Complexes of polyadenylic acid and 7-methyl-xanthine and their optical rotatory dispersion

Polyadenylic acid forms a 2:1 complex with 7-methyl-xanthine at pH 7.0, 0.15 M Na⁺, and a 1:1 complex at pH 6.0, 0.15M Na⁺. The complexes have been characterized by equilibrium dialysis and by ORD measurements. Both complexes are laevorotatory at wavelengths greater than 286 nm, and the 1:1 complex is remarkably optically active: [m] = ?154,000° at 292 nm. The properties and structures of the complexes are discussed.     

D-pantolactone as a circular dichromism (CD) calibration.

A critical study of circular dichromism (CD) measurement at the wavelength region around 220 nm was made. Test sample methods gave deviations of abou 20%, at most, between instruments calibrated at 290 nm with D-10-camphorsulfonic acid. A calculation of the CD from optical rotatory dispersion (ORD) data was made for d-pantolactone which gave a CD maximum at 219 nm, [θ]₂₁₉²⁵ = ?17.3 × 10³ in water. A proposal is made to use this value for calibration of CD in the 220-nm region.     

Optical properties and viscosity of hyaluronic acid in mixed solvents: Evidence of conformational transition

Circular dichroism, optical rotatory dispersion, and viscosity of hyaluronic acid at various solvents compositions, concentrations, and pH values have been studied. The data show a large change in the molecular properties in organic/water solvents such as ethanol, p-dioxane, or acetonitrile/water at pH ? pK_a. At this pH range of aqueous solution, hyaluronic acid shows a CD minimum near 210 nm whereas in the presence of organic solvent it exhibits a strong negative dichroism (below 200 nm) and a positive band near 226 nm. It undergoes a sharp, cooperative transition with respect to pH and solvent. The observed CD features are assigned to the π-π* and n-π* transitions of the amide and carboxyl chromophores. The ORD results show a gradual blue shift of trough at 220 nm with increasing magnitude of rotation when the organic solvents and hydrogen ion concentrations are increased. A one-term Drude's equation was used to analyze the ORD data, and the result show a variation of dispersion parameters with different solvents in accordance with the observed CD changes. The intrinsic viscosity of hyaluronic acid in mixed solvent at pH 2.6 is lower than that of aqueous solution. All the observed property changes of hyaluronic acid are reversed on addition of foramide in mixed solvents indicating that the hydrogen bonds are involved in this transition. The observed spectroscopic and hydrodynamic features are attributed to a conformational change of hyaluronic acid in a mixed solvent involving intramolecular hydrogen bonding between the acetamido and carboxyl groups. The possible conformational state of hyaluronic acid in solution under various conditions is discussed in terms of the reported helical structure of hyaluronic acid from x-ray diffraction studies.     

Polypeptides. LVI. Effect of lithium bromide and of temperature on the conformation of a copolymer of glutamic acid and lysine

By using optical rotatory dispersion measurements, the helix content of poly Glu⁵⁰Lys⁵⁰ has been investigated and compared with that of poly Glu²⁰Lys²⁰Ala⁶⁰ in aqueous solutions. Measurements were made at pH 3 and at pH 8 in various concentrations of lithium bromide. Various factors affecting helix stabilization are considered and their perturbation by lithium bromide is related to the shape of the observed transition curves. A residual helix content of 12% in 8M LiBr, based upon a b₀ of +100 for a fully random conformation, was observed for poly Glu⁵⁰Lys⁵⁰ at pH 3 and 8. The loss of helix content of poly Glu⁵⁰Lys⁵⁰ as a function of temperature is also reported. ΔH is approximately ?6.9 kcal./mole for the overall transition, compared to ?6.5 kcal./mole for poly Glu²⁰Lys²⁰Ala⁶⁰. The midpoint of the broad transition is near 40°C. at pH 3, but much lower, at ?10 to 0°C., at pH 8. These results are discussed in terms of the stabilizing factors for the partial helix content of the polypeptides.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

Specific aggregation of poly(α-L-glutamic acid) and hysteresis effects in aqueous solutions. I. Influence of temperature-dependent aggregation on the optical rotation of PGA

