bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of water with poly‐α‐amino acids. I. Relationships between conformation and relaxation processes
Authors: Shiraishi H. , Hiltner A. , Baer E.
Published: December 2004

The relaxation behaviour of the sodium salt of poly (L‐glutamic acid) in the solid state has been examined by means of dynamic mechanical spectroscopy. Bound water was found to exert a profound influence on the relaxation behaviour and on a bulk property, the rigidity. Certain features of the loss spectrum have been identified with the hydration‐dependent β‐to‐α conformational transition. Thus two side‐chain relaxations are observed below ambient temperature, one associated with the β from (β1β) and a second at a lower temperature associated with the α form (β1α). The greater rigidity of the α form below the relaxation temperature and the larger rigidity drop accompanying the β1α can be explained in terms of the structural differences of the two conformations.
Heats of binding protons to globular proteins
Authors: Shiao D. D. F. , Sturtevant J. M.
Published: December 2004

The heats of binding of protons ΔHb to three globular proteins, lysozyme, chymotrysinogen A, and oxidized cytochrome c, from pH 11‐2 or lower at 25°C were determined by flow microcalorimetry. In addition, the acid–base titrations of chymotrypsinogen A and oxidized cytochrome c were investigated under conditions similar to those used in the calorimetric experiments. The results of the calorimetric experiments and the acid–base titrations for lysozyme and chymotrypsinogen A in the neutral and alkaline pH regions are in accord with the Linderström‐Lang model with a pH‐independent electrostatic factor. However, in order to interpret the results on oxidized cytochrome c in the same pH region, it is necessary to assume the protonation of two unidentified groups; one of these groups, with ΔHb = −18 kcal/mol and pKa = 9.4 was detected in previous work [Watt, G. D. & Sturtevant, J. M. (1969) Biochemistry 8, 4567]. The normal heats of protonation for carboxyl, ϵ‐amino, phenolic, α‐amino, and imidazole groups on globular proteins as deduced from the study are 0, −10.5, −6.3, −10.0, and −6.3 kcal/mol, respectively. ΔHb of chymotrypsinogen A between pH 4.5 and 1.3 is +30 kcal/mol. This value, which is undoubtedly too large to be accounted for by the protonation of normal carboxyl groups, leads to the conclusion that this protein undergoes a pH‐induced conformational change in this pH region. The same idea can be applied to explain the abnormal ΔHb observed for oxidized cytochrome c in the alkaline pH region.
Ultracentrifugal compression of gels
Authors: Bloomfield Victor A.
Published: December 2004

It has been observed that the amount of water associated with casein micelles is markedly higher when measured by intrinsic viscosity than by water content of the pelleted casein. A possible explanation of this discrepancy is that water is squeezed out of the pellet by the ultracentrifugal field. We have calculated the potential magnitude of this effect by considering a simple model system: an elastic gel swollen by solvent and compressed by the centrifugal field. Equilibrium is reached when the sum of the ultracentrifugal, elastic, and mixing free energies is a minimum. The equilibrium degree of swelling is calculated as a function of rotor speed, thickness of the unsolvated gel material, and enthalpy of mixing of solvent and solute. Sizable compressions can occur for highly swollen gels, if the enthalpy of mixing is moderately positive. Casein micelles from intrinsic viscosity measurements have a “hydration” of about 3.7 g water/g protein, corresponding to a swelling ratio of 6.3 relative to the dry protein. The observed pellet hydration is 1.9 g/g. Under the conditions of the experiment, pelleting at 25,000 rpm is predicted to decrease the swelling ratio to 4.9, and the hydration to 2.7 g/g, about half the observed decrease, if the enthalpy of mixing is 0.5 RT/mol segment. These calculations may be relevent to the deterination by pelleting of the solvent content of other biopolymer gels.
Spectroscopic properties of complexes of acridine orange with glycosaminoglycans. II. Aggregated complexes—evidence for long‐range order
Authors: Salter Mary K. , Abrahamson E. William , Rippon W. Barton
Published: December 2004

Aggregated complexes of acridine orange with dermatan and chondroitin sulfates have been studied in aqueous solution by absorption and circular dichroism spectroscopy. Aggregation was found to be favored at high‐dye and glycosaminoglycan concentrations, and in solutions where anionic sites of the glycosaminoglycan are effectively complexed with dye. The aggregates can be removed from solution by centrifugation at 27,000 × g for 1 hr or by filtration through a membrane containing pores of 0.1 μm diameter.

The aggregated complexes exhibit large‐magnitude‐ellipticity circular dichroism bands. In addition, the circular dichroism spectrum observed for a solution containing aggregated acridine orange/chondroitin 4‐sulfate complexes is nearly a mirror image of that obtained for aggregated acridine orange/dermatan sulfate complexes.

