ALBERT

Notes: We have measured the ultraviolet CD spectra for covalently closed and linear forms of phage PM2 DNA in solution. We find that increased concentrations of salt or ethanol (up to 50% ethanol by weight) depress the long-wavelength positive CD bands in the spectra of both forms of DNA, although the spectrum of the native covalently closed DNA always has a slightly larger magnitude of these bands than does the spectrum of the linear DNA. In addition, both DNAs are equally capable of undergoing a transition to the A conformation in 70-80% ethanol at low Na+ concentrations. Thus, the constraint imposed by the covalent closure of a DNA molecule does not seem to hinder its conformational response to these changing solution conditions. Lang [(1973) J. Mol. Biol. 78, 247-254] has found by electron microscopy that T7 DNA has an inherent ability to condense into compact particles, suggested to be supercoils of multiple order. Both covalently closed and linear forms of PM2 DNA also become condensed when the DNA, in 0.2M ammonium acetate and 1 mM EDTA, is exposed to ethanol and subsequent drying on specimen grids [Lang, D., Taylor, T. N., Dobyan, D. C. & Gray, D. M. (1976) J. Mol. Biol. 106, 97-107]. Under similar conditions, in solutions of 0.2M ammonium acetate and 1 mM EDTA to which ethanol is added, we have measured the CD spectra of both covalently closed and linear forms of DNA. Below ethanol concentrations at which the DNA obviously precipitates, the CD spectra of both forms have reduced long-wavelength positive CD bands.

Notes: The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.

Notes: The low-temperature dynamic mechanical relaxation of ox Ligamentum nuchae elastin, swollen with water and ethylene glycol at various swelling ratios, has been investigated by means of the Rheovibron DDV II viscoelastometer, at frequencies from 3.5 to 110 Hz. The peak location on the temperature scale, its intensity and activation energy have been studied as a function of solvent content and it has been found that the two solvents influence the dynamic mechanical behavior in a quite different way. A similar interaction of the solvent molecules with the backbone peptide groups is assumed for both solvents, and the differences in the mechanical spectra, which are more marked the higher the solvation degree, are explained in terms of the different size of the protein-solvent relaxing units.

Notes: The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.

Notes: The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m2′)] and poly[A(e2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ〈280 nm (τα) are different from those found at λ 〉280 nm (τβ), indicating the presence of more than two conformational states. The time constants τα increase in the series poly(dA), poly[A(m2′)], constants τβ/τα is approximately 2.5, except in poly(dA) where τβ/τα ≈ 9. Relaxation measurements with r(A)n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D2O reveal a solvent isotope effect for τα of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τβ. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar-phosphate chain. The absence of a seprate relaxation process (corresponding to τβ) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar-phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.

Notes: An algorithm has been developed that permits one to find all possible conformations of the sugar-phosphate backbone for any given disposition of DNA base pairs. For each of the conformations thus obtained, the energy of the helix was calculated by the method of atom-atom potentials. Several isolated regions in the space of the bases′ parameters (Arnott's parameters) have been found for energetically favorable helical structures. Two parameters, the distance of a base pair from the helix axis, D, and the windling angle, τ, allow one to subdivide possible conformations into the families of closely related forms. Two regions (ravines) on the (D, τ) map correspond to the know A and B families. In the B family a continuous transition has been obtained in which the double helix undergoes increasing winding, while the base pairs are moving toward the major (nonglycosidic) groove.Interrelationships between the variables, characterizing the spatial structure of the double helix, D, τ, TL and χ, when going along the bottom of the B ravine, were also obtained.Besides the Known A and B families, several new ones were found to be energetically possible. Among these the strongly underwound helices with the negative D values, as well as the forms with the C4-C5 angle in a trans position, should be mentioned.Biological roles of the different double-stranded conformations, in particular, in protein-nuclei acid interaction are discussed.

Notes: The theoretical basis for the statistical mechanical deconvolution of a thermally induced macromolecular melting profile is presented. It is demonstrated that all the thermodynamic quantities characterizing a multistate macromolecular transition can be obtained from the average excess enthalpy function, 〈ΔH〉, of the system, without any assumption of the particular model or mechanism of the reaction.Experimentally, 〈ΔH〉 is obtained from scanning calorimetric data by direct integration of the excess apparent molar heat capacity function, ΦCp. Once 〈ΔH〉 is known as a continuous function of the temperature, the partition function, Q, of the system can be calculated by means of the equation \documentclass{article}\pagestyle{empty}\begin{document}$$Q_{\left( T \right)} = \exp \smallint _{T_0}^T {\left( {\frac{{\left\langle {\Delta H} \right\rangle }} {{RT^2}}} \right)} dT $$\end{document} From the partition function all the thermodynamic quantities of the system can be obtained. It is shown that the number of discrete macroscopic energy states, the enthalpy and entropy changes between them, and the relative population of each state as a function of temperature can be calculated in a recursive form.

Notes: The high-resolution 1H-nmr study of the ferrichrome cyclohexapeptides, in d6-DMSO solutions, has been extended to the amide NH spectral region. A total of ten diamagnetic analogues of ferrichrome that differ in the coordinated metal ion (Al3+, Ga3+ or Co3+), the primary structure, the nature of the bidentate hydroxamate moiety, or the isotope compositions (14N, 15N) have been investigated. The 3JαNH values reflect regiorous conformational isomorphism throughout the complete suite of analogues, quite independent of the residue occupancy of each site. Totally resolved amide multiplets have been obtained in most cases and the four-line (doublet of doublets) appearances of glycyl NH resonances has been observed for the first times; these data enabled stereospecific assignment and accurate analysis of the NH-CαH proton spin systems. The high resolution was made possible by the use of a suitable spectral deconvolution shceme at 360 MHz. The fine structure, extraordinarily well displayed in the 15N-peptide spectra, provides a series of parameter values whose consistency has been checked by computer simulation. Since the crystallographic structure for two of the ferric peptides is known to 0.002-Å resolution, a 3J vs θ correspondence could be confidently established. A Karplus curve was derived from the combined x-ray and nmr data: \documentclass{article}\pagestyle{empty}\begin{document}$$ ^3 J_{\alpha {\rm NH}} = 5.4\cos ^2 \theta - 1.3\cos \theta + 2.2{\rm Hz} $$\end{document} It is suggested that seriously nonplanar amides can exhibit 3JαNH values higher than predicted by the ferrichrome curve.

Notes: The general formulas describing the low-angular dependence of polarized (Vv) and depolarized (Hv) light-scattering intensity by a chain macromolecule of arbitrary form with anisotropic polarizability are derived. It is shown that the value dHv/dtHvo (where t = [〈R2〉S/L]2, S being the scattering vector) is an indication of the chain-flexibility mechanism. This permits one to distinguish between the models of a wormlike chain, regular zigzag, or statistical zigzag. The results of numerical calculations useful for the interpretation of experimental data are presented.