The instability of aqueous solutions of poly(α-L -glutamic acid) (PGA) at low pH is due to two distinguishable phenomena: precipitation, favored above 40°C., and aggregation, favored below 20°C. The aggregated form of PGA can be isolated by gel permeation chromatography. Both aggregation and precipitation increase with decreasing pH, i.e., with decreasing ionization of the side chain carboxyl groups. Temperature-induced aggregation and disaggregation give rise to a reproducible hysteresis loop which can be followed by optical rotation, light scattering, sedimentation, viscosity, and chromatography. Hysteresis has been observed with different PGA samples, and in several aqueous buffered or unbuffered solvents and organic-aqueous solvent mixtures and in the pH range 4.1–4.5. Aggregation manifests itself as an increase in negative optical rotation in the visible and ultraviolet spectral range. The specific relation at 233 mμ is sensitive to aggregation and also reflects the hysteresis. Measurements of optical rotatory dispersion indicate that a₀ reflects the hysteresis but b₀ does not, the latter revealing only reversible changes with aggregation and disaggregation. The helix-coil equilibrium is apparently unperturbed by aggregation, as is the thermal stability of the helix structure. For fully aggregated PGA it is estimated that a₀ increases by about 300 degrees, which suggests that a₀ may be a sensitive parameter to measure aggregation in other systems. The rate of aggregation increases with decreasing temperature. The disaggregation, upon heating, is more rapid. However, kinetics measurements have not yet been done. The temperature M at which all aggregates are disrupted increases with decreasing pH, but is independent of total PGA concentration, at constant pH. No molecular weight dependence of M was detected in the range 20–100 × 10³. The shape and size of the hysteresis loop depends upon pH and molecular weight, which is interpreted as a dependence on the extent of aggregation. One branch of the loop, representing the helix–coil transition of isolated molecules, is reversible, while the others, representing the formation and disruption of the aggregates, are not. The system exhibits both ascending and descending scanning curves, which are typical of a true hysteresis.     

Nature of Cu(II)-poly(glutamic acid) complex in aqueous solution

Complex formation of poly(glutumic acid) with cupric ions in aqueous solution was investigated by three different methods: optical spectroscopy, optical rotatory dispersion, and electron spin resonance. Formation of a characteristic complex was found to occur in a pH region suitable for the helix-coil transition. An analysis of the ESH spectrum of the complex is given, and the results of calculation of bonding parameters suggest that the bond between copper and nitrogen atoms had an appreciably covalent character. The change in the secondary structure of the polymer as a result of complex formation is discussed.     

Optical Rotatory Dispersion and Circular Dichroism of Amino Acid Hydantoins

Optical rotatory dispersion (ORD) and circular dichroism (CD) of 17 amino acid hydantoins were measured between 190 and 600 nm. Most of hydantoins exhibited the negative Cotton effect which showed the trough between 238 and 245 nm. The negative trough of CD was also observed between 212 and 236 nm. The Cotton effect of hydantoins was attributable to n→π^* transition of carbonyl group at C-4 of hydantoin ring.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Conformation of poly-S-carboxyethyl-L-cysteine in aqueous solutions

Poly-S-carboxyethyl-L -cysteine, a higher side-chain homolog of poly-S-carboxymethyl-L -cysteine, has been prepared from poly-S-carbobenzoxyethyl-L -cysteine with hydrogen bromide in chloroform or acetic acid. The polymer is found to be in the β-conformation of an antiparallel arrangement of polypeptide chains in solid films, both in acid and salt forms, when examined by infrared spectra. Aqueous solutions of t he polymer have been investigated by measurements of rotatory dispersion and circular dichroism as well as by infrared spectra in D₂O. These properties show sharp changes around pH 5.5, as the pH of solution is varied. At higher ionization the polymer is randomly coiled, but at lower ionization it is in the β-conformation. Dependence of the rotatory properties upon polymer concentration as well as on ionic strength has been observed even at the lowest degree of ionization attained, and this has been attributed to the formation of intermolecular β-conformation in solutions. The β-structure is characterized by a negative circular dichroic band at 223 mμ and a positive dichroic band at a wavelength lower than 200 mμ, and furt her by a negative b_o value, ?140°. The pH-induced coil-β transition of the polymer is compared with that of poly-S-carboxymethl-L -cysteine.     

