Henryk Eisenberg

THERMODYNAMIC ANALYSIS OF MULTICOMPONENT SOLUTIONS.

Publisher Summary This chapter discusses thermodynamic analysis of multicomponent solutions. An exhaustive consideration of the properties of solutions and theory of electrolytes requires a treatise far beyond the scope of this chapter, in which one shall be concerned primarily with only certain thermodynamic problems specifically implied by the presence of at least three components, some diffusible and some nondiffusible. Because the classic equilibrium thermodynamics of fluid systems can be regarded as complete science, now in no way tentative and not expected to conceal as yet unrevealed profundities, it is not unrealistic to expect to formulate statements that are precise and definitive, so far as thermodynamics is concerned, of relations among experimental quantities important in the study of dilute solutions. Another situation of considerable importance arises when there are several diffusible and several nondiffusible solutes, but the relative amounts of the latter are kept unchanged as their total concentration is altered at constant potential of nondiffusible components.

Ribonucleic acid from Escherichia coli; preparation, characterization and physical properties.

A method has been developed for the isolation of RNA from E. coli “protoplasts”, by extraction with a phenol-water mixture. The RNA preparation contained no detectable DNA and only traces of proteins and polysaccharides. Sedimentation in the ultracentrifuge yielded three boundaries. The two faster moving components were separated from the slower one by (NH4)2SO4 precipitation from a phenol-saturated water solution. From sedimentation, viscosity and light-scattering data the molecular weight was estimated to be of the order of one million. The RNA preparations in their viscosity behavior closely resemble coiling synthetic polyelectrolytes, and behave quite unlike DNA. The viscosity behavior, birefringence of flow, and potentiometric titration data are discussed in terms of a single contractile coil model. The ϵ(P)260 mμ values of the isolated material in 1 M phosphate (pH 7.1) were about 7400. Alkaline hydrolysis or polyribonucleotide phosphorylase action brought about an increase in absorption ranging from 56 to 59% while pancreatic ribonuclease caused a 28–31% increment. At very low concentrations of RNAase the change in optical density remained constant, while viscosity decreased rapidly with time.

Biochemical, structural, and molecular genetic aspects of halophilism.

Publisher Summary The study of halobacteria relates to a better understanding of evolutionary relationships. Halophilic enzymes are very unstable in low salt concentrations. Because some of the important fractionation methods in protein chemistry, such as electrophoresis or ion-exchange chromatography, cannot be applied at high salt concentrations, the available fractionation methods are limited. The existing purification procedures fall into two groups: the nonhalophilic approach and the halophilic approach. This chapter reviews the developments in the molecular characterization of halobacterial proteins, starting with the methodology of their purification. The biochemical and biophysical structural analyses of some enzymatic systems for which extensive knowledge is accumulated is described.. Macromolecular structures from halophilic bacteria are discussed to improve the understanding of the molecular mechanisms of adaptation to high salt concentration environments by considering genome organization, genetic tools, isolation of genes, or transcript organization, and structure. The structural aspects of halophilism can be determined by ribosomal subunits, surface layers, purple membrane, or halophilic malate dehydrogenase.

Nucleosome core particle stability and conformational change: Effect of temperature, particle and NaCl concentrations, and crosslinking of histone H3 sulfhydryl groups☆

We have studied the reversible dissociation of core size DNA from chicken erythrocyte nucleosome core particles in solutions containing 0 X 1 M to 0 X 6 M-NaCl. Dissociation increases with increasing NaCl concentration, increasing temperature and decreasing particle concentration. At high particle concentrations, no free DNA is observed below 0 X 3 M-NaCl, whereas above 0 X 3 M-NaCl a lower limit of dissociation is reached. A theoretical analysis based on the migrating-octamer mechanism of Stein is in disagreement with his conclusions concerning dependence of core particle dissociation on particle concentration, but provides a good explanation for our observations, and those of others, using salt concentrations up to 1 M-NaCl. It appears that the core particle is not stabilized primarily by electrostatic interactions. DNA length is not critical for core particle stabilization. The conformation of remaining intact nucleosome core particles changes only moderately within the range of NaCl concentrations studied. Crosslinking by copper phenanthroline of the Cys110 histone H3 single sulfhydryl groups in the intact nucleosome core particle leads to a decrease in stability, yet essentially unchanged hydrodynamic properties are maintained at 0 X 6 M-NaCl, confirming conclusions derived from the behavior of the native core particles. Values for density increments of nucleosome core particles over a range of NaCl concentrations are also given. A method is described for studying binding of histones to nucleosome core particles in the ultracentrifuge by scanning at 230 and 260 nm.