Cooperative alterations (sharp transitions) in the circular dichroism ellipticities of the aggregates occur at elevated temperatures, and result in spectroscopically distinct aggregates upon cooling. The circular dichroism properties and temperature effects are attributed to a supramolecular ordering of acridine orange/glycosaminoglycan complexes within the aggregates, which can be reorganized to a more stable form at high temperatures.

Mixed aggregates, containing two different glycosaminoglycans, can be formed. The circular dichroism properties of the mixed aggregates also indicate the existence of long‐range order in the arrangement of the complexes. Mixed aggregates containing dermatan sulfate and either chondroitin 4‐sulfate or chondroitin 6‐sulfate resemble pure dermatan sulfate aggregates in circular dichroism characteristics.
Rapid characterization of protein molecular weights and hydrodynamic structures by quasielastic laser‐light scattering
Authors: McDonnell Milton Eddy , Jamieson A. M.
Published: December 2004

Two methods for the characterization of protein molecular weights from their diffusion coefficients are discussed. These measurements can be made quickly and reliably at low concentrations using quasielastic light‐scattering techniques. First, an empirical calibration of the diffusion coefficient at infinite dilution of denatured random coils against molecular weight is reported. The second method combines the measurement of D0 with the intrinsic viscosity [η]. This D0–[η] relationship proves to be very insensitive to polymers structure or solvent type. The data indicate that the ratio of the hydrodynamic radius measured by viscosity to the hydrodynamic radius measured by diffusion is about 15% smaller than that predicted by theoretical models. The nature of the molecular‐weight average obtained for polydisperse systems is defined for a Schulz distribution. These hydrodynamic methods have also been used to demonstrate the presence of chain branching in the glycoprotein ovomucoid. In addition, a method is proposed by which the effective segment length and an excluded volume parameter for random coils may be evaluated for diffusion measurements.
Interpretation of energy‐transfer experiments by theoretical studies of model compounds using semiempirical potential functions. I. Three‐linked aromatic peptide units
Authors: Guillard R. , Englert A.
Published: December 2004

It is shown that theoretical conformational analysis, based on the evaluation of semiempirical potential functions, can be used to compute the quantities relevant to the interpretation of energy‐transfer experiments. The relevant properties are computed for a segment of a polypeptide chain with the sequence Tyr‐Tyr. In particular, the average value of the orientation factor 〈κ2〉 and its distribution ƒ(κ2) are examined. It appears that the degrees of freedom for rotation of the side chains are not sufficient to randomize completely the orientation factor of the transition dipoles. Two additional degrees of freedom, namely the torsion angles around the valence bonds of the backbone, ψ1 and ϕ2, bring 〈κ2〉 close to the value that corresponds to randomly oriented transition dipoles.
Proton and phosphorus‐31 magnetic relaxation studies on the interaction of polyriboadenylic acid with Mn2+
Authors: Yamada Atsuko , Akasaka Kazuyuki , Hatano Hiroyuki
Published: December 2004

Proton and phosphorus‐31 nuclear spin–lattice relaxation times T1 and spin–spin relaxation times T2 have been measured on the single‐stranded polyriboadenylic acid [poly(A)]–Mn2+ system in a neutral D2O solution in the temperature range 10°–90°C at 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T1 have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn2+ to H2, H8, H1′, and the phosphorus nucleus to be 4.7, 4.1, 5.2, and 3.0 Å, respectively. The electron spin of Mn2+ penetrates into the phosphorus nucleus, giving 31P hyperfine coupling of more than 106 Hz. Evidence of penetration of the electron spin into H8 and H2 is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn2+ Ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn2+ ion added is bound directly to two phosphate groups with a Mn2+–phosphorus distance of 3.3 Å, while a part of the Mn2+ ions are simultaneouly bound to the adenine ring. It is estimated that 39 ± 13% and 13 ± 5% of Mn2+ are coordinated by N7 and N3 (or N1), respectively. The motional freedom of poly(A) in the environment of the Mn2+ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn2+–free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn2+–free poly(A), and are found to be 8.3, 8.5, 6.1, and 8.7 kcal/mol for H8, H2, H1′, and phosphorus, respectively. T2 of phosphorus is determined by the dissociation rate (k−1) of Mn2+ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn2+ from the adenine ring are also estimated from proton T2 values below 50°C.
The β association of (DLLys)10–(LVal)20–(DLLys)10: An nmr study
Authors: Schwartz Arnold M. , Fasman Gerald D.
Published: December 2004

(DLLys)10–(LVal)20–(DLLys)10, a copolymer of a valine block in the β conformation with randomly coiled flanking blocks of lysine [Kubota, S. & Fasman, G. D. (1975) Biopolymers 14, 605–631], was studied by nuclear magnetic resonance. The peak areas of the NH and CH resonances of valine and lysine are concentration‐dependent with little chemical shift change. A model is presented describing the aggregation of the copolymer. Additional evidence, obtained by monitoring the spin‐lattice relaxation times of amino, amide, γ‐methyl‐valine, and ϵ‐methylene‐lysine, is consistent with the aggregation model. Hydrodynamic studies are presented which show monomer → oligomer aggregation of four or five subunits.
Viscosity at low shear and circular dichroism studies of heparin
Authors: Chung M. Ching Ming , Ellerton Nerida F.
Published: December 2004

The viscosity of heparin solution was investigated under conditions of low shear stress between 0.0193 and 0.222 dyne cm−2, in water, in the presence of various cations (Na+, K+, Cs+, Mg2+, Ba2+, Cu2+) and at several pH's. The viscosity was found to decrease with in creasing shear stress. Shear dependence was greatest in the absence of added salts, and decreased as the ionic strength increased.