Notes: The kinetics of the fluorescence decay of the energy donor in a homologous series of oligopeptides each containing at its ends a donor and an acceptor of electronic excitation energy was investigated in solvent mixtures of different viscosities. The repeating unit in the peptides was N5-(2-hydroxyethyl)-L-glutamine and the chromophores used as donor and acceptor were naphthalene and dansyl, respectively. The number of units in the peptides studied varied from four to nine. The solvents used were mixtures of glycerol and trifluoroethanol in various proportions. The decay rate of the donor fluorescence increases when the solvent viscosity decreases. This behavior is due to the disturbance of the equilibrium end-to-end distribution of distance of the excited molecules by the energy transfer process, which is more favorable foe short than for long distances. The subsequent rearrangement towards the equilibrium distribution by diffusion of the molecular ends relative to one another enhances the efficiency of the energy transfer. Assuming a modified Fick equation to describe this diffusion motion, the fluorescence decay data were analyzed in terms of a diffusion coefficient describing the Brownian motion of the molecular ends. The diffusion coefficients thus evaluated increase systematically upon decreasing the solvent viscosity. For example, for the oligopeptides studied it changes from unmeasurably small values in glycerol solution to values varying between 10-8 to 10-7 cm2/sec at room temperature in a glycerol trifluoroethanol solvent mixture of viscosity of 8 centipoise. The values obtained for the diffusion coefficient are smaller by about an order of magnitude than the values expected for the diffusion coefficients of the free chromophores in solvents of comparable viscosity. It is thus concluded that the backbone of the polymeric chains possesses appreciable internal friction which exerts resistance to the Brownian motion of the polymer chains. The diffusion coefficient of the end-to-end motion is systematically smaller for the shorter than for the longer chains. For example, at room temperature in a solvent mixture of 8 centipoise it is 3×10-8, 5×10-8, 7.6×10-8, and 8.5×10-8 cm2/sec for oligomers containing four, five, eight, and nine N5-(2-hydroxylethyl)-L-glutamine repeating units, respectively. The internal friction thus impedes the motion of the molecular ends more effectively in the shorter chains than in the longer ones.Analysis of the energy-transfer experiments in solvents spanning a wide range of viscosities shows that the orientational factor appearing in Förster's equation for energy transfer does not interfere perceptibly with the evaluation of the end-to-end distances from the fluorescence decay data when naphthalene and dansyl serve as donor and acceptor, respectively. This is due to the fact that both the donor emission and acceptor absorption in the range of spectral overlap are characterized by more than one transition dipole moment, as is evident from the corresponding polarization data.

Notes: Raman spectroscopic studies have been carried out on polymers of L-valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40-1750 cm-1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L-alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L-valine.

Notes: The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λmax) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λmax, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λmax of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.

Notes: Group-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4KM KCl and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times at different concentrations using incident wavelengths of 632.8 nm from a He-Ne laser and of 488 nm from an argon-ion laser yield information on aggregation properties, molecular weight (Mr), radius of gyration 〈r0g〉1/2z, translational diffusion coefficient 〈D〉0z, and second virial coefficients A2 and B2 of C-1 polysaccharide.At relatively high ionic strength (0.04M KCl + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution Mr = 5.15 × 105, 〈r2g〉1/2z = 345 Å, A2 = 1.25 × 10-4 ml/g, 〈D〉0z = 1.16 × 10-7 cm2/sec with a corresponding Stokes radius of 240 Å and B2 = 4.4 ml/g. A2 and B2 are the second virial coefficients from intensity- and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils. Viscosity and sedimentation studies further confirm our conclusions that the fractioned C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum at ∼0.2M KCl. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors.

Notes: We extend fluorescence correlation spectroscopy to systems that undergo translation or laminar flow in a sample cell. We include theoretical and experimental results; we consider uniform and nonuniform velocity profiles. Concentration correlation analysis extracts microscopic rate parameters from measurements of the spontaneous concentration fluctutations, which occur even at equilibrium. Fluorescence is one of the most sensitive means of monitoring these fluctuations. Analysis of flowing or translating systems (1) offers a method of measuring number concentrations of selected species, for example, of aggregates or polymers, (2) provides a nonperturbing velocity probe, (3) sometimes allows one to circumvent photolytic degradation, (4) has proved extremely helpful in testing and aliging apparatus for fluorescence correaltion measurement and in verifying theoretical analyses, and (5) may be required for interpretation of results obtained on systems in motion, even though that motion is undesired or initially unsuspected. We include both theoretical and experimental results for combined Poiseuille flow and diffusion in the geometry which is of most practical interest. Theoretical expressions for the much simpler cases of nondiffusive Poiseuille flow as well as uniform flow or translation with or without diffusion constitute limiting cases which are displayed explicitly.

Notes: In the preceding article1 we demonstrated that the partition function of a system is experimentally accessible from scanning calorimetric data. In this article the general results of the deconvolution theory are applied to the general case of cooperative transitions in macromolecules. It is demonstrated that, in the limit of very large systems, all the relevant molecular averages and molecular distribution functions can be directly obtained from the experiment. In doing this, the method of the grand partition function is used. It is shown that in the case of homopolymers, only one parameter, the stability constant, needs to be explicitly specified for a complete description of the system. Since the partition function is directly evaluated from the experiment, no special assumptions or artificial constraints directed to obtain a mathematically solvable model are required. This result offers the unique opportunity of having direct experimental access to statistical averages of systems in which the partition function cannot analytically be solved. Consequently, the theory can be extended to cooperative transitions occurring in two and three dimensions by introducing cluster distribution functions.

Notes: The pyridoxal Schiff bases of the polypeptides poly(L-lysine), poly(L-ornithine), and poly(L-α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L-α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.

Notes: The protein turkey-heart cytochrome c is used as a model protein to study charge effects in sedimentation equilibrium experiments in three-component solutions. Data are given for the dependence of the apparent M (1-υρ) on ρ in solutions of KCl, RbCl, CsCl, and triethylamine hydrochloride. The results show the Donnan effect to have a significant influence on the apparent molecular weight, found by extrapolation of the data to a solution density of one. The apparent molecular weights are for protein at infinite dilution. A theoretical treatment is presented where the magnitude of this effect can be predicted accurately from the formal net charge of the protein as computed from the amino acid composition. The results are shown to be important in computing the preferential hydration of the protein in concentrated salt solutions. For such systems the Donnan effect should be subtracted from the total interaction coefficient for multicomponent system in order to obtain the preferential hydration.

Notes: The cupric complexes of poly(Nε-acetoacetyl-L-lysine), [Lys(Acac)]n′ poly(Nδ-acetoacetyl-L-ornithine), [Orn(Acac)]n′ and poly(Nγ-acetoacetyl-L-diaminobutyric acid), [A2bu-(Acac)]n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.

Notes: The Mettler/Paar precision density meter DMA-02D has been used to determine the concentration of saturated solutions of amino acids at 20.0, 25.0, and 29.8 °C. The technique has proven itself an elegant and precise method. The solubilities of all of the amino acids with the exceptions of proline, lysine, and cystine have been measured. The Gibbs free energies of transfer from saturated water solution to 1M Na2SO4 and to 1M Gu·HCL along with the van't Hoff heats and entropies have been calculated. The van't Hoff heats have been compared with the calorimetrically determined heats for some of the amino acids. The Lumry-Rajender relation between the entropy and heats has been observed. The process of transfer of the amino acids from water to the solvents is primarily enthalpic rather than entropic.

Notes: The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10-6-10-1M at the ionic strengths of 10-3M Na+. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg++ increases the DNA stability, the effect being observed mainly in the concentration range 10-6-10-4M. At[Mg++]〉10-2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ∼10-5M and then decreases to 7-8°C at the ion content of 10-3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na+] = 10-3M) and [Mg++] ≤ 10-5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA-Mg++ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA.