Molecular conformation of poly-S-carboxymethyl-L-cysteine in aqueous solutions

Poly-S-carboxymethyl-L -cysteine has been prepared by debenzylation of poly-S-carbobenzoxymethyl-L -cysteine with hydrogen bromide in acetic acid. By the infrared spectroscopic method the polymer is found to be in the extended β-conformation with an antiparallel arrangement of polypeptide chains in solid film, if it has been regenerated from dimethyl sulfoxide solution. Aqueous solutions of the polymer have been investigated by measurements of optical rotatory dispersion and viscosity. Various properties sharply change around pH 5 at different ionic strengths. By combining these with infrared studies in D₂O solutions, it has been shown that the polymer exists in the random coil conformation at higher ionization but associates into the intermolecular β-conformation at lower ionization. At the lowest pH attainable in solution, the β-form is partly converted into the random coil as the temperature is raised. The rotatory dispersion of the polymer is described by the Moffitt equation. While the random coil form has a large negative a₀ value and a zero b₀ value, the β-form is characterized by a positive a₀ value and a negative b₀ value, ?130°.     

Fluctuation of an -helix structure. Difference between the central and terminal portions

Four samples of poly-l-glutamic acid with different degrees of polymerization, 30, 60, 73 and 96, have been prepared. By optical rotatory dispersion and infrared absorption measurements, the random-coil and α-helix contents were estimated in heavy water plus dioxane solutions with various pD values. In the same solvent, the kinetics of hydrogen-deuterium exchange in each sample was followed by an infrared absorption measurement. It was found that the rate of exchange depends appreciably on the degree of polymerization; the rate is lower for the polymers with higher degrees of polymerization, so that the rate constant is considered to become lower on going from the terminals towards the inner portions of the polymer chain. An analysis of this lowering has been made by taking the random-coil content estimated by the optical rotatory dispersion measurement into account. It is suggested that the rate constant of the helix-coil fluctuation in the central portions of the chain is as low as 4 × 10⁻⁶ sec⁻¹.     

Kinetics of formation of the 4,4′-bis-dimethylaminodiphenyl carbonium ion (BDC+) and its reaction with sulfhydryl residues

The use of 4,4′-bis-dimethylaminodiphenylcarbinol (BDC-OH) as an analytical reagent for sulfhydryl residues and as a specific chemical modification reagent for proteins is dependent upon the unique properties of the BDC⁺ cation present in aqueous buffers below a pH of 6.5. In the presence of aqueous buffers, pH 5.1, BDC⁺ exhibits a λ_max of 606 nm with an apparent molar absorption coefficient of 10,000 m^?1 cm^?1. Upon the addition of 4m guanidine hydrochloride this apparent coefficient is enhanced to 70,800 m^?1 cm^?1. The true molar extinction coefficient for BDC⁺ was determined to be 128,000 m^?1 cm^?1. The reaction of BDC⁺ with sulfhydryl residues of proteins or simple thiols is rapid and leads to a complex devoid of visible color. In the pH range 3.0–7.0, a complex equilibrium is established among the three species BDC-OH, BDC⁺, BDCH⁺⁺. The formation of this equilibrium is proton mediated, and is discussed in terms of the equilibrium, rate, and acid dissociation constants.     

Helix-coil transition of poly-gamma-N-carbobenzoxy-L-alpha, gamma-diaminobutyrate and poly-delta-N-carbobenzoxy-L-ornithine

The helix–coil transition of poly-γ-N-carbobenzoxy-L -α,γ-diaminobutyrate (PCLB) and poly-δ-N-carbobenzoxy-L -ornithine (PCLO) in chloroform–dichloroacetic acid mixtures was followed by optical rotatory dispersion. PCLB displays a “normal” temperature-induced transition, but PCLO an “inverse” one. The thermodynamic parameters for helix formation of the two polymers were determined using the Zimm-Bragg theory. The enthalpy for adding an amide residue to a helical region, ΔH, and the initiation factor σ were ΔH = ?180 cal/mole and σ = 9.2 × 10^?5 for PCLB and ΔH = +490 cal/mole and σ = 1.9 × 10^?5 for PCLO.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.     