Differences in viscosity in the presence of various cations appear to be related to the binding affinity of these cations to heparin. Viscosity studies of the periodate oxidation of heparin confirmed that heparin contains vicinal hydroxyl groups in its primary structure.

Circular dichroism spectra of the same heparin solutions were also studied. The binding process between Cu2+ and heparin appears to be different from that of other divalent ions. A reduction in the pitch of the helix would qualitatively explain the conformational changes that occur on binding Cu2+ to heparin. These changes are reversible on removal of Cu2+ and replacement with Na+. The circular dichroism spectrum was virtually lost for periodateoxidized heparin.
An electrostatic model for collagen fibrils. The interaction of reconstituted collagen with Ca++, Na+, and Cl−
Authors: Li Shu‐Tung , Katz Elton P.
Published: December 2004

A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å.

The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α‐amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect.

The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3–5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M−1, respectively.

The model also predicts that an average 4‐mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties.

The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility.
Geometry of DNA–dye intercalation complexes from the study of linear dichroic spectra of stretched films
Authors: Kelly Gregory R. , Kurucsev Tomas
Published: December 2004

Films of DNA–dye complexes were combined with films of pure DNA deposited on poly(vinyl alcohol) support and stretched. Reproducible dichroic spectra were obtained after equilibration of the stretched films at 93% relative humidity. Dye diffusion into the supporting poly(vinl alcohol) matrix was eliminated. The long axis of intercalated acriflavine is perpendicular to the DNA helix; proflavine deviates slightly and 9‐aminoacridine significantly from such an intercalation geometry. The dichroism of two mutually perpendicularly polarized transitions of 9‐aminoacridine enabled us to determine both the angles of tilt and twist of the plane of the dye relative to the DNA helix in the complex.
Effect of europium(III) on the thermal denaturation and cleavage of transfer ribonucleic acids
Authors: Rordorf B. F. , Kearns D. R.
Published: December 2004

The interaction of tRNA with Eu(III) has been studied by optical and gel electrophoretic tecfhniques. At low levels (less than six metals per tRNA), Eu(III) stabilizes yeast tRNAPhe against thermal denaturation; however, at higher levels (about eight to ten Eu/tRNA) the tRNA is destabilized by Eu(III). This may have important implications regarding recent attempts to grow crystals of tRNA from solutions containing europium. Comparative studies of the effects of Mg(II) and Eu(III) on tRNA structure confirm that the first four Eu(III) ions are more strongly bound than Mg(II). At slightly elevated temperatures (50°C, pH 7) the binding of Eu(III) catalyzes the hydrolysis of the tRNA backbone. From an analysis of the fragments produced by the hydrolysis and of the variation in the rate of cleavage as a function of the metal per tRNA ratio, we conclude that (i) the addition of Eu(III) to the tRNA is sequential, (ii) the first Eu(III) is bound in close proximity to the two dihydrouridine residues, and (iii) the rate of hydrolysis depends on the number of Eu(III) free in solution. Metals bound at sites 2–4 are relatively much less active in promoting the cleavage but the metal bound at site 5 is again active. The initial cleavage products are shown to be identical with those obtained using magnesium, zinc, or lead.
Effect of concentration on the aggregation of poly(γ‐benzyl‐L‐glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates
Authors: Gupta Avadhesh K.
Published: December 2004

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ‐benzyl‐L‐glutamate) in dioxane have been carried out over a range of concentration 10−4–10−2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (≃ 10−3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration.

Maximum length of linear head to tail type aggregates for poly(γ‐benzyl‐L‐glutamate) in dioxane as determined from these results correspond to an α‐helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.
Racemization in the synthesis of sequential polypeptides using N‐hydroxysuccinimide esters
Authors: Rapaka Rao S. , Bhatnagar R. S. , Nitecki D. E.
Published: December 2004

Racemization in the synthesis of peptide intermediates and their polymers was investigated, using L‐amino acid oxidase. The formation of N‐hydroxysuccinimide esters from N‐protected peptide acids yielded optically pure products in contrast to p‐nitrophenyl and pentachlorophenyl active esters. The racemization in the polymerization step was found to be base sensitive. Partially racemized polymer can result from optically homogeneous monomer. Thus, the optical integrity of active monomer species carries no guarantee for that of the polymer.
A study of DNA denaturation in the ultracentrifuge
Authors: Wiesehahn Gary , Cech Thomas R. , Hearst John E.
Published: December 2004

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region.