Notes: Studies on the cross-linking of a tripeptide (t-butyloxycarbonyl-L-alanyl-D,L-2-amino-6-heptenoyl-L-alanine methyl ester) have shown that it is possible to form specific cross-links in good yields through Schiff base formation of the ε amino group of lysine. The heptenoic acid residue has been ozonized to an aldehyde and condensed with the ε amino of lysine in the compounds alpha-t-butyloxycarbonyl-L-alanyl-L-lysine methyl ester and alpha-t-butyloxycarbonyl-L-lysine methyl ester to form the cross-link, lysinonorleucine. This compound has been stabilized by reduction with sodium borohydride and quantitated on the amino acid analyzer. This technique converts from 60 to 98% of the available aldehyde to lysinonorleucine.

Notes: The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit 〉 bovine ≫ ovalbumin; with anti-ovalbumin, it is ovalbumin ≫ rabbit. Since the frequencies of the amide I band appear near 1655 cm-1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.

Notes: The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H2O and D2O at pH 3-7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10-2 to 5 × 10-4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10-3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (-143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.

Notes: The Raman spectra of poly(L-lysine) with various structures, ionized poly(L-glutamic acid), and deuterated N-methylacetamides have been observed using visible and the 257.3-nm laser lines as the light source. Most of the Raman bands with significantly enhanced intensities in the uv-excited spectra of the polymers have been assigned to the vibrations associated with the C=O and C-N stretching modes, the amide I, II, III, I′, II′, and III′, with reference to the results obtained for simple amide molecules including the deuterated N-methylacetamides. Several amide frequencies have been newly identified and the structures of the polymers have been discussed through the comparison of the Raman and ir amide frequencies.

Notes: Real and imaganiry parts of complex dielectric constant of dilute solutions of DNA in 10-3M NaCl with molecular weight ranging from 0.4 × 106 to 4 × 106 were measured at frequencies from 0.2 Hz to 30 kHz. Dielectric increments Δε were obtained from Cole-Cole plots and relaxation times τD from the loss maximum frequency. The τD of all samples agrees well with twice of the maximum viscoelastic relexation time in the Zimm theory, indicating that the low-frequency dielectric relaxiation should be ascribed to be the rotation of DNA. The rms dipole moment, which was obtained from Δε, agree well with that calculated from the counterion fluctuation theory. The dielectric increment was found to be greatly depressed in MgCl2, which is resonably interpreted in terms of a strong binding of Mg++ ions with DNA.

Notes: Ultravoilet photoelectron spectroscopy has been employed to exmamin the valence electronic structure of 5-fluorouracil, 5-chlorouracil, 5-bromouracil, and 5-iodouracil. Photoelectron bands associted with the three highest π orbitals and the two oxygen atom lone-pair orbitals were assigned by a comparison to similar bands observed in the photoelectron spectrum of uraciul. Bands arising from the halogen atom lone-pair orbitals were assigned by comparing the present results with photoelectron spectra measured for halobenzenes, and by considering the linear dependence of halogen atom lone-pair ionization potentials upon halogen atom electronegativities. The present spectroscopic results have been compared with results from studies of association constants of 5-halouracil-adenine complexes. This examination in dicates that the complex association constants incresase as the ionization potentials of the highest occupied π orbital and the halogen atom lone-pair orbitals of th halouracils decrease.

Notes: Electric dichroism measurements on poly(A) in low-ionic-strength solution demonstrate that below a molecular weight of 130,000 the double-stranded polymer is hydrodynamically rigid and above that molecular weight becomes increasingly flexible. At 500,000 it is considerably more flexible than DNA of the same molecular weight, with a mean end-to-end distance of about 1150 Å compared to approximately 1600 Å for DNA. The fully extended length for both DNA and poly(A) of this molecular weight is about 2750 Å. It is further shown that the orientation of these polyelectrolytes in an electric field is consistent with theoretical treatments of the counter-ion distribution and a preliminary model based on the additivity of classical valence charge anisotropy and counter-ion polarization is postulated for the orientation mechanism. Single-stranded pol (A) is shown not only to retain its base stacking in the presence of the electric field but to extend the persistent regions of stacked bases so that it attains a rodlike structure very similar to the one in the double-stranded polymer is found to be less than that expected from consideration of the x-ray structure. An explanation for this result is sought in the electric asymmetry of the helical polymer.

Notes: The effect of hydration on the solid-state conformation and relaxation behavior of the alkaline earth salts of poly (L-glutamic acid) has been examined. The calcium and strontium salts take the β-conformation and the magnesium salt takes the α-form. No hydration-dependent conformational transitions were observed. The amount of bound water, as measured by differential scanning calorimetry, appears to depend on the size of the cation, while conformation has little or no effect. A relative measure of the barrier to side-chain reorientation was obtained from the temperature of the side-chain (β1) relaxation. It was found that the barrier is determined by electrostatic forces and increases with increasing cation surface charge density. Side-chain motion also appears to be more hindered in the β-conformation than in the α-conformation.

Notes: An optimized potential function for base-stacking interaction is constructed. Stacking energies between the complementary pairs of a dimer are calculated as a function of the rotational angle and separation distance. Using several different sets of atomic charges, the electrostatic component in the monopole-monopole approximation (MMA) is compared to the more refined segmented multipole-multipole representation (SMMA); the general features of the stacking minima are found to be correctly reproduced with IEHT or CNDO atomic charges. The electrostatic component is observed to control the location of stacking minima. The MMA, in general, is not a reliable approximation of the SMMA in regions away from minima; however, the MMA is reliable in predicting the location and nature of stacking minima. The attractive part of the Lennard-Jones 6-12 potential is compared to and parameterized against the expression for the second-order interaction terms composed of multipole-bond polarizability for the polarization energy and transition-dipole bond polarizabilities for approximation of the dispersion energy. The repulsive part of the Lennard-Jones potential is compared to a Kitaygorodski-type repulsive function; changing the exponent from its usual value of 12 to 11.7 gives significantly better agreement with the more refined repulsive function. Stacking minima calculated with the optimized potential method are compared with various perturbation-type treatments. The optimized potential method yields results that compare as well with melting data as do any of the more recent and expensive perturbation methods.

Notes: The 1H-nmr spectra (270 MHz) of cyclic di- and tri-L-azatidine-2-carboxylic acid [cy-clo(L-Aze)2 and cyclo(L-Aze)3] were determined in CDCl3 and D2O and computer simulated. The spectral results were compared with those obtained with cyclo (L-Pro)2 and cyclo (L,-Pro)3. In CDCl3 and D2O solution, the four membered ring of cyclo (L-Aze)2 is puckered with the α-proton in a pseudo-axial position, and the φ angle is smaller in absolute value than -60°, as found for cyclo (L-Pro)2,. The puckering of the four-membered ring of cyclo(L,-Aze)3 in CDCl3 has the α-proton in a pseudo-equatorial position and φ angle larger in absolute value than -60°, in agreement with cyclo(L-Pro)3. In D2O, cyclo(L-Aze)3 was found to interconvert rapidly between different conformers. In the azetidine cyclic peptides studied, the range of values found for the φ angles was smaller than in the related proline cyclic peptides, indicating greater rigidity in the four-membered ring.

