The effect of ionic strength on the entropy-driven polymerization of tobacco mosaic virus protein: Contributions of electrical work and salting-out

The entropy-driven polymerization of tobacco mosaic virus protein is favored by an increase in ionic strength, μ, and by a decrease in pH. The effect of ionic strength is interpreted in terms of salting-out and electrical work, a function of charge and, therefore, of pH as well as of μ. The extent of polymerization is measured in terms of a characteristic temperature, $\text{T}{}^1$, corresponding to a characteristic value of the equilibrium constant, $\text{K}{}_{\mathrm{<mtext>c</mtext>}}{}^{\mathrm{T1}}$ is measured at an early stage in the polymerization process where the optical density increment from light scatter is 0.01. The theory developed encompassing both salting-out and electrical work terms relates $\text{1}\text{T}{}^1$ to μ approximately according to the equation, $\text{1}\text{T}{}^1\text{= C + Bμ ?}\text{A}\text{μ}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where C is the ratio of entropy to enthalpy, B is proportional to the salting-out constant divided by enthalpy, and $\text{A}\text{μ}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ depends upon the square of the charge and is proportional to the electrical work contribution divided by the enthalpy. Data in which μ varied from 0.025 to 0.150 at three pH values, 5.95, 6.35, and 6.50, were fitted to this equation and the parameters C, B, and A were evaluated. Experiments were also carried out at a constant μ of 0.10 at pH values in increments of 0.1 between 5.9 and 6.8. The theory predicts that, at constant μ, $\text{1}\text{T}{}^1$, corrected for the electrical work contribution, is a linear function of pH with a negative slope proportional to the number of hydrogen ions bound per protein unit during polymerization, divided by the enthalpy. The data obtained fit two straight lines with different slopes above and below pH 6.3. Independent experiments carried out by the method of Stevens and Loga show that the number of hydrogen ions bound per protein unit also differs above and below pH 6.3 and the ratio of these is the same as the ratio of the above mentioned slopes. The data, therefore, make it possible to evaluate the enthalpy to be 24.8 kcal/mol of associating A protein and, with this value, the parameters C, B, and A can be interpreted. Standard entropies range from 86 e.u. at pH 6.5 to 88.5 at pH 5.95 and the salting-out constant, K_S^′, is 2.2 at all pH values studied. At μ = 0.10, the values of the electrical work contribution at pH 5.95, 6.35, and 6.50 are +0.298, +0.455, and +0.534 kcal/mol, respectively. Theoretical calculations from models predict values in agreement within a factor of less than two.     

Association of myelin basic protein with detergent micelles

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being $\text{3.58 ± 0.12}\text{and}\text{2.30 ± 0.15}$ for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively, $\text{1.34 ± 0.10}$ for deoxycholate (at 0.12 ionic strength) and $\text{4.0 ± 0.5}$ for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strength in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and detergent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single polypeptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

Further studies of the relationship between cation-induced chlorophyll fluorescence and thylakoid membrane stacking changes

Salt-induced changes in thylakoid stacking and chlorophyll fluorescence do not occur with granal membranes obtained by treatment of stacked thylakoids with digitonin. In contrast to normal untreated thylakoids, digitonin prepared granal membranes remain stacked under all ionic conditions and exhibit a constant high level of chlorophyll fluorescence. However, unstacking of these granal membranes is possible if they are pretreated with either acetic anhydride or linolenic acid.Trypsin treatment of the thylakoids inhibits the salt induced chlorophyll fluorescence and stacking changes but stacking of these treated membranes does occur when the pH is lowered, with the optimum being at about pH 4.5. This type of stacking is due to charge neutralization and does not require the presence of the 2000 dalton fragment of the polypeptide associated with the $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b}$ light harvesting complex and known to be lost during treatment with trypsin (Mullet, J.E. and Arntzen, C.J. (1980) Biochim. Biophys. Acta 589, 100–117).Using the method of 9-aminoacridine fluorescence quenching it is argued that the surface charge density, on a chlorophyll basis, of unstacked thylakoid membranes is intermediate between digitonin derived granal and stromal membranes, with granal having the lowest value.The results are discussed in terms of the importance of surface negative charges in controlling salt induced chlorophyll fluorescence and thylakoid stacking changes. In particular, emphasis is placed on a model involving lateral diffusion of different types of chlorophyll protein complex within the thylakoid lipid matrix.     