An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM‐2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all‐or‐none transition with no equilibrium bands at partially denatured densities.

The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The Tm does not show a molecular‐weight dependence in the range 1.2 × 106–1.5 × 107 daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late‐melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.
Effect of ligands on the optical rotatory dispersion of hemoglobin. I. Ferrihemoglobin cyanide, ferrihemoglobin azide, ferrihemoglobin hydroxide, and carbonmonoxyhemoglobin
Authors: Beychok Sherman
Published: December 2004

The optical rotatory dispersion curves of ferrihemoglobin at pH 6 and 9.5, ferrihemoglobin cyanide, ferrihemoglobin azide, and carbon monoxyhemoglobin have been measured over the wavelength interval 500–190 mμ. Helix contents, based on the measured rotations at 233 and 200 mμ are estimated for the ferrihemoglobin derivatives on the basis of comparison with the rotations of the helical poly‐α,L‐glutamic acid. For the latter, a mean value of −14,200° is found for [m′]233 and 65,000° for [m′]200. Within experimental error, all the ferrihemoglobins examined have the same helix content, the value of which is 65% based on rotations at 233 mμ and 72% based on rotations at 200 mμ. Thus, the addition of ligands to ferrihemoglobin which enormously stabilize the molecule to acid denaturation and involve heme–heme interaction has no effect on the number of amino acid residues which are in α‐helical segments. Change of pH from 6.8 to 5.0, through which interval the molecular weight decreases to that of the dimer, also leaves the helix content essentially unchanged, although at pH 5 heme–heme interaction in the binding of cyanide ion to ferrihemoglobin is absent. In addition to the Soret band Cotton effect observed for all the hemoglobin derivatives examined, there is at least one (and possibly two) other heme Cotton effects at wavelengths intermediate tot he Soret Cotton effect near 400 mμ and the n‐π* peptide bond transition near 225 mμ. The Soret band Cotton effect is different in magnitude in all the derivatives examined, the most notable difference being in the pronounced intensification of this Cotton effect in CO‐hemoglobin relative to the ferrihemoglobin derivatives. Similar differences have been observed by others in different myoglobin derivatives as well. While the changes are not so large as observed with hemoglobin, this indicates that these altered magnitudes are not directly related to the specific details of heme environment which give rise to differences in affinity for oxygen in the two proteins and result in a Bohr effect in hemoglobin but not in myoglogin.
Polarographic studies of ion binding in solutions of pepsin. I. Binding of cadmium ions
Authors: Lapanje Savo
Published: December 2004

The results of the application of the modified polarographic method to the study of binding of cadmium ions in solutions of pepsin are reported. Isoionic solutions of pepsin were converted by suitable treatment with a cation‐exchange resin to solutions of cadmium pepsin. As in previous cases, i.e., in solutions of synthetic polyelectrolytes, the extent of ion binding was obtained by assuming that only free, i.e., unbound, ions contribute to the diffusion and migration currents, respectively. It has been furthermore shown that cadmium ions are bound to pepsin ionically as well as covalently, and the relative amounts of cadmium in either state of binding have been determined.
Stabilizing effectiveness of alkali metals toward ribonucleic acid from the bacterial virus R17
Authors: Gordon Julius A.
Published: December 2004

The addition of alkali metal cations (0.10–1.00M) stabilizes the macromolecular structure of R17‐RNA as evidenced by a shift of the optical density–temperature profiles to higher temperature. This effectiveness is found to decrease in the order Li+ > Na+ > K+ > Rb+ > Cs+ and the midpoints of each profile (Tm) are found to increase linearly for each salt with the log of the salt concentration. The differences in effectiveness in this series can be best interpreted as a decreasing specific interaction of Li+, Na+, K+, and Rb+ with the phosphate groups of RNA as suggested by known binding constants to phosphate groups and the failure of adenine, adenosine, adenine monophosphoric acid, and uracil to show specific solubility differences in these salts. The importance of this feature is that the thermal stability of RNA in the presence of alkali metals shows specific ion requirements in addition to general electrostatic interactions. Furthermore, the more nonspecific, ionic strength‐dependent stabilization of native RNA also involves a decrease in adenine solubility (e.g., increased activity) and an increase in phosphate solubility in the aqueous salt solutions in addition to the well recognized Coulombic shielding of the negatively charged phosphates.
Studies on amylose and its derivatives. Part VI. Estimation of cohesive energy densities of some amylose derivatives
Authors: Cowie J. M. G.
Published: December 2004