Notes: The study of the Cu(II)-hyaluronate complexes by absorption and CD spectra, as well as by acid-base titration and viscosity, provides information about the nature of ligands and the conformation of the polymer. Three different complexes have been identified. The first (complex I), which is formed between pH 3 and 6, involves mainly the carboxyl groups of the polymer as ligands and is characterized by a strong absorption band at 238 nm. In this complex formation, the CD properties of hyaluronate do not charge appreciably. The second (complex II) forms between pH 6 and 8 bad shows a major change in CD properties. The changes include (1) a new positive CD band at 250 nm and a strong negative on in the π → π* amide transition region and (2) the disappearance of the negative n → π* amide CD band near 210 nm. A sharp increase in absorbance at 238 nm from complex I to II has been attributed to a conformational transition which is also manifested in the CD features of hyaluronate. Complex II involves, in addition to the carboxyl group, the nitrogen atom of the deprotonated acetamido group coordinated to Cu(II). The absorption at 230-280 nm is associated with the optically active charge-transfer transitions involving ligands to metal ion. At higher concentrations of the polymer or at higher pH, complex II aggregates to a gel, complex III. Chondroitin, differing from hyaluronic acid in the C-4 hydroxyl group configuration of the glucosamine moiety, does not show any CD change in the presence of Cu(II).The results provide further support to our fourfold helical structure of Cu(II)-hyaluronate complex at pH between 6 and 8. Intrinsic viscosities of hyaluronate in the presence of the cupric ion is lower than in the presence of other monovalent or bivalent cations, indicating a compact conformation of the polymer when it is complexed with Cu(II).

Notes: The far-uv CD spectra of eight lectins are presented. These eight lectins, as well as others previously studied, are grouped into three classes according to their particular CD spectra. The eight lectins studied appeared to have secondary structure dominated by β-pleated sheet, which so far has been true of all lectins. An attempt was made to quantitate the three structural components (α-helix, β-pleated sheet, and aperiodic) in the lectins using the data for reference proteins given by Chen et al. [Biochemistry 13,3350 (1974)]. For lectins in two of the classes, this method proved excellent and values for the three components are given. However, for the third class of lectins, which includes most of the lectins as well as Concanavalin A, this method of analysis proved to be unsatisfactory. This inadequacy was due to two factors: (1) the reference proteins used by Chen and (2) the unusual CD spectra of these lectins manifested by considerable ellipticity above 225 nm in a region where there are no known peptide electronic transitions.

Notes: In an attempt to better our understanding of the conformational stabilities in RNAs, an intensive theoraticl study has been carried out on one of its dimeric subunits, ApA, using an improved set of atom-atom interaction energy parameters and an improved version of energy-minimization technique. The C(3′)0endo and the C(2′)-endo sugar ApA units were sperately considered and 38 probable conformations have been analyzed in each case. The total potential energy, comprising nonbonded, electrostatic, and torsional contributions, was minimized by varying all seven relevant dihedral angles simumtaneously. The result reveal that 17 conformations in the case of C(3′)-endo sugar ApA and 7 confomations in the case of C(2′)-endo sugar ApA unit, the lowest energy conformation corresponds to a nonhelical structure and the A-RNA and the Watson-Crick-yype conformations lie at energy levels of about 0.5 and 1.0 Kcal/mo., respectively, above the lowest energy found. For ApA with the lops of different types in the backbone and they all differ in energies by about 3.5 Kcal/mol with refrence to the lowest energy founs. It is noted that the order ofmprefrence of the base stacking is observed in the A-RNA and the Watson-Crick type conformers. The ApA unit with C(2′)-endo sugar is forced to assume phosphodiester conformations with large deviations fom the expected staggered conformations compared to the ApA unit with C(3′)-endo sugar. The result obtained for ApA are discussed with refrence to those previously obtained for the dApdA unit. Te theoretical predictions are compared with the experimental data on the tRNAPhe crystal, as well as those on fibrous RNAs and RNa subunitlike crystal structures. This study brings out many important aspects of the conformational stability of ApA which have been missed by studies made by others on this system.

Notes: The synthess of 18 co-oligopeptides of L-methionine and glycine is reported. A series of oligomers, Boc-Gly-Metn-OMe (n = 1-6), and six hexamers-containing five methionyl and one glycyl residue were synthesized using the mixed anhydride procedure. Polarimetric studies give evidence that oligomers in the Boc-Gly-Metn-Ome series are essentially disordered in hexafluoroacetone sesquihydrate but begin forming secondry structures at n 〉 4 in trifluorethanol. Difference in the molar rotation values found for the six hexamers in hexafluoroacetone sesquihydrate may indicate that these compounds, while primirily disordered, are present in slightly different conformations.

Notes: Monodisperesed, N-and C-Protected homo-oligopeptides [number (n) of resides from 2 to 5] of L-valine, L-isoleucine, and L-phenylalnine were studied by ir absorption spectroscopy between 1200 and 350 cm-1 at various solvents. The solvents and chain-length effects were examined for non-hydrogen-bonded peptide groups. The frequencies of the self-associated species are consistent with a model derived from the amide data. Self-association species are consistent with a model derived from the amide data. Self-association is favored by higher values of n = 2, the peptide is insoluble when more than two chains are bonded. For n = 3, 4, several chains may be associated by sliding along one another and remain soluble. For n = 2, the peptide is insoluble when more than two chains are bonded. For n = 3, 4, several chains may be associated by sliding along one another and remain slouble. For n = 5, the effect of n is to favour a model in which two chains exactly face each other so that the peptide precipitates at relatively low concentration.

Notes: Possible conformations of X-D-alanyl4-D-alanine5 and its analogs X-L-analyl4-D-alanine5 X-D-alanyl4-L-alanine5, X-D-butyl4-D-alanine5, X-D-alanyl4-D-butyric acid5, X-D-valyl4-D-alanine5, and X-D-alanyl4-D-valine5 have been analyzed by theoretical methods. These studies suggest that L-alanine and D-valine at the 4 or 5 postion of the pentapeptide moiety of peptidoglycan will drastically reduce the cross-linking in peptidoglycan biosynthesis, whereas the effect of D- butyric acid will be marked at the 4 postition and modeate at the 5 position. This is in good agreement with experimental results. The cross-linking enzyme transpeptidase requires a specific confroamation for the 4th and 5th residues for optimal binding.

Notes: Infrared dichroism measurements of oriented films of poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)] have been made under the conditions of low salts content and high humidity for which the geometry is known. The angles which the transition moments make with the helix axis are compared with the orientations of the corresponding bonds. Except for the in-plane base model of poly[(A-T)]·poly[d(A-T)], there is no agreement. This may imply either that a model which assumes bonds and transition moments to be colinear is not acceptable or that x-ray data are inaccurate. These possibilities are discussed especially with respect to phosphate group orientation. An appendix gives the derivations of dichroic-ratio expressions for helical molecules of different symmetry types.