The redox potential of cytochrome c-552 from Euglena gracillis: a thermodynamic study.

The redox potentials for cytochrome c-552 at different ionic strengths, pH 7, have been determined, together with the thermodynamic parameters of the redox reaction. The effects of the electrostatic media on the redox potential of cytochrome c-552 do not depend on the nature of the ions employed. At 25 °C and pH 7 the observed potentials depend on the ionic strength, I, according to the equation: $\text{E}{}_{\mathrm{obs}}{}^{\mathrm{o}}\text{= 0.280 + .525 (I}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(I + I}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{))}$. The significance of the ionic strength dependence of the redox potentials and their derived thermodynamic parameters are discussed and compared to those of mammalian cytochrome c. It is concluded that the redox potentials for ionic strength approaching zero are not affected by the overall net charge of the proteins; at finite ionic strengths, the protein charges play a very important role in determining the observed redox potentials.     

Charge-induced pretransition in phosphatidylethanolamine multilayers. The occurrence of ripple structures

The influence of pli and ionic strength on the phase transition behaviour of 1,2-dihexadecylphosphatidylethanolamine was studied calorimetrically. In the range of ionic strength from 0.75 to 1.5 M NaC1 at $\text{pH}\text{? 13}$, where the amino group of the phosphatidylethanolarnine is in the deprotonated state, resulting in one negative charge per lipid molecule, the calorimetric scan shows a pretransition before the main transition. Accompanying freeze-fracture electron microscopic studies on these preparations in the temperature range between the pre- and main transitions show a regular surface, the so-called ripple structure. These are comparable with the structures seen in phosphatidylcholine-water systems af temperatures between the pre- and main transition.     

Comparative kinetic-ionic strength study of two differently charged cytochrome C: effects are limited to overall charge.

The reduction kinetics of two differently charged cytochromes $\text{c}$, horse cytochrome $\text{c}$ and $\text{Rhodosprillum rubrum}$ cytochrome $\text{c}{}_2$, by ferrous EDTA^2? were studied as a function of ionic strength. Since both proteins have nearly the same heme edge region, but have very different overall surface charge, this comparative study served as a direct test of the utility of small nonbinding non-physiological redox agents in the study of the charge of electron transfer sites of redox proteins. Calculations based on the ionic strength-kinetic data yielded protein charges of +10 and +2.3 for cytochrome $\text{c}$ and cytochrome $\text{c}{}_2$ respectively and compared well with values of +9 and +3 for the overall charge of the proteins based on acidic and basic amino acid residues. It is concluded that ionic strength effects upon the redox kinetics with such nonbinding nonphysiological redox agents reflect the influence of the overall protein charge and not the localized charge of the presumed site of electron transfer.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome $\text{c}{}_1$ and cytochrome $\text{c}$ have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome $\text{c}{}_1$ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome $\text{c}$ is rather insignificant. The formation of a complex between cytochromes $\text{c}$ and $\text{c}{}_1$ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome $\text{c}$ upon mixing with cytochrome $\text{c}{}_1$ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome $\text{c}$ was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: Structural and functional properties

The bivalve mollusc Scapharca inaequivalvis contains in the coelomic fluid erythrocytes with a dimeric (HbI) and a tetrameric (HbII) hemoglobin like the other members of the arcid family. The tetrameric protein is made up from two types of polypeptide chain, while the dimeric protein is made from a single type of chain which differs from the other two in terms of molecular weight and isoelectric point.The optical and circular dichroism spectra show that the heme environment in HbI and HbII resembles that of vertebrate hemoglobins, although distinctive features are present in the deoxygenated derivative. p]The dimeric HbI in the pH range 6 to 9 does not change its association state upon deoxygenation, while the tetrameric HbII polymerizes as indicated by the appearance of a fast peak in the sedimentation velocity patterns. The dependence of the areas and sedimentation coefficients of the fast and slow peaks on protein concentration is characteristic of a rapidly established association-dissociation equilibrium between tetramers and polymers higher than octamers. The pH, ionic strength and temperature dependence of polymer formation indicate that both hydrophobic and ionic interactions stabilize the polymers.The functional properties of HbI and HbII differ. HbI shows co-operative oxygen binding (h = 1·5) and a constant oxygen affinity ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7.8}\text{mm Hg}$) over the pH range 5.5 to 9.5. HbII likewise shows co-operativity in oxygen binding (h = 2·0). Its oxygen affinity at neutral and alkaline pH values is slightly lower ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.1}\text{mm Hg}$) than that of the dimeric protein, but becomes higher at pH values below 6.5 due to the presence of an acid Bohr effect. At high protein concentrations, under conditions of extensive polymerization of the deoxygenated derivative, the oxygen affinity is lowered and co-operativity slightly increased. Both phenomena require that the oxygen affinity of the polymer be lower than that of the tetramer, consistent with the predictions of linkage theory.     