The cohesive energy densities (CED) of three amylose derivatives have been estimated from viscosity measurements in a range of solvents, by using methods proposed by Mangaraj and Bristow and Watson. The values assigned to the triacetate, (2.5) nitrate, and tricarbanilate are 92 ± 2, 90 ± 2, and 87 ± 2 cal./cc., respectively. The CED of unsubstituted amylose has been measured and found to be 154 ± 4 cal./cc. The results are discussed in relation to solvent power, structure, and possible correlation with the internal pressure of the polymer.
Effect of temperature on the buoyant density of bacterial and viral DNA in CsCl solutions in the ultracentrifuge
Authors: Vinograd Jerome , Greenwald Robert , Hearst John E.
Published: December 2004

The effect of temperature on the buoyant density of CsDNA in CsCl at equilibrium in the ultracentrifuge has been measured between 5 and 60°C. The temperature coefficients of the byoyant density for three DNA's of varying guanine‐cytosine (G‐C) contents were, within experimental error, +4.2 ± 0.3 × 10−4 g./ml.°C. This coefficient may be used to normalize results obtained between 5 and 60°C. to 25°C. The results are explained in terms of a loss of water from the hydrated CsDNA complex. It is estimated that about one‐third per cent of the water preferentially hydrated by the DNA is liberated per degree elevation in temperature. Significant errors in buoyant density determinations are made if the thermal expansion coefficient of CsCl solutions is ignored.
Ultraviolet light‐induced change in collagen macromolecules
Authors: Fujimori Eiji
Published: December 2004

Viscosity measurements and calculated rotary diffusion constants show that collagen undergoes photopolymerization when irradiated with ultraviolet light of 2537 A. Fibril formation at the same time is inhibited. The results are correlated with the aromatic amino acid content of the dangling peptides protruding from the rigid portion of the macromolecule.
Studies of the conformation of carboxymethyl amylose in aqueous solutions
Authors: Rao V. S. R. , Foster Joseph F.
Published: December 2004

In an attempt to clarify certain questions of the configuration of amylose in aqueous solution, carboxymethyl groups were introduced into corn amylose (degree of substitution ca. 0.8) and certain properties of the derivative (CMA) studied. The intrinsic viscosity exhibits a strong increase between pH 2 and 6 but remains constant at higher pH. A pH 6 and above, CMA exhibits typical polyelectrolyte behavior. CMA does not form a complex with iodine at pH 6 or above but does form a violet‐colored complex at low pH. The absorption maximum of the CMA complex lies at a somewhat lower wavelength (550 mμ) than that of the complex formed by the parent amylose. The reduced viscosity and specific optical rotation of the CMA–iodine complex remains constant up to a concentration of 1.2 mg. iodine/100 mg. CMA, then both properties decrease with further increases of iodine content. All of the above results and hydrogen ion titration behavior are in agreement with the hypothesis that CMA exists as a coil of short helical segments in acid solution and that these helical segments break down on ionization of the carboxyl groups. The specific optical rotation of CMA increases between pH 2 and 6, remains constant between pH 6 and 12, and decreases above pH 12. This later result shows that the similar decrease in rotation seen in amylose cannot be attributed, at least entirely, to a helix–coil transition. There must be another effect on optical rotation, direct or indirect, of the ionization of hydroxyl groups.
Dependence of the melting temperature of DNA on salt concentration
Authors: Schildkraut Carl , Lifson Shneior
Published: December 2004

Data on the decrease of the DNA melting temperature Tm with the salt concentration M are reported and discussed. The electrostatic free energy change in the helix–coil transition, ΔFe is related to the potential, ψ, which represents the electrostatic repulsion between the phosphate charges; ψ is calculated as a function of M and of the distances between the charges of the two strands. The Debye‐Hückel approximation is shown to overestimate ψ. It is suggested that the high local concentration of the counterions in the immediate vicinity of the fixed charges screen these charges from interacting with other fixed charges, to the extent that the system behaves as if the fixed ions carry a reduced charge. The notion of a reduced charge represents in a single parameter the deviation of the Debye‐Hückel approximation from the true potential. A plot of Tm versus ΔFe gives a straight line as predicted. ΔH0 is calculated from the slope and found to be consistent with experimentally determined values. Our calculations support the hypothesis that the change of Tm with salt concentration is due to changes in the screened interactions between the fixed phosphate charges. In analyzing the results of these calculations, we are able on the one hand to indicate some of the limitations of the theoretical model and, on the other hand, draw some conclusions about the order of magnitude of the nonelectrostatic interaction energy of formation of the double helix.
Helix–coil transition in poly‐ε‐carbobenzoxy‐L‐lysine
Authors: Karasz F. E. , O'Reilly J. M. , Bair H. E.
Published: December 2004

The reversible helix‐to‐random coil conformational change which occurs in solvated poly‐ϵ‐carbobenzosy‐L‐lysine, a synthetic polypeptide, has been studied. The course of this cooperative transition, which in many ways resembles a first‐order phase transition, was followed both by measuring changes in optical rotation as a function of temperature, and calorimetrically by determining the heat capacity of the solution through the transition region. From the latter measurements, the enthalpy of transition was calculated. From the results it has been shown directly, for the first time, that in a statistical thermodynamical sense, the transition in this system is significantly more cooperative than that undergone by poly‐γ‐benzyl‐L‐glutamate, a closely analogous polypeptide. Thus a previously held concept that the degree of cooperation might be quantitatively similar for a wide range of such systems has been shown, for at least one important example, not to be tenable.
Magnesium binding to poly(L‐aspartic acid)
Authors: Jacobson A. L.
Published: December 2004