Light-induced currents from oriented purple membrane: I. Correlation of the microsecond component (B2) with the L-M photocycle transition

When irradiated, purple membrane from Halobacterium halobium oriented in a polyacrylamide gel produces a photocurrent. The correlation of the microsecond component (B2) of the photocurrent with the L-M optical transition was studied. It was found that the lifetimes of B2 and the L-M transition are identical over the entire pH range from 2.4 to 11.0 when measured in high salt (>5 mM CaCl₂ or >40 mM KCl). Changing the temperature from 10 to 35°C, or replacing the H₂O with D₂O maintains this correlation. The amplitude of B2 and the L-M transition are also correlated over the pH range where both of them can be represented as a single exponential. At high pH (>8), three exponentials were required to fit both the optical and photocurrent signals. Two of them are the previously described fast and slow components of M formation, but a new intermediate with a very fast lifetime, 0.3 μs, was observed both in absorption (λ = 410 nm) and photocurrent measurements. The lifetimes of all three were found to be pH independent. This would exclude models for the L to M portion of the photocycle that explained its complex pH-dependent behavior as being due to a single pH-dependent rate constant. The area of B2, which is proportional to the number and the distance the charge moved during the transition, is almost constant from pH 5.0 to pH 8.0. It decreases to almost zero at pH 2.0 and pH 10.6 with pKs at 2.8 and 9.1. Because B2 is thought to normally reflect proton release from the membrane, this suggests at very low and high pH the photocycle does not pump protons. The pK at high pHs for the formation of the nonpumping photocycle is probably related to the formation of a new photocycle featuring the fast rising form of M.     

Potentiometric titration of poly-S-carboxymethyl-L-cysteine in aqueous NaCl solution

pH titration measurements of poly- S-carboxymethyl-L -cysteine were undertaken in the aqueous Nacl solution in relation to the β form–random coil transition. The titration curves show a marked molecular weight dependence because of the shortened chain length of materials. Comparison of the optical rotatory dispersion parameter a₀ with the titration curve reveals that the titration curve apparently reflects a β structure–random coil transition. The β form of this polymer is assumed to be an intramolecular β form, rather than a β structure stabilized by an intermolecular hydrogen bond, at least in the polymer concentration range considered here. The standard free energy change per amino acid residue for the transition from un-ionized random coil to un-ionized β form is estimated to be about ?750 cal/mole residue in the range of 0.005–0.2M NaCl concentration.     

Conformational study of calf brain tubulin

The conformation of calf brain tubulin has been monitored by circular dichroism, optical rotatory dispersion, and spectrophotometric titration as a function of pH, temperature, ligand concentrations, and denaturants. At pH 7, calf brain tubulin maintains its structural integrity between 5 and 37 °C as determined by circular dichroism. Furthermore, the presence of MgCl ₂ up to 1.6 × 10^?2m does not induce any observable changes in the circular dichroism spectra, nor does 10^?4m CaCl₂. With increasing pH, the spectral data can best be described as a gradual loosening of the secondary structure between pH 7 and 9. Both spectral and titrimetric data suggest a major unfolding of tubulin between pH 9 and 10. The apparent pK of tyrosine shifts from 10.85 to 9.98 upon transferring from buffer to 6 m guanidine hydrochloride, indicating that at least 14 of the 15 tyrosine groups are not fully accessible to protons in the native protein. The single disulfide bridge in calf brain tubulin helps to maintain a domain which is highly resistant to unfolding by denaturants.     

Kinetics of charge translocation in the passive downhill uptake mode of the Na/H antiporter NhaA of Escherichia coli

The Na⁺/H⁺ antiporter NhaA is the main Na⁺ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li⁺ and Na⁺ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na⁺ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K_m values were 11 mM for Na⁺ and 7.3 mM for Li⁺ at basic pH and 180 mM for Na⁺ and 50 mM for Li⁺ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K_m as well as turnover (V_max).     

Interaction of magnesium ions with poly A and poly U

The binding of Mg⁺⁺ to poly A and poly U has been measured quantitatively by using the metallochromic indicator calmagite. The method is described in detail. It is shown that there is electrostatic interaction between the binding sites, viz., the phosphate groups, and the intrinsic association constant, for the specific binding can be determined. After extrapolation to zero ionic strength we find that, for the binding of Mg⁺⁺ to poly A, k_int = 4 × 10⁴ and for that, to poly U, k_int = 3 × 10⁴. The intrinsic enthalpy of association is negative. The effect of Mg⁺⁺ on the secondary structure of poly A and poly U has been studied by measuring the ultraviolet absorbance, optical rotatory dispersion and viscosity as a function of the amount of added Mg⁺⁺ ions. It was found that Mg⁺⁺ promotes the formation of a more ordered secondary structure by neutralizing or screening the negative charges. It is concluded from the absorbance measurements that for poly A at pH ? 7 and for poly U at pH >xs 9 this ordering involves stacking of the bases. Likewise, in solutions of UDP with a pH around 10, base stacking occurs on addition of Mg⁺⁺.