Polymerization studies on protein from the dahlemense strain of tobacco mosaic virus: Light scattering and related studies

Light-scattering and related studies on protein of Dahlmense strain of tobacco mosaic virus (DTMV) show that its polymerization characteristics are considerably different from those of TMV protein. At pH 6.0 in phosphate buffer (I = 0.1), the extent of polymerization of DTMV protein is greater than that of TMV protein, they are nearly the same at pH 6.25, and that of DTMV protein is less than that of TMV protein at pH 6.5. At pH 7.0 and 7.5, DTMV protein polymerizes more readily than TMV protein. Similar studies in phosphate buffer (I = 0.05) show that the extent of polymerization for DTMV protein is less than that of TMV protein at pH 6.0 and almost negligible at pH 6.25. Acid-base titration studies show that, upon temperature-mediated polymerization, about 2 H⁺ ions are bound per monomer of DTMV protein at pH 6.O.Electron microscope studies show that DTMV protein exists at room temperature as double discs and polymerized rods in phosphate buffer at pH 7.5, I = 0.1; at pH values below 6.5, DTMV protein is entirely in the form of polymerized rods. Velocity sedimentation studies of DTMV protein at room temperature are in agreement with these findings. At low temperatures, except at pH 7.5, most of the material sedimented with an s value of around 25 S. Thus, at low temperatures, except at pH 7.5, DTMV protein in solution is in the form of particles the size of double discs with an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 596,000 g/mole or even larger. Therefore, temperature-mediated polymerization of DTMV protein at pH values below 6.5 in phosphate buffer (I = 0.1) and below 6.25 in phosphate buffer (I = 0.05) involves particles at least as large as double discs as the starting material.     

Occurrence in mammalian liver of a protein which replaces the B protein of E. coli quinolinate synthetase.

$\text{Escherichia coli}$ contains two proteins (A and B) which together convert dihydroxyacetone phosphate and aspartate to quinolinic acid, a precursor of NAD. Although mammalian liver homogenate does not catalyze this reaction it contains a protein which will replace the B protein of the $\text{E. coli}$ system. The behavior of the liver protein on Sephadex G-75 suggests it is much smaller than the $\text{E. coli}$ B protein. Liver B protein also appears to contain tightly bound FAD while FAD is easily removed from the $\text{E. coli}$ B protein. The pH optimum for the hybrid system $\text{E. coli}$ A protein-liver B protein is 9.0 while in the pure $\text{E. coli}$ system the optimum is pH 8.0. The hybrid system is inhibited by NAD to the same extent as the pure $\text{E. coli}$ system.     

Conformation and dynamic interactions in hyaluronate solutions

In hyaluronate solutions, the polysaccharide chains behave as random coils with minor but important deviations; (1) there is an extra degree of stiffening that could have its origin in inter-residue hydrogen bonding, (2) chain-chain contact induces the transient development of tertiary and higher levels of structure, which are enhanced by suppression of electrostatic repulsions on increasing ionic strength or lowering pH, melted out on heating, and inhibited by competition with short chain segments. The deviations are important because they account for viscoelastic properties that are believed to be relevant to the role in biological fluids and the intercellular matrix; thus the chain-chain interactions are favoured entropically by the inherent stiffness of isolated segments and contribute appreciably to the dynamic network structure.Viscoelastic and flow properties have been measured over a wide range of conditions using both oscillatory and steady shear techniques, and systematised in terms of recent rheological theory. The onset of intermolecular coupling occurs at an unusually low degree of coil overlap, and shows no systematic variation with pH, molecular weight or ionic strength. At higher concentrations, however, dynamic intermolecular interactions are enhanced by increasing ionic strength and suppression of molecular charge on lowering pH. Breakdown of the resulting transient intermolecular network on shearing is systematised in terms of the generalised shear parameter $\text{β = γ(η}{}_0\text{? η}{}_{\mathrm{<mtext>solvent</mtext>}}\text{) ×}\text{M}\text{cRT}$. The large anomalous decrease in intrinsic viscosity under alkaline conditions that parallels similar effects seen in nuclear magnetic resonance relaxation, small-angle X-ray scattering and viscoelastic behaviour, is ascribed to an increase in chain flexibility, which may result in part from the ionisation of hydroxy groups involved in hydrogen bonding.     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Surface charges on chloroplast membranes as studied by particle electrophoresis