The optical rotatory dispersion of poly(L‐aspartic acid) in magnesium chloride and tetramethylammonium chloride solutions has been measured to determine helix content. At pH 4, helix content estimated by using the Moffitt bo coefficient is about 20%. Optical rotation has been used to determine changes in conformation with polymer ionization. The changes in rotation are very sensitive to the relative concentration of added salt. At very low salt concentrations no ordered structure appears to be present. The effect of magnesium ion binding on the dissociation of side‐chain carboxyl groups in poly(L‐aspartic acid) has been measured by a modified titration technique. The changes in ionization of the carboxylic acid side chains were measured as a function of both the degree of polymer preionization and of added magnesium chloride. These changes have been related to possible changes in the polymer conformation as a function of degree of polymer ionization. The effect of ionization on magnesium ion binding to poly(L‐aspartic acid) and to poly(L‐glutamic acid) have been compared with essentially equivalent polymer concentrations at equivalent salt additions. pH titrations have been used to compare ionization in the two polyacids. The pH titration method proposed by Wada has been used to determine the extent of the helix‐‐coil transition region in this polymer concentration range. Poly(L‐aspartic acid) appears to be more easily ionized than poly(L‐glutamic acid). For both polyacids, a helical conformation appears to favor ion binding. However, in this region, binding to poly(L‐aspartic acid) is approximately double that observed with poly(L‐glutamic acid) at a corresponding degree of ionization. In the coil region the trends observed with magnesium binding are similar to those observed with other extended polyelectrolytes.
Optical rotatory dispersion of mucopolysaccharides and mucopolysaccharide–dye complexes
Authors: Stone Audrey Larack
Published: December 2004

Anomalous optical rotatory dispersion (ORD) is induced in symmetric dyes when bound in metachromatic complexes with chondroitin sulfates (ChS) and kerato sulfate (KS) from mammalian and shark origin as well as with heparin (H) providing inference that secondary structures exist in these biopolymers. The induced rotation occurs in the absorption band of the bound dye and differs in shape and/or spectral position from the classical Cotton effect seen with single optically active chromophores. The AO:ChS complex has an induced ORD curve similar to that of the exciton band of a helical polymer. Strength of the induced rotations with methylene blue increases with the degree of metachromasy of the 1:1 complex. The sense of the MB Cotton effects may depend upon the conformations of the repeating dimeric units, since MB complexes with ChS (alternating 1‐3,1‐4 β‐glycosides) give (+) effects while MB:H (1‐4 α‐glycosides) have (−) effects in the γ band region. Preliminary evidence for the occurrence of Cotton effects with the mucopolysaccharides themselves in the ultraviolet region was obtained. The sign of the Cotton effect for ChS‐C is (−), trough about 45.9 kK., crossover about 48.8 kK. whereas that for H is (+), peak about 50.4 kK.
Kinetics of polypeptide denaturation
Authors: McQuarrie D. A. , McTague J. P. , Reiss H.
Published: December 2004

The irreversible kinetics of polypeptide denaturation and renaturation is discussed in terms of the Zimm‐Bragg equilibrium model. An exact solution is presented for situations in which the back reaction is negligible. The solution is given in terms of parameters which are rate constants for the elementary processes in denaturation or renaturation. The formalism is also applicable to reactions of linear polymers in which the reactivity of a given site depends on the state of reaction of its neighbors.
Cyclodextrin–polyethylenimine conjugates for targeted in vitro gene delivery
Authors: Forrest M. Laird , Gabrielson Nathan , Pack Daniel W.
Published: December 2004

Many human gene therapies will require cell‐specific targeting. Though recombinant viruses are much more efficient than nonviral vectors, the latter, especially polymers, have the advantage of being targetable via conjugation of cell‐specific ligands, including sugars, peptides, and antibodies, which can be covalently attached to the polymer using a variety of chemistries. Cyclodextrin, which forms inclusion complexes with small hydrophobic molecules, has been incorporated into a gene‐delivery polymer and may provide a facile and versatile attachment site for targeting ligands. Polyethylenimine (PEI) was derivatized with β‐cyclodextrin on ∼10% of the polymer's amines (termed CD‐PEI). Human insulin was also derivatized with a hydrophobic palmitate group (pal‐HI), which could anchor the protein to CD‐PEI/DNA polyplexes. CD‐PEI was essentially nontoxic to HEK293 cells at concentrations optimal for gene delivery and mediated nearly 4‐fold higher gene expression than unmodified PEI, which is relatively toxic to these cells. More importantly, addition of the pal‐HI to CD‐PEI enhanced gene expression by more than an order of magnitude compared to unmodified PEI, either with or without the pal‐HI. Because of the relative ease with which CD‐binding moieties may be attached to various types of ligands, CD‐PEI may be a generally useful material for testing novel cell‐specific targeting compounds. © 2004 Wiley Periodicals, Inc.
Synthesis of galacto‐oligosaccharide from lactose using β‐galactosidase from Kluyveromyces lactis: Studies on batch and continuous UF membrane‐fitted bioreactors
Authors: Chockchaisawasdee Suwimol , Athanasopoulos Vasileios I. , Niranjan Keshavan , Rastall Robert A.
Published: December 2004

