 – 2.9 units. At higher ΔpH values, the rate of electron flow increases much more dramatically than the internal H⁺ concentration. ATP (0.1 mM) prevents this increase. It is suggested that at pH 8.1 but not at pH 7.0, the conformation of coupling factor 1 is altered at high ΔpH values. Its altered conformation may result in an increased efflux of H⁺ from the chloroplasts. This notion is supported by the effects of ATP on electron flow and ΔpH as well as the effect of external pH and light intensity on the reactivity of coupling factor 1 to N-ethylmaleimide.     

Measurement of flow birefringence at very low velocity gradients

Instrumentation and associated techniques are described for the measurement of flow birefringence and extinction angles of high molecular weight polymer systems at extremely low velocity gradients. Precision of less than one degree in the extinction angles is obtained for birefringence smaller than 10^?11, based upon studies of very dilute high molecular weight DNA solutions over a range of velocity gradients from 0.1 to 3 sec^?1. Extinction angles approaching 44° are also observed with at least this degree of reliability for these systems. A relatively simple method of data reduction is outlined which permits explicit correction for solvent effects and for residual instrument birefringence and background; only the latter are found to be necessary at the shear rates reported in this communication. Applicability of the techniques to other macromolecules of biological interest is discussed.     

Electron microscopic visualization of RecA-DNA filaments: Evidence for a cyclic extension of duplex DNA

As visualized by electron microscopy, RecA protein binds in a highly cooperative manner to single-stranded fd DNA in solutions of 0.01 M Tris (pH 7.5). The resulting nucleoprotein filament loops are 1.25 μm in length, have a fiber diameter of 12 nm and show an indication of a 4.5 nm repeat along the axis of the compact fibers. RecA binds to linear duplex fd DNA in solutions of 0.01 M Tris (pH 7.5) to yield chains of beads which, in the presence of Mg²⁺ and ATP, coalesce into smooth filaments with a length of 1.9 μm (the length of protein-free fd duplex DNA) and have a fiber diameter of 12 nm. In solutions containing Mg²⁺ and ATP-γ-S, however, RecA binds to duplex DNA in a highly cooperative manner to yield rigid filaments 3.0 μm in length. These filaments are 12 nm in diameter and show a very clear 7.5 nm axial repeat. This extension of DNA to 150% of its usual length in the apparent absence of any single-stranded components suggests that the DNA helix must also be highly unwound and provides new insights into the mode of RecA action.     

Monitoring the orientation of myosin bridges on two-dimensional maps of birefringence in a single muscle fiber

Two-dimensional maps of birefringence in sarcomers of a single fiber of rabbit m.psoas were obtained by an automated interference microscope developed at our laboratory. The changes in birefringence of muscle fibers reflect the movement of myosin cross-bridges. The orientation of cross-bridges was modified by varying the pH (pH 7.0, 6.0, 8.0) and ionic strength (mu = 0.115, 0.085, 0.235) of the bathing rigor solution. The maximum value of total birefringence in the rigor state was observed at neutral pH. Total birefringence markedly decreased (by 40%) as pH was changed from 7.0 to both 8.0 and 6.0. No significant changes in light phase shifts were found at a 1.5 reduction of ionic strength in the rigor solution. The calculated birefringence values were 45% higher in rigor solutions of a high (mu = 0.235) ionic strength. The results observed are discussed in terms of changes in the orientation of cross-bridges due to the movement of the alpha-helical subfragment-2 away from the filament shaft (pH 8) or coming closer to it (mu = 0.235). The available data do not allow one to explain the results obtained at pH 6.0.     

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.     

Heat aggregation studies of phycobilisomes, ferritin, insulin, and immunoglobulin by dynamic light scattering.

Dynamic laser light scattering studies on the heat aggregation behavior of phycobilisomes (PBS), ferritin, insulin, and immunoglobulin (IgG) in dilute aqueous solutions has been reported. Except for PBS, results are reported for heat aggregation trends in these proteins for three different pH environments (4.0, 7.5, 9.1). For PBS, studies were performed only in the neutral buffer medium (pH 7.5). The experiments were performed in the very dilute concentration regime (between 0.23 and 1.8 gL-1). For all these samples heat aggregation and dissociation trends were found to be linear with temperature. Upon temperature reversal (self-cooling), hysteresis-like behavior observed in insulin was found to be predominantly large at pH 7.5. PBS, ferritin, and IgG showed no such behavior at any of three pH values, and retraced their path of aggregation while dissociating on temperature reversal. Heat aggregation and dissociation processes in ferritin were found to be independent of pH. The IgG samples showed smooth aggregation tendency only up to 35 degrees C in the buffer media pH 4.0 and 9.1, whereas for pH 7.0 the same could be observed until 60 degrees C. Low polydispersity in the correlation spectra was observed in case of all these samples.     