Notes: Measurements of fluorescence depolarization on fumarase labeled with the dye pyrene-butyryl were used to test for previously reported structural changes in this enzymes. These apparent conformation changes were of interest because they seemed to correlate with variation in catalytic activity provoked by changing temperature or pH, or by the presence of a competitive inhibitor. In the present studies, the bound dye pyrene-butyryl and the enzymes were investigated systematically to ensure that simple interpretation of fluorescence depolarization results would be meaningful. This analysis showed that carefully controlled experimental condition were necessary to eliminate a dye component with a short fluorescence lifetime and that it was essential to allow for small variations of lifetime with temperature. Contrary to the previous report, a constant rotational relaxation time of the magnitude expected for a nearly spherical molecule of fumarase was found. No changes were detectable by fluorescence depolarization in the size or shape of pyrene-butyryl-fumarase under the solution conditions tested that caused variation in enzyme activity.

Notes: Several cyclic hemopeptides containing L-azetidine-2-carboxylix acid (Aze) - an imino acid homologous with proline but containing one less methylene group in its cyclic side chain - have been prepared. The peptides reported include (Aze)2, cyclo(Aze)3, and cyclo(Aze)6. The synthesis and spectral characterization of these cyclic peptides are described, and the results discussed in terms of the rigidity and steric constraints attributable to Aze-containing peptides. CD spectra of these materials in several solvents are reported and compared with those of proline analogs; the similarity between the CD spectra of cyclo(Aze)3 and cyclo (Pro)3, is noted.

Notes: The application of rotating frame spin-lattice relaxtion to the determination of the intramolecular motions in peptides is discussed, and results are presented on the application of 13C T1p to peptide microdynamics in solution. The effective molecular rotational reorientation times at the amide and amino nitrogens may be derived from appropriate data onT1p of the carbons adjacent to them. We also show by theoretical caculations that 1H and 13C T1p experiments of suitable 2H and 15N substituted peptides will allow intramolecular main- and sidechain motions, characterized by times in the range 10-1-10-6 sec, to be investigated.

Notes: A Monte-Carlo method including long-range interactions is used to oligopeptide chains in random-coil state. The chains are composed of 4, 9, or 14 repeating units and are labeled with the luminopheres tyrosine or tryptophan. Interactions with a solvent (water) are taken into account in the calculations through modifications of the semiempirical potential-energy functions. The chains represent oligopeptides composed of hydrophobic or hydrophilic amino acid residues. Various properties relavent to the interpretaiton of nonradiative enrgy-transfer experiments, such as the average value of the orientation factor for dipole-dipole interaction of the luminophores, 〈k2〉, the distribution function of the distances between the luminophores f(rl), the efficiences of energy transfer in the static and dyamic averaging regimes, 〈T〉s amnd 〈T〉d, as well as the fluorescence decay I(t) of the donor luminophore in various averaging conditions, are computed. It is shown that, for all chains considered, 〈k2〉 is not vary far form 0.67 and that 〈T〉s and 〈T〉d have completely different values. Due to the small extent of correlation between the distances rl and the mutual orientations of the lumninophores, the decay kinetics 〈I(t)s corresponding to a static averaging regime can be expressed in terms of distribution functions f(rl). These results are in agrrement with those obtained previously for the unperturbed chain model.

Notes: For a better understanding of the interactions between DNA and various acridine dyes, the binding of acridine (Acr) and 10-methylacridine (MeAcr) to native and heat-denatured calf-thymus DNA was studied in the pH range between 4 and 10 by the equilibrium dialysis and spectroscopic methods. The binding between DNA and the dyes was predominantly electrostatic. The amount of bound Acr varied with pH, mixing ratio (P/D), and the DNA conformation, and reached a maximum at pH = 5.2. The amount of bound MeAcr was constant in the pH range 5-9. The apparent binding constants of these dyes were obtained at some pH, and they were found to vary with P/D for native DNA-dye complexes. The pure spectra of bound Acr and MeAcr could be unmasked. The bound Spectra were bathochromic and hypochromic relative to the spectra of free days. Acridine bound to native DNA was shown to undergo structural changes from an acridiniumlike to a neutral acridinelike form as the pH of solutions was varied. The pK value for the transition between the bound forms was evaluated to be 7.3. The extrinsic Cotton effects of the bound dyes were observed in the DNA-Acr and-MeAcr complexes and varied with pH and the conformation of DNA.

Notes: An x-ray structural study of the 1-methylcytosine H+ complex reveals asymmetrically hydrogen-bonded dimers which are further stacked such that the molecular overlaps are between charged and neutral bases. This study supports the assignment of the asymmetry in the interbase hydrogen bonding in the cytidine-cytidine H+ hydrogen-bonded complex. Furthermore, it is suggested that hemiprotonated, helical polyribocytidylic acid has as its translational repeat unit a set of highly stacked pairs of asymmetrically hydrogenbonded bases. The asymmetry in the interbase hydrogen bonding is stimulated, at least in part, by base-stacking considerations.

Notes: The theory of adsorption of semistiff chains on a planar surface developed by the authors previously has been used to consider the helix-coil transition in single-stranded macromolecule interacting with an adsorbent plane. The cases of nonselective interaction when the adsorption energy is independent of the unit conformation (a) and selective interaction with only helical (b) or coiled (c) sequences active in adsorption were investigated. In case (b) the existence of secondary structure favors chain bonding to the surface. This leads to the increase in the stability of the helical state and complete polypeptide chain spiralization. The profile of the conformational helix-coil transition acquires an asymmetrical shape inherent to the second-order phase transition.In case (c) the bonding of a partially helical chain to the surface is similar to the adsorption of Gaussian coils and is accompanied by the destruction of secondary structure, this destruction being appreciable even if the helical state in space was favorable.

Notes: Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double-strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10-5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single-strand breaks and a smaller number of double-strand breaks, also increased the initial rate. However, this increase was due only to the double-strand breaks, that is, single-strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 105 erg/mm2, suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.

Notes: A combination of the DeVoe and Kirkwood polarizability concepts is developed to calculate CD spectra of nucleic acid monomers. The method is perfectly general and applies to any system where the constituents have absorption properties which are widely separated in terms of frequency. The theory is applied to calculate the CD spectra of adenosine and 2′-deoxyadenosine conformers. Bond polarizabilities are evaluated for the ribosyl moiety of adenosine, as a function of glycosidic rotational angles and polarizability anisotropies. It is found that a wide range of C-C and C-O bond polarizabilities give similar CD results. Isotropic atom polarizabilities are also evaluated. It is found that the CD results using these polarizabilities do not differ significantly from those obtained with bond polarizabilities. The CD spectra of adenosine and 2′-deoxyadenosine are calculated for three x-ray diffraction determined geometries: A-form RNA, B-form DNA, and C-form DNA. The results indicate that the monomer CD spectra are strongly dependent on the precise geometry and appear to be of importance in understanding the spectra of oligomers and polymers. The deoxyadenosine conformers are found to have calculated CD spectra which are less intense than those of the ribosyl conformers. These results indicate that the measured differences between the CD magnitudes of ribo- and deoxyriboadenosine are due to the presence or absence of the 2′-hydroxyl. Weighted averaged adenosine CD spectra are calculated with the aid of probability distributions from conformational energy calculations. The results suggest a new method for obtaining empirical monomer parameters for use in optical calculations. The calculations in this paper indicate for the first time that DeVoe theory, in combination with the Kirkwood theory, provides a useful method for the calculation of the CD spectra of nonpolymeric molecules.