A study of galacto‐oligosaccharides (GOS) synthesis from lactose with β‐galactosidase from Kluyveromyces lactis (Maxilact® L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40°C and pH 7, in a stirred‐tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non‐competitive inhibitor. In a further study, a laboratory‐scale reactor system, fitted with a 10‐kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross‐flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40°C, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAE‐PAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal®GOS). © 2004 Wiley Periodicals, Inc.
Studies on the response of activated sludge to shock–loadings
Authors: Krishnan P. , Gaudy A. F.
Published: December 2004

The response of heterogeneous populations to shockloading was studied under a variety of operational conditions using synthetic wastes consisting of glycerol, glucose, and a mixture of glucose and glycerol. In all experiments, the cells were acclimated to glycerol. The addition of glucose as a shockloading at various times during the aeration period caused an immediate cessation of glycerol metabolism, and glycerol was not again actively metabolized until all glucose had been assimilated. Experiments conducted at various initial sludge concentrations showed that this effect was not dependent upon biological solids level. Glycerol metabolism was also blocked when glucose was applied as a shockloading to a system operating under severe nitrogen deficiency, further reenforcing the conclusion that there is a blockage mechanism for catabolic pathways which operates to suppress enzyme function rather than enzyme synthesis. It was also found that considerable amounts of metabolic intermediates or end products were released during glucose metabolism regardless of the presence or absence of glycerol. In systems containing a source of nitrogen, these products were metabolized after dissimilation of glucose. In resting cell suspensions, the metabolic intermediates were not subsequently removed, indicating that their metabolism depended upon the synthesis of new enzymes. Using old cell sludge, concurrent rather than sequential removal of glycerol and glucose was observed. The results using two component (C6C3) carbon sources were in general the same as for C6C6 synthetic wastes. The results provide further evidence for the generality of sequential substrate removal, and suggest that the metabolite suppressing catabolism of glycerol lies below the triose level.
Production of Bacillus cereus exopenicillinase on a pilot‐plant scale
Authors: Miller G. , Bach G. , Markus Z.
Published: December 2004

A substrain of Bacillus cereus 569/H produced above 10,000 units of penicillinase/ ml. when grown in a pilot‐plant fermentor using a medium containing Casamino acids techn. (Difco) or N‐Z‐Amine type B (Sheffield), and salts. Simplified purification and concentration procedures give an overall yield of 50‐65% enzyme. The freeze‐dried enzyme preparation had a good storage stability in vacuumsealed ampules kept at 4, 30, and 37°C. In vials containing air in the head space, partial inactivation occurred in two months at 30 and 37°C. The freezedried preparation showed satisfactory performance in the production of yoghurt fermented milk.
Controlled pH and oxidation–Reduction potential with a new glass tissue‐culture fermentor
Authors: Daniels William F. , Parker David A. , Johnson Roger W. , Schneider Louis E.
Published: December 2004

A 1‐l. fermentor was designed and tested for use as a tissue‐culture vessel. It features a temperature control device, impeller agitation without the necessity of a shaft seal, and a means of measuring, recording, and controlling both pH and oxidation‐reduction potential (ORP). Tests have shown the ORP to change fairly rapidly with impeller speed variations under conditions of a continuous carbon dioxide‐air overlay. Working with strain L mouse fibroblasts (Earle), cell counts of more than 1.25 ×106 ml., without centrifugation and medium renewal, were achieved, and cell counts were maintained above 1 ×106 for more than 30 hr. With the vessel studied, pH control was ±0.05, the ORP control was ±10 mV. Controlled environments for tissue‐cell metabolic studies are entirely feasible with this system.
A model system for evaluating shear in the design of stirred fermentors
Authors: Midler Michael , Finn R. K.
Published: December 2004

A model system, utilizing shear‐sensitive protozoa, has been developed for characterizing the disruptive forces in agitated systems. The model system gives a measure of the maximum shear stresses in the apparatus being tested, and is particularly useful when tissue fragility is a factor in fermentor design. The time dependency of protozoan disruption is shown and discussed. Breakdown data in conventional stirred vessels and a laminar shear device are presented and discussed.
The effect of equipment scale and degree of mixing on continuous fermentation yield at low dilution rates
Authors: Hansford G. S. , Humphrey A. E.
Published: December 2004