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts.2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge.3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface.4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues.5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting $\text{chlorophyl}\text{a}\text{chlorophyll}\text{b}\text{pigment · protein complex}$.6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts.7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory.8. Calculations of the ζ-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500–2000 Ų.9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

Ultracentrifugation studies on early stage polymerization of tobacco mosaic virus protein

The effects of absolute temperature (T), ionic strength (μ), and pH on the polymerization of tobacco mosaic virus protein from the 4 S form (A) to the 20 S form (D) were investigated by the method of sedimentation velocity. The loading concentration in grams per liter (C) was determined at which a just-detectable concentration (β) of 20 S material appeared. It was demonstrated experimentally that under the conditions employed herein, an equilibrium concentration of 20 S material was achieved in 3 h at the temperature of the experiment and that 20 S material dissociated again in 4 h or less to 4 S material either upon lowering the temperature or upon dilution. Thus, the use of thermodynamic equations for equilibrium processes was shown to be valid. The equation used to interpret the results, log ($\text{C?β) =}\text{constant}\text{+ (}\text{ΔH}{}^1\text{2.3RT}$) + ($\text{Δ}\text{W}{}^1{}_{\mathrm{<mtext>el</mtext>}}\text{2.3RT}$) ? K′_sμ + ζpH, was derived from three separate models of the process, the only difference being in the anatomy of the constant; thus, the method of analysis is essentially independent of the model. $\text{ΔH}{}^1$ and ΔW¹_el are the enthalpy and the change in electrical work per mole of A protein (the trimer of the polypeptide chain), K^′_s is the salting-out constant on the ionic strength basis, ζ is the number of moles of hydrogen ion bound per mole of A protein in the polymerization, and R is the gas constant. The three models leading to this equation are: a simple 11th-order equilibrium between A₁ (the trimer of the polypeptide chain) and D, either the double disk or the double spiral of approximately the same molecular weight, designated model A; a second model, designated B, in which A₁ was assumed to be in equilibrium with D at the same time that it is in equilibrium with A₂, A₃, etc., dimers and trimers, etc., of A₁ in an isodesmic system; and a phase-separation model, designated model C, in which A protein is treated as a soluble material in equilibrium with D, considered as an insoluble phase. From electrical work theory, $\text{Δ}\text{W}{}_{\mathrm{el}}{}^1\text{/T}$ was shown to be essentially independent of T; therefore, in experiments at constant μ and constant pH the equation of log (C ? β) versus 1/T is linear with a slope of $\text{ΔH}{}^1\text{/2.3R}$. The results fit such an equation over nearly a 20 °C-temperature range with a single value of $\text{Δ}\text{H}{}^1$ of +32 kcal/mol A₁. Results obtained when T and pH were held constant but μ was varied did not fit a straight line, which shows that more than simple salting-out is involved. When the effect of ionic strength on the electrical work contribution was considered in addition to salting-out, the data were interpreted to indicate a value of $\text{Δ}\text{W}{}^1{}_{\mathrm{el}}$ of 1.22 kcal/mol A₁ at pH 6.7 and a value of 4.93 for K_s^′. When μ and T were held constant but pH was varied, and when allowance was made for the effect of pH changes on the electrical work contribution, a value of 1.1 was found for ζ. This means that something like 1.1 mol of hydrogen ion must be bound per mole of A₁ protein in the formation of D. When this is added to the small amount of hydrogen ion bound per A₁ before polymerization, at the pH values used, it turned out that for D to be formed, 1.5 H⁺ ions must be bound per A₁ or 0.5 per protein polypeptide chain. This amounts to 1 H⁺ ion per polypeptide chain for half of the protein units, presumably those in one but not the other layer of the double disk or turn of the double spiral. When polymerization goes beyond the D stage, as shown by previously published data, additional H⁺ ions are bound. Simultaneous osmotic pressure studies and sedimentation studies were carried out, in both cases as a function of loading concentration C. These results were in complete disagreement with models A and C but agreed reasonably well with model B. The sedimentation studies permitted evaluation of the constant, β, to be 0.33 g/liter.     