Notes: The heats of ionization of protons, ΔHi, of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C were determined using a titration calorimeter which simultaneously afforded the potentiometric titration curve. Reproducibility of the thermal titrations is within 2%, and evaluation of the heats observed after the heat loss corrections is estimated to be within 5%. A single titration of oxidized cytochrome c from pH 11 to 3 is in excellent agreement with the thermal titration of this protein obtained with flow calorimetry. The thermal titration, however, is not reversible, due in part to the loss of titratable group(s) in this pH region and to the heat contribution of the acid and alkaline conformational changes which occur. Although of lesser magnitude, the reduced form also indicates similar thermal transitions. These differences are due solely to conformational contributions to the thermal process, since the potentiometric curves are reversible. The nature of the irreversibility for oxidized cytochrome c appears to involve the loss of a group with pK′ 8.9 and the shift of two groups from pK′ 5.6 to 4.8. Thermal difference curves for this process indicate that heats of -7.8 and -24.1 kcal/mol are liberated which are centered at pH 9.3 and 3.9, respectively.

Notes: The preferential orientations of the purine bases in dinucleoside monophosphates such as ApA, ApG, and GpA in 10-2M neutral aqueous solutions have been investigated by proton relaxation at 250 MHz. These orientations are deduced from computer simulations of the magnetization recovery curves following a 180° nonselective pulse. The distances between the H(8) proton of a base and the ribose ring protons which are used in these calculations are obtained by minimization as a function of the glycosyl torsion angle ϒ of the standard deviation between the isotropic reorientation correlation times τR derived from the relaxation rates of these protons. The average H(1′) - H(8) distance obtained by this procedure may be readily verified from the reduction of the H(1′) relaxation rate when H(8) is substituted by a deuteron. The limits of validity of the assumption of a single correlation time τR governing the proton relaxation have been estimated, taking into account several possible internal motions, e.g., the rotation of the base, of the methylene exocyclic group and the N ⇄ S interconversion of the ribose ring. For 10-10 〈 τR 〈 2 × 10-10 sec, it appears that the influence of these motions on the proton relaxation becomes perceptible when the jump rates among equilibrium positions exceed ca. 109 sec-1.The whole of the experimental results show that for the ribose ring N conformer, the orientation of the bases is found in the ranges 60° 〈 ϒ 〈 80° (syn) and 180° 〈 ϒ 〈 210° (anti). For ribose S conformer, it is observed that this orientation is mainly syn with 5° 〈 ϒ 〈 90°. The average H(1′) - H(8) distance provides semiquantitative information on the overall syn or anti orientations of the base in each nucleoside moiety. At 298 K the population of the anti conformer is found to increase in the order A- pG 〈 Ap-G ∼ Gp-A 〈 Ap-A 〈 A-pA 〈 G-pA. A more detailed analysis of relaxation data shows that the maximum possible fraction of the stacked form of dinucleotides, due to the occurrence of N-anti conformers in both nucleoside moieties, is in the order ApG 〈 GpA 〈 ApA, in agreement with previous works, with however smaller values.Lastly the deuteron linewidth in position 8 of the bases indicates a syn-anti transition rate of the order of 109 sec-1 at room temperature, without noticeable effects therefore on the proton relaxation.

Notes: In a recent paper, we developed a thermodynamic theory on the complex coacervation in the absence of low molecular ions, under the assumption that the coacervation is a condensation phenomenon of aggregates of polyanion and polycation in the aqueous solution, by obtaining the interaction potential US between these aggregates on the basis of Flory's method. In this paper, we have extended the theory to a more complicated phenomenon of the counterion-containing solutions. This treatment has led the interaction potential having an additional contribution to US resulting from an entropy increase by the counterion distribution. The phase diagram between solution (sol) and separated phase has been obtained as a function of the difference of charges between polyanion and polycation. It has been found that the presence of counterions sensitively suppresses the coacervation.

Notes: The effects of salts (NaCl, LiCl, Me4NCl, AgNO3, MgCl2, CuCl2 and MnCl2) and dyes (acridine orange and methylene blue) on the low-frequency dielectric relaxation (0.1 Hz-30 kHz) of dilute aqueous solutions of DNA were investigated with varying salt or dye concentrations. Both the dielectric relaxation time τD and the rotational relaxation time τ estimated from the reduced viscosity decrease in quite parallel ways with increasing M/P (M/P being the normality ratio of cation to phosphate residue), reflecting the contraction of DNA molecule due to electrostatic shielding and cation binding. The agreement between τD and τ through the whole range of M/P supports our previous conclusion that the low-frequency relaxation of DNA arises from rotation of the molecule. The dielectric increment Δε also decreases with increasing M/P on account of both the contraction of DNA and the decrease in effective degree of dissociation of DNA. Δε as a function of M/P is interpreted in terms of a quasi-permanent dipole due to counterion fluctuation. These effects of cations are the strongest for divalent cations and rather weak for Na+, Li+, and Me4N+. Effects of dye on τD and Δε are also well explained by the rotation of DNA molecule with a quasi-permanent dipole due to counterion fluctuation on the basis of intercalation of dye at D/P 〈 0.2 (D/P being the molarity ratio of dye to phosphate residue) and external binding at 0.2 〈 D/P 〈 1.0.

Notes: Rotational and wobbling diffusion coefficients for spherical and long-chain molecules in membranes are calculated using a simple hydrodynamic model. Estimates of the contributions to the diffusion coefficients arising from hydrodynamic interactions between molecules and membrane interfaces are obtained and found to be small. For molecules containing polar head groups, we show that the presence of a membrane interface can produce a significant reduction in the wobbling diffusion coefficient over what would be obtained in an isotropic fluid.

Notes: The nonlinear electrical properties of DNA solutions were measured when different monovalent cations were added to DNA. The influence of different parameters has been examined: fundamental frequency, field strength, and concentration. A linear relationship between the harmonic current Ih and the DNA concentration is shown, even for higher concentration values (400 mg/l.). The frequency dispersion of Ih has the same shape for all the cations and the low-frequency amplitude of Ih increases in the following order: Li+ 〈 Na+ 〈 K+ 〈 NH4+ 〈 Cs+. The nonlinear polarizability values are compared with the linear ones determined using the very low field electric birefringence technique. Both linear and nonlinear values are of the same order of magnitude. It is thought that the nonlinear electrical property of high-molecular-weight DNA mainly results from the deformation of the DNA coils by the electric field.

Notes: Quasielastic light scattering methods were used to study calf thymus DNA in solutions of LiCl, NaCl, NH4Ac, and NH4Cl. Plots of the reciprocal relaxation time (1/τ) vs sin2(θ/2), where θ is the scattering angle, exhibit two linear regions, in accordance with theories for semiflexible polymers based on the t → 0 approximation. In these theories the slope of the linear region at low angles is associated with the translational diffusion coefficient (Dt), whereas the slope of the linear region at high angles is associated with the segmental diffusion coefficient (Ds = kT/ƒs). The midpoint of the “transition” between these two linear regions is associated with the mean displacement between segments (b). Data presented here indicate that the Rouse-Zimm parameters b and ƒs are significantly different for DNA in 0.4M NH4Cl relative to the other salts at comparable ionic strengths. It is suggested that this difference reflects local solvent structure and that both b and Ds are sensitive to the local water structure.