Equation for the Refractive Index of Water

The observations that β−1 (∂ lnf/∂P)T=B is a constant (of order unity, independent of temperature), and —γ−1 (∂ lnf/∂T)P=B+Cγ−1, lead to an equation f(n)≡(n2−1)/(n2+2)=AρB exp(−CT), which describes the refractive index n of water between 0° and 60°C (at any given wavelength in the visible spectrum) to within a few digits in the seventh decimal. Here ρ is the density, β=(∂ lnρ/∂P)T and γ=—(∂ lnρ/∂T)P. The constant C=—(d lnf/∂T)V reflects the change in the structure of water at different temperatures. The temperature of maximum index [(∂n/∂T)P=0] corresponds to γ=—C/B, and the minimum in the Lorenz—Lorentz specific refraction (n2−1)/(n2+2)ρ corresponds to γ=C/(1—B). The values of the parameters A, B, and C are independent of pressure up to 1100 bar in the temperature range examined. It is suggested that refractive index, or dielectric, measurements at higher pressures and temperatures may yield further information on the structure of water in terms of specific models related to the variation of B and C with...

Isolation and physical studies of the intact supercoiled: The open circular and the linear forms of CoIE1-plasmid DNA

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

Halophilic proteins and the influence of solvent on protein stabilization

Competition between protein-solvent and protein-protein interactions is arguably the most important contributing factor to polypeptide folding in general. A study of halophilic proteins, correlating their stability and solution structures in different conditions, focuses on the effects of a high salt solvent. A mechanism is proposed to explain how these proteins have adapted to such an extreme environment.

Multicomponent Polyelectrolyte Solutions. Part I. Thermodynamic Equations for Light Scattering and Sedimentation

Equations are derived, in terms of the derivative of the osmotic pressure with respect to concentration, for light scattering and sedimentation (transport and equilibrium) of polyelectrolyte systems comprising a solvent component 1, a macromolecular component 2, and an added simple electrolyte component 3. It is shown by thermodynamic arguments that, when suitable restrictions are applied to the chemical potential of component 3, the equations for the three‐component systems become formally identical with the equations for two‐component systems. A striking similarity is achieved between the equations for light scattering and sedimentation, by direct use in the latter of the derivative of the solution density with respect to concentration, instead of the more familiar buoyancy terms. The extension of the equations to more than one supporting electrolyte is immediate, and the extension to a macromolecular component inhomogeneous with respect to molecular weight may be achieved by known methods.

Bovine Liver Glutamate Dehydrogenase

Publisher Summary This chapter discusses the current ideas on the catalytic reaction of glutamate dehydrogenase, including its mechanism and regulation, substrate inhibition or activation, the binding of coenzymes to the enzyme, the effect of purine nucleotides, and the mechanism of ligand-induced structural changes. The chapter also presents chemical modification of sulfhydryl groups, lysine, tyrosine, and histidine residues; however, most of the discussion will be restricted to the properties of mammalian mitochondrial enzyme. The enzyme glutamate dehydrogenase from bovine liver presents a particularly interesting example of self-assembly, because some of the control mechanisms for assembly are understood and the assembly process has been characterized at several different levels of molecular organization. But, there are still many general aspects of the mechanism of both the deamination and amination reactions that are not understood. Although, the recent rapid kinetics and difference spectroscopy studies have provided new and valuable information on intermediates of the enzymic catalysis, work along the lines developed by authors should lead to a better understanding of the catalytic action of glutamate dehydrogenase and its regulation by metabolic effectors. The chapter proposes that improved physical methods and imaginative design of experiments may lead, in the future, to a more detailed understanding of the structure and function of this enzyme.

Biophysical study of halophilic malate dehydrogenase in solution: revised subunit structure and solvent interactions of native and recombinant enzyme

In previous work, malate dehydrogenase from Haloarcula marismortui(hMDH), a halophilic enzyme that is only stable in high concentrations of certain salts, was characterized by different biophysical methods as a dimer of molar mass 87 000 g mol–1 with solvent interactions in multimolar NaCl solutions that are significantly larger than for non-halophilic proteins. A model was proposed in which hMDH is stabilized by different mechanisms in different salt solvents (cf. Eisenberg et al., Adv. Protein Chem., 1992, 43, 1). Recently, the gene coding for hMDH was isolated and sequenced and the recombinant protein expressed in E. coli and renatured (Cendrin et al., Biochemistry, 1993, submitted). A subunit molar mass of 32 638 g mol–1 was calculated from the sequence and confirmed by mass spectrometry. The present study was undertaken in order to resolve the discrepancy between this value and the previously proposed dimer solution structure. The absorption coefficient of the protein was redetermined by amino acid analysis. New densimetry measurements in a large range of salt concentrations, ultracentrifugation and light scattering experiments were performed on the native and recombinant enzymes, and previous ultracentrifugation, X-ray and neutron scattering data were re-analysed. Control experiments on bovine serum albumin (as a model for non-halophilic proteins) were also repeated under similar conditions. All the data are now in agreement with a tetrametric solution structure for hMDH, with solvent interactions (e.g. in multimolar NaCl or KCl solutions: ca. 0.4 g water and 0.1 g salt per g protein) that show water binding comparable to non-halophilic proteins yet an order of magnitude more salt binding than for non-halophilic proteins. The stabilization model for hMDH remains valid. The complementarity and accuracy of the different methods for solution structure analysis are discussed critically in the light of this study.