Structure and properties of hemocyanins. XIII. Dissociation of Helix pomatia alpha-hemocyanin at alkaline pH

Helix pomatia α-hemocyanin can dissociate stepwise into $\text{1}\text{2}$-size, $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules. Both $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules can occur in two isomeric forms, differing considerably in sedimentation behaviour.The effects of pH, ionic strength, temperature, Ca²⁺ concentration and oxygen pressure on these dissociation and isomerization steps were investigated systematically by sedimentation analysis.Dissociation and isomerization are favoured by increasing pH or temperature. Changes in ionic strength affect each step differently. Calcium ions are extremely effective in preventing dissociation and isomerization at low ionic strength, but this stabilizing ability diminishes at higher ionic strengths. Oxygen binding shifts the pH-dependent dissociation of whole into $\text{1}\text{2}$-size molecules to higher pH. Oxygen has no effect on the other dissociation steps. Intermolecular interactions appear to be predominantly electrostatic.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

New insight on beta-lactoglobulin binding sites by 1-anilinonaphthalene-8-sulfonate fluorescence decay

The fluorescence time decay parameters of the beta-lactoglobulin-1-anilinonaphthalene-8-sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a "surface" site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye-Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the "surface" site mainly responds to the presence of these local titrating charges while the "internal" site more closely follows the overall protein net charge.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.     

Hydrogen ion uptake upon tobacco mosaic virus protein polymerization.

To determine the stage at which H⁺ ions are bound during the entropy-driven polymerization of tobacco mosaic virus protein, acid-base titrations were carried out at a concentration of 5 mg/ml in 0.1 m-KCl from pH 8 to pH 5.2 and back to pH 8 at 4, 10, 15 and 20 °C. The titration was always completely reversible when the addition of acid or base was so slow that the experiment required seven hours in each direction. When the titration was started at pH 7 and performed down and up twice as rapidly, a hysteresis loop, indistinguishable from one previously published, was obtained at 20 °C.Ultracentrifugation experiments were carried out at selected pH values at the four temperatures. H⁺ ion uptake, as determined from the reversible titration curves, is correlated with the disappearance of the 4 S component and is independent of whether the polymerized species is in a 20 S or higher state of aggregation. At pH 7, approximately 1 mole of H⁺ ion is bound per mole of monomer. At pH values between 6.56 and 6.05, 1.5 moles of H⁺ ion are bound per mole of monomer upon polymerization. At pH 6.05, 0.5 mole of H⁺ ion is bound before any polymerization takes place.Tobacco mosaic virus protein at 20 °C in an unbuffered 0.1 m-KCl solution at pH 7.18 at a concentration of 41 mg/ml, largely in the 20 S state, was depolymerized entirely to the 4 S state by dilution with 0.1 m-KCl adjusted to the same pH. Under these conditions, there was no pH change, indicating that no H ⁺ ions are released.These seemingly contradictory findings can be explained by assuming that the 4 S component polymerizes to form either double discs without binding H⁺ ions, or, alternatively, two-turn helices accompanied by the binding of H⁺ ions. Both double discs and two-turn helices sediment at approximately 20 S. Whether polymerization in the neighborhood of pH 7 leads to helices or discs depends upon the availability of H⁺ ions.     

Effect of temperature and pH on lipid accumulation by Trichoderma reesei

Summary The fungal micro-organism Trichoderma reesei was grown in batch culture with excess glucose at pH values between 2.7 and 4.5 and temperatures between 25°C and 35°C. A maximum lipid concentration of 16.9% of the cell dry weight was achieved at pH 3.2 and a temperature of 27°C. Lipid concentration was shown to be correlated with a calculated maximum specific growth rate (µ _mc ) and the maximum lipid value occurred at µ _mc = 0.10 h^–1. Fatty acid analysis was carried out and found to change with changing pH and temperature. Palmitic (16:0) acid and unusually high proportions of stearic acid (18:0) were commonly present. A conversion of fatty acids to palmitoleic acid (16:1) occurred following an unidentified nutrient limitation other than nitrogen depletion after 70 h of culture. Offprint requests to: D. E. Brown