Notes: Sodium ion activity was measured using a Na-glass electrode in a solution of poly(S-carboxymethyl-L-cysteine) with no added salt at various degrees of neutralization and various concentrations for samples of different molecular weights. The conformational change from random coil to the β-structure was detected from the activity coefficient of counterions, as well as from CD. At a constant degree of neutralization, the activity coefficient is insensitive to a concentration change not only in the random-coil state, but also in the range of conformational change if the concentration is below about 3 × 10-2 monomolal. At high concentrations of about 5 × 10-2 monomolal, however, the activity coefficient becomes low, probably due to the occurrence of the stacking of the pleated sheets.

Notes: 1H-nmr spectra for a series of Boc-L-(Met)n-OMe (n = 2-9) homo-oligopeptides have been observed in the helix-supporting solvent trifluoroethanol (TFE) at millimolar concentrations. Interfering solvent peaks were eliminated using two decoupling frequencies to selectively remove the methylene and hydroxyl protons of the solvent. Comparisons with specifically α-deuterated homo-oligopeptides gave complete assignments of the NH region of the Boc-Metn-OMe oligomers up to the heptapeptide. Analysis of chemical shifts, coupling constants, and temperature dependence of chemical shifts suggests that up to the hexapeptide, similar structures exist in deuterochloroform and TFE. In contrast, nmr parameters at the heptapeptide for several internal residues differ in these solvents. These results suggest that a C7 to α-helix transition may occur in TFE as the chain length of the methionine oligopeptides increases.

Notes: The natural-abundance 15N-nuclear magnetic resonance (nmr) spectrum of the cyclic decapeptide gramicidin S has been measured and assigned in the solvents dimethyl sulfoxide, methanol, and 2,2,2-trifluoroethanol. Three methods have been investigated to distinguish between peptide groups which are exposed to or shielded from the solvent. The solvent dependence of the 15N chemical shift is correlated with the two types of peptide group in gramicidin S—those with the carbonyl group exposed or shielded from the solvent. The second method monitors the lability of the N—H proton (via the collapse of the reduced 15N-1H coupling) in the presence of added base used to promote intermolecular exchange - peptide protons shielded from the solvent exchange more slowly. The third method looks at the temperature dependence of the 15N chemical shifts in dimethyl sulfoxide. Here the data are not so distinctive as to allow the differentiation between solvent-exposed or shielded N—H bonds at all peptide groups.

Notes: Small changes in the apparent radius of gyration of native cytochrome c were measured as a function of pH in solutions of 0 and 50 mM ruthenium hexicyanide. The forward scattering was also measured as a function of pH under the same conditions to ascertain the contribution of the Ru ion shell to the forward scattering and concomitantly to the radius of gyration. The forward scattering and small alterations in the radius of gyration were then measured as a function of Ru counterion concentration. These results are then interpreted in terms of the mechanisms which influence the measured apparent radius of gyration. It is shown that the Debye-Hückel theory of counterion distributions can be used to describe the data in situations where scattering from the bound Ru ions contributes less significantly than scattering from the ion cloud.

Notes: Fluorine nmr experiments carried out at 51.0 and 94.1 MHz have been used to explore the interaction of the probe molecule p-fluorocinnamate with conjugates formed from α-chymotrypsin and poly(N-acryloyl-β-alanine). The data obtained include enzyme-induced chemical-shift effects, spin-lattice (R1) and transverse (R2) relaxation rates, and the rate constant for dissociation of the fluorocinnamate-enzyme complexes. Analysis of the results indicates that while overall molecular tumbling of the enzyme molecule is not greatly changed by attachment of polymers of various sizes, conjugated polymer can appreciably affect the structure of the p-fluorocinnamate binding site. The important variable involved in such structural changes appears to be the amount of polymer present per mole of protein.

Notes: Expressions for zeroth, first, and second spatial moments are obtained for diffusing macromolecules A and B that move due to an external field and undergo reversible isomerization, switching back and forth according to first-order kinetics. In addition, expressions for third and fourth moments are derived for the special case of equal diffusion coefficients, equal rate constants, and equal but opposite velocities. The initial conditions are arbitrary amounts of A or B concentrated in an infinitesimally narrow region. The moments are computed from derivatives of the Fourier-transformed concentration profiles of A and B. The moments are used in an expansion in term of Hermite polynomials, the Gram-Charlier expansion, to construct the concentration profiles of A or B or A and B together. The examples presented show that a few terms of the expansion, for which explicit expressions are given, can give tolerable accuracy if the velocity is not too large and the rate constants and diffusion coefficient are not too small. The expansion can be used to determine when the profiles are unimodal.

Notes: The thermal triple helix-coil transition of covalently bridged collagenlike peptides with repeating sequences of (Ala-Gly-Pro)n, n = 5-15, was studied optically. The peptides were soluble in water/acetic acid (99:1) and were found to form triple-helical structures in this solvent system beginning with n = 8. The thermodynamic analysis of the transition equilibrium curves for n = 9-13 yielded the parameters ΔH°s = -7.0 kJ per tripeptide unit, ΔS°s = -23.1 J deg-1 mol-1 per tripeptide unit for the coil-to-helix transition, and the apparent nucleation parameter σ ≃ 5 × 10-2. It was suggested through double-jump temperature experiments that the rate-limiting step during refolding is not only influenced by the difficulties of nucleation, but also by cis-trans isomerization of the Gly-Pro peptide bond.

Notes: Poly(hydroxyalkyl-L-glutamine) (alkyl = ethyl, propyl, butyl) solutions have been studied by CD as functions of temperature and activity of calcium chloride and sodium perchlorate. Helical content is altered by changes in salt activity and temperature. The helicity of poly(hydroxybutyl-L-glutamine) and poly(hydroxypropyl-L-glutamine) falls to zero in a monotonic fashion with increasing calcium chloride activity. A nonzero helicity reappears at activities in excess of 5-50 mol kg-1. Poly(hydroxypropyl-L-glutamine) is much more sensitive to calcium chloride than is poly(hydroxybutyl-L-glutamine), and both polypeptides are more sensitive to calcium chloride than are typical proteins. Markedly different behavior is observed with sodium perchlorate. This salt acts as a helix stabilizer at low activities but becomes a destabilizer at activities higher than 0.3-1.0 mol kg-1. In this respect the effect of sodium perchlorate on nonionic poly(hydroxyalkyl-L-glutamines) resembles that seen with cationic poly(L-lysine) and poly(L-arginine). Helix stabilization at low sodium perchlorate activity is moderate for poly(hydroxybutyl-L-glutamine) and large for poly(hydroxypropyl-L-glutamine) and poly(hydroxyethyl-L-glutamine).