Liquid crystallinity in collagen solutions and magnetic orientation of collagen fibrils

Studies of the optical birefringence of solutions of acid-soluble collagen from rat-tail tendon at 22°C in the pH range 1.0–6.0 show that collagen exhibits an isotropic to mesophase transition only between pH 2.4 and 3.0 at 10% weight concentration. Such liquid crystalline order is probably essential for the orientation of collagen in a magnetic field. When solutions of neutral salt-soluble collagen were precipitated at pH 7.0 by warming to 37°C (“heat gelling”) in a magnetic field of ca. 20 kG, the resulting fibrils wee oriented perpendicular to the direction of the field. Heat gelling is shown to be a useful technique for maintaining the orientation induced in precursor solutions even after the sample is removed from the magnetic field.     

Effects of short term iron citrate treatments at different pH values on roots of iron-deficient cucumber: A Mössbauer analysis

Alkaline pH values and bicarbonate greatly reduce the mobility and uptake of Fe, causing Fe deficiency chlorosis. In the present work, the effects of pH and bicarbonate on the uptake and accumulation of Fe in the roots of cucumber were studied by Mössbauer spectroscopy combined with physiological tests and diaminobenzidine enhanced Perls staining. Mössbauer spectra of Fe-deficient cucumber roots supplied with 500 μM ⁵⁷Fe(III)-citrate at different pH values showed the presence of an Fe(II) and an Fe(III) component. As the pH was increased from 4.5 to 7.5, the root ferric chelate reductase (FCR) activity decreased significantly and a structural change in the Fe(III) component was observed. While at pH 4.5 the radial intrusion of Fe reached the endodermis, at pH 7.5, Fe was found only in the outer cortical cell layers. The Mössbauer spectra of Fe-deficient plants supplied with Fe(III)-citrate in the presence of bicarbonate (pH 7.0 and 7.5) showed similar Fe components, but the relative Fe(II) concentration compared to that measured at pH values 6.5 and 7.5 was greater. The Mössbauer parameters calculated for the Fe(II) component in the presence of bicarbonate were slightly different from those of Fe(II) alone at pH 6.5–7.5, whereas the FCR activity was similarly low. Fe incorporation into the root apoplast involved only the outer cortical cell layers, as in the roots treated at pH 7.5. In Fe-sufficient plants grown with Fe(III)-citrate and 1 mM bicarbonate, Fe precipitated as granules and was in diffusely scattered grains on the root surface. The “bicarbonate effect” may involve a pH component, decreasing both the FCR activity and the acidification of the apoplast and a mineralization effect leading to the slow accumulation of extraplasmatic Fe particles, forming an Fe plaque and trapping Fe and other minerals in biologically unavailable forms.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.     

Calorimetric and optical characterization of sickle cell hemoglobin gelation.

We have used two techniques to characterize the gelation of deoxyhemoglobin S, a high sensitivity heat-flow calorimeter to measure the heat of gelation and a simple light-transmission method to measure the optical birefringence resulting from the alignment of deoxyhemoglobin S fibers in the gel. A theory for the interpretation of the birefringence measurements is presented. We combine the results of the calorimetric and optical measurements with those of sedimentation experiments to obtain enthalpy changes for gelation. The enthalpy change obtained from scanning and isothermal calorimetric measurements (0.25 m-potassium phosphate, 0.05 m-sodium dithionite, pH 6.9) varies from 4000 to 2200 cal mol⁻¹ hemoglobin between 16 and 25 °C. There is a large apparent heat capacity change of −130 to −190 cal deg.⁻¹ mol⁻¹. The apparent enthalpy change estimated from solubility measurements and birefringence melting experiments is 2200 ± 500 cal mol⁻¹ in qualitative agreement with the calorimetric results. Analysis of the time dependence of the calorimetric and optical progress curves at 20 °C leads to a rough estimate of 1800 to 4000 and −800 to 1500 cal mol⁻¹ hemoglobin for the enthalpies of polymerization and alignment of fibers, respectively. The small magnitude of the observed enthalpy change is in accord with the view that no large conformational change takes place in the deoxyhemoglobin S molecule upon gelation.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Chromatography of human prothrombin from Nitschmann fraction III on Q Sepharose Fast Flow using axial and radial flow column