Previously, the degree of mixing was not felt to be an important consideration in fermentor design. In this study on the continuous propagation of Baker's yeast, it was found that at low dilution rates, i.e., 0.02hr−1, the degree of mixing achieved does effect the cell yield. At low dilution rates, appreciable quantities of sugar can be utilized for endogenous respiration in comparison to that utilized for making cell mass. Poor distribution of the sugar aggravates the balance of sugar utilized for each process. Yields at these low dilution rates can be improved to a limited extent by using a multiple feed‐distribution system and better mixing.
Studies with a multiple‐rod mixing impeller
Authors: Steel R. , Maxon W. D.
Published: December 2004

The performance of a multiple‐rod mixing impeller was compared to that of conventional turbine impellers in viscous novobiocin beers. The advantages of the multiple‐rod impeller were found to be: (1) the power requirement was independent of changes in apparent viscosity of the fermentation beer; and (2) it gave the same novobiocin yield and oxygen‐availability rate at about one‐half of the power required by turbines.
Design and operation of a simple continuous‐culture apparatus
Authors: Saeed Mohammed , Moss Frank J.
Published: December 2004

A versatile and simple continuous‐culture apparatus is described. With this apparatus, independent control of limiting growth factors and other nutrilites is possible and the conditions of each experiment are reproducible. In view of the synchronized speed of the feeder syringes, flow variation troubles are not encountered. The device allows the performance of growth experiments at different dilution rates simultaneously in a single run which makes the comparison of the results more reliable. The operation of the device has been tested successfully with a study of adenine deaminase induction in yeast.
The production of bacteriophage μ2
Authors: Sargeant K. , Yeo R. G.
Published: December 2004

In three series of experiments, 3‐l., 20‐l., and 150‐l. bacterial cultures were grown in stirred, deep culture vessels to average bacterial cell densities of 71 × 108, 63 × 108, and 43 × 108 viable organisms per milliliter, respectively, and then infected with phage. The average yield of progeny phage in each case was ca. 3000 mpfu (minimum plaque‐forming units) per cell. Thus, the average mass of phage obtained in the 3‐l. experiments was not less than 124 mg./l., calculated from the plaque counts, assuming a particle size of 3.6 × 106 Daltons for the μ2 phage. This is about twentyfold higher than is obtainable by conventional methods in aerated, shaken culture flasks. The actual phage yields are probably much higher than the minimum values calculated from plaque counts. For example, in the case of one of our culture lysates which was purified at King's College, the efficiency of plating was shown to be only 19%. The carbon dioxide evolution rate of cultures was measured and used as a guide to the time at which phage should be added. In this way, greater control of cultural conditions was obtained than is possible in shaken flasks. For the best yield of phage per milliliter of culture, the optimum time for phage infection was such that bacterial lysis just prevented the carbon dioxide evolution rate from reaching its potential maximum. The major factor influencing the phage yield per milliliter of culture was the aeration capacity of the culture vessel used. All had maximum aeration capacities much higher than those obtainable in shaken culture flasks. Cultures grown and infected in 3‐l. Vessel operated under conditions of low aeration gave poor yields of phage. The reason for this are discussed.
Isolation, characterization, and growth of basidiomycetes
Authors: Schuytema Eunice C. , Hargie Martha P. , Merits Ilmar , Schenck Jay R. , Siehr Donald J. , Smith Muriel S. , Varner Estie L.
Published: December 2004

Our program on the mass cultivation of basidiomycetes was designed to determine whether these organisms, being more highly differentiated than bacteria, molds, and yeasts, would in turn carry out markedly different reactions on natural products. A discussion of our methods of isolating and characterizing a representative collection of basidiomycetes and of our difficulties in obtaining pure cultures which grew well is presented. Some information we obtained on the growth and types of products that these organisms produced in a synthetic medium is discussed. Our techniques for examining fermentation beers for transformation products from indole compounds and from progesterone are explained, and the types of products we obtained are described.
Mass culture of the protozoa Colpoda steinii
Authors: Proper George , Garver J. C.
Published: December 2004

Growth of the ciliate, Colpoda steinii, was studied in shake flasks, 30‐l., stirred jars, and a conventional 50‐gal. stainless‐steel fermentor. This organism was growth with either washed or unwashed Escherichia coli cells as the sole food source. Generation times of 3–4 hr. were obtained at 30°C. With average yields of 78% (0.78 g. of protozoa/g. of bacteria). In a 100‐l. fermentation, a dry weight concentration of 12.5 g./l. (1.04 × 107 protozoa/ml.) was reached by means of periodic addition of E. coli paste. The influence of bacterial concentration on the growth rate of the protozoa was also investigated. It was found that this relationship could be represented by a Michaelis‐Menten equation with a maximum specific growth rate of 0.23 hr. −1 (3‐hr. generation time) and a bacterial concentration for half‐maximum growth rate of 6.0 mg. (dry wt.)/l.