Notes: The structural properties of uracil photohydrates at the monomer and dimer level in aqueous solution have been examined in detail by nmr spectroscopy. Based on such evidence, the absolute configurations of the two possible diastereomers have been assigned, and the conformational perturbations induced by photohydration have been evaluated. In all instances, photohydration shifts the 2E ⇌ 3E puckering equilibrium of the sugar ring of the uridylyl fragment towards 2E (from 12-18%). In addition, for both dimers examined in detail, ho6UpA and Apho6hU, the effect of dimerization on sugar pucker is such that the 3′-terminal unit shows a clear increase in the percentage of 3E (relative to the appropriate 5′-mononucleotide), whereas the percentage 3E of the 5′-terminal unit shows no change. This is contrary to the findings in the normal dinucleoside monophosphates, where an increased preference for 3E pucker occurs in both residues on dimerization and increased base stacking. Significant base-base interactions were observed in both hydrated dimers despite the loss of the planar π-system in the uracil fragment. In addition, the rate of photohydration for a particular dimer pair (e.g., ApU and UpA or GpU and UpG) is shown to be inversely dependent on the amount of base stacking in the parent dimer. This latter parameter has also been correlated with the ratio of the two possible diastereomers formed in the reaction and is associated with a preferential attack at one face of the pyrimidine base ring. The shift of the sugar puckering equilibrium towards 2E has been compared with similar shifts observed when adenosine and guanosine are methylated at N(1) and N(7), respectively. The possible biological significance of the above-mentioned conformational aspects is discussed.

Notes: A simplified model of a polypeptide chain is described. Each residue is represented by a single interaction center. The energy of the chain and the force acting on each residue are given as a function of the residue coordinates. Terms to approximate the effect of solvent and the stabilization energy of helix formation are included. The model is used to study equilibrium and dynamical aspects of the helix-coil transition. The equilibrium properties examined include helix-coil equilibrium constants and their dependence on chain position. Dynamical properties are examined by a stochastic simulation of the Brownian motion of the chain in its solvent surroundings. Correlations in the motions of the residues are found to have an important influence on the helix-coil transition rates.

Notes: Measurements of the uv absorption and melting profiles of poly(rC) at low-salt conditions confirm the existence of a double-stranded complex at low pH and imply, but do not prove, the structure proposed by Akinrimisi et al. (1963) Biochemistry 2, 340-343. Electric dichroism and birefringence provide additional supporting evidence and indicate that the complex is one in which the bases are tilted considerably from the major molecular axis. The base-stacking tendency of single-stranded poly(rC), unlike poly(rA), is found to be insufficient to maintain a rodlike structure; the persistence length of stacked bases in poly(rC) must be very small. The existence of an out-of-plane transition in poly(rC) in the vicinity of 300 nm is conclusively demonstrated by both dichroism and birefringence measurements.

Notes: The effect of deuterium substitution of exchangeable hydrogen atoms on the reduction potential of Clostridium pasteurianum 2(4Fe-4S) ferredoxin has been studied. The studies were conducted to determine if NH⃛S hydrogen bonds to the iron-sulfur cluster are dominant in the mechanism of influence of the protein on cluster reduction potential, as has been proposed [Carter, C. W. (1977) J. Biol. Chem. 252, 7802-7811]. Deuteration of the slowly exchangeable hydrogen atoms, however, yields essentially no shift in the reduction potential (-0.2 ± 0.8 mV), suggesting that NH⃛S bonds are not important modifiers of cluster reduction potential in this protein.

Notes: The theoretical kinetics are analyzed for reactions between torsionally stressed DNA and another compound which reacts at different rates with portions of the substrate molecule which are in different conformational states. These reactions are assumed to obey Michaelis-Menten kinetics, so no cooperative effects occur. The DNA is regarded as being susceptible to a stress-induced local conformational transition as described recently by Benham. Briefly, alterations in torsional stress consequent on superhelicity effectively change the relative concentrations of the two conformational states of the substrate, thereby influencing the course of the reaction. This theory is developed for transition between the B-form helix and the single-stranded, random-coil states. To illustrate the influence of posited stress-induced melting on kinetics, calculations are made on simple models of two biochemical phenomena. First, the variations in initial nicking rates of single-strand-specific endonucleases with substrate superhelicity are interpreted as arising from changes in the concentration of (stress-induced) single-stranded binding sites. Second, the observed dependence of the transcription rate of RNA polymerase core enzyme on substrate superhelicity is interpreted in terms of a model in which the rate-limiting step in the initiation event is the formation of a complex between the enzyme and a single-stranded region. Related experimental results are shown to be qualitatively consistent with the suggestion that sufficiently supercoiled DNA contains locally melted regions.

Notes: A statistical mechanical calculation of the binding properties of DNA bis-intercalators is presented, based on the sequence-generating function method of Lifson. The effects of binding by intercalation of one or both chromophores of a bifunctional intercalating agent are examined. The secular equation for a general model that includes the effects of neighbor (nearest and non-nearest) exclusion and/or cooperativity in the binding of both singly and doubly intercalated ligands is derived. Numerical results for binding curves are presented for a more restricted model in which each type of bound ligand rigorously excludes its nearest neighbor and the total number of sites covered by a doubly intercalated ligand is variable. At low values of free ligand concentration bis-intercalation dominates the binding process, while at high value of free ligand concentration, intercalation of only one chromophore per ligand becomes significant due to the unavailability of contiguous free sites required for bis-intercalation. Also, depending on the binding parameters, the free energy of the system can be lowered by a loss of doubly intercalated ligands in favor of singly intercalated ones. Corresponding to this transition in binding mode, the average number of sites occupied by a bound ligand decreases from that characteristic of bis-intercalation to that characteristic of mono-intercalation as free ligand concentration increases. An analysis of Scatchard plots describing bis-intercalation is presented.

Notes: The statistical mechanical model for the binding of bifunctional intercalating ligands to duplex DNA described in the preceding paper is applied to the example of echinomycin-DNA interactions. This is the only system for which binding curves have been obtained under conditions leading to binding by both bis-intercalation and mono-intercalation simultaneously. Binding parameters and Scatchard plots are calculated for a variety of conditions. A detailed comparison of these calculations with the results from the previous analysis of the same binding data in terms of the McGhee-Von Hippel theory, assuming only one mode of binding, is presented. The results of our calculations are consistent with the model of bis-intercalation requiring the two bound chromophores of a bifunctional ligand to be separated by two base pairs. It is not necessary to assume violation of the nearest-neighbor exclusion principle, as occurred in the original analysis.

Notes: We describe the capabilities of a method for obtaining high-resolution melting profiles of short, homogeneous DNAs using a thermo-differential absorbance technique. The absorbance difference of two identical DNA solutions, raised linearly in temperature and maintained at a constant temperature difference, is monitored using a double-beam spectrophotometer. A specially constructed temperature controller and cell holder enable the temperature of the DNA samples to be controlled and monitored directly. A heating rate of 6.75°C/hr has been found to give reproducible results at ionic strengths 〉 0.01M. A method of reconstructing the true derivative from experimental data using a Taylor series expansion is described and shown to work well when the difference in temperature between samples is in the range of 0.2°C. Reconstructed derivative profiles are further analyzed by deconvolution into distinct Gaussian components. The melting profile of PM-2 DNA is shown to consist of 14 components, while the much longer lambda DNA yields 55. Related techniques such as data management and analysis for the fractional G·C content of specific domains are also described.

Notes: Neue technische Entwicklungen, Änderungen im Rohstoffangebot oder bei den Rohstoffpreisen, aber auch Forderungen des Umwelt- und Arbeitsschutzes führen dazu, daß die technische Herstellung von anorganischen und organischen Basisprodukten weit stärkeren Veränderungen unterworfen ist, als die Lehrbücher vermuten lassen. „Chemie in unserer Zeit“ setzt mit dem vorliegenden Beitrag eine lockere Folge von Berichten über aktuelle Verfahren zur Herstellung industrieller Basischemikalien fort.