An axial column (Hitrap Q 5 ml, 2.5 2 1.6 cm) and a radial flow column (3.5 2 5 cm) packed with Q Sepharose Fast Flow media had been evaluated for the separation of human prothrombin. Nitschmann fraction III dissolved in buffered saline (0.10 M sodium chloride buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Effects of sample flow rate of the two columns were screened. Under radial flow conditions using the radial column, sample flow rate up to 15 ml/min (i.e. 18 bed volumes/h) was achieved and the operating pressure was below 0.2 MPa eventhough the elution velocity was 30 ml/min. Breakthrough capacity was determined by analyzing the total protein and prothrombin activity of the target protein-containing fraction under subsaturating conditions and both columns had almost the same breakthrough capacity per ml media, indicating that the sample loading was independent of radial column geometry. It was concluded that the radial column is an attractive alternate to traditional axial packed bed column, exhibiting very good potential for use in the separation of human prothrombin.     

The uptake of ferulic and p-hydroxybenzoic acids by Cucumis sativus

The uptake of ferulic acid (FA) and p-hydroxybenzoic acid (p-HBA) from solutions (0.1–1.0 mM, pH 4.0–7.0), was determined for intact and ex     

Aerobic photolysis of alkylcobalamins: quantum yields and light-action spectra

Quantum yields (φ) for the aerobic photolysis of 5′-deoxyadenosylcobalamin (dAB₁₂), methylcobalamin (MeB₁₂), propylcobalamin (PrB₁₂), and ethylcobalamin (EtB₁₂) were determined as a function of the irradiation wavelength. φ Determinations were made for both the base-on and base-off forms of each compound (except base-off dAB₁₂) at incident wavelengths from 250 nm to 570 nm. As a rule, the φs were high (0.1–0.5) and they varied significantly with respect to the irradiation wavelength. In general, each alkylcobalamin at pH 7.0 displayed a quantum yield spectrum distinct from its base-off form at pH 1.0. Across most of the spectrum, the φs of the base-off form were appreciably smaller than the base-on φs of the same compound. An exception to this generality was MeB₁₂ for which the φs at pH 1.0 were about the same as, or slightly greater above 450 nm than those at pH 7.0. At pH 7.0 and in the visible region the trend of the φs was dAB₁₂ < MeB₁₂ < PrB₁₂ < EtB₁₂. Under neutral conditions each compound showed a broad quantum yield peak in the 450–470 nm region.From the quantum yield and absorption spectra, photolysis spectra were calculated for 5.0 × 10^?5m solutions of each compound. The light-action spectra accurately give the relative rates/μ Einstein that these solutions photolyze at each wavelength. Thus, for example, MeB₁₂ photolyzed faster at pH 7.0 versus pH 1.0 in 510 nm light, but it photolyzed slower at pH 7.0 versus pH 1.0 in 450 nm light. Solutions of each compound photolyzed faster in the ultraviolet region as opposed to the visible (e.g., 310 nm versus 510 nm).Our findings show that the previously reported photolysis rates estimated by others with tungsten lamps provide no valid information about the intrinsic photolability of various alkyl-cobalt bonds. This also applies to the relative white-light photolysis rates reported for the base-on versus the base-off form of MeB₁₂. All such relative rates are artifacts which represent only the extent of overlap between the true action spectrum and the light emission spectrum of an incandescent lamp.     

Isolation and characterization of cytochrome c550 from the methylamine-oxidizing electron-transport chain of Thiobacillus versutus

The isolation and purification of cytochrome c550 from the methylamine-oxidizing electron-transport chain in Thiobacillus versutus is reported. The cytochrome is a single-heme-containing type I cytochrome c with a relative molecular mass of 16 +/- 1 kDa, an isoelectric point of 4.6 +/- 0.1, a midpoint potential of 272 +/- 3 mV at pH less than 4 and 255 +/- 5 mV at pH = 7.0, and an axial coordination of the Fe by a methionine and a histidine. The midpoint potential decreases with increasing pH due to the deprotonation of a group tentatively identified as a propionate (pKa = 6.5 +/- 0.1 and 6.7 +/- 0.1 in the oxidized and reduced protein, respectively) and a change in the Fe coordination at pH greater than 10. The electron-self-exchange rate appears to depend strongly on the ionic strength of the solution and is relatively insensitive to changes in pH. At 313 K and pH 5.2 the electron-exchange rate amounts to 0.7 x 10(2) M-1 s-1 and 5.3 x 10(2) M-1 s-1 at I = 40 mM and I = 200 mM, respectively. Amino acid composition and molar absorption coefficients at various wavelengths are reported. Resonances of heme protons and the epsilon H3 group of the ligand methionine of the Fe have been identified in the 1H-NMR spectrum of the reduced as well as the oxidized cytochrome.