ALBERT

Notes: The two-state theory of protein denaturation, in which it is assumed that a protein exists as two species, native and denatured, is examined by three approaches in this paper. First, the point is made that denaturation of an isolated molecule involves a continuous shift in the probabilities of occurrence of many states generated by the partition function for the protein. It is argued that the maximum term approximation does not lead to a two-state mechanism of denaturation, and that the extent of cooperation implied by the two-state theory should give very much sharper transitions than are actually found in proteins. Second, the two-state theory is applied to the various model systems treated in this series of papers, and is found to be inadequate. Since the detailed behaviour of the models is known, it is possible to deduce the effect of the incorrect application of the two-state theory to a system that “denatures” by gradual unwinding. This exercise is useful when, thirdly, we examine experimental data that seem to depart radically from a two-state interpretation. We conclude that a mechanism of gradual unwinding is the most generally valid assumption, and that the two-state theory has no theoretical or experimental support.

Notes: Three types of band-forming centerpieces useful in band centrifugation in the analytical ultracentrifuge are described. The mode of filling and the advantages and disadvantages of each type are discussed.

Notes: The ability of periodate-oxidized amylose to form aggregates in aqueous solution was studied by sedimentation, light scattering, and viscosity analyses. Ultracentrifuge schlieren patterns show that aggregation can be appreciable at pH 3.0 and 1.0. The hydroxyl ion-catalyzed degradation of the oxidized amylose is faster at pH 3.0 than at pH 1.0. Viscosity and sedimentation analyses conducted at pH 3.0 show that a minimum in the degree of aggregation of the oxidized molecules is obtained at 15-25% oxidation. Solubulity studies and x-ray diffraction patterns on retrograded amylose show that maximum solubility of the retrograded amylose is obtained by oxidizing to the extent of 25-35%. It was therefore concluded that in the general range of 20-30% periodate oxidation, the oxidized amylose has a minimum ability to form intermolecular hydrogen bonds. Outside of this range, oxidized amylose readily forms aggregates in aqueous solutions.

Notes: Calorimetric measurements of the heat of the addition of the second strand of poly U to poly (A + 2U) to form the three-stranded poly (A + 2U) complex in 0.1M and 0.5M NaCl at 24 and 37°C. are reported. A value of ΔH = -3800 cal./mole of poly (A + 2U) formed was found to be fairly insensitive to the experimental conditions employed. The heat of the addition of the third strand to the preformed poly (A + U) helix is considerably less exothermic than the heat of reaction between poly A and poly U to form poly (A + U). The insensitivity of the heat of addition of the third strand (poly U) to changes in salt concentration and temperature lends qualitative support to the earlier hypothesis that the major portion of the variation of the ΔH of the poly A and poly U reaction with experimental conditions arises from differences in the conformation of poly A. Combining the information obtained in this study for the ΔH of the reaction forming poly (A + 2U) with data for the ΔH of the formation of poly (A + U) indicates that the conversion of poly (A + U) to poly (A + 2U) is opposed by an enthalpy change which increases with temperature. Extrapolation of these values to 52°C. where poly (A + U) is transformed to poly (A + 2U) in 0.5M NaCl leads to a value of ΔH = +3800 cal./mole (A + 2U) formed and ΔS = 11.5 cal./mole of (A + 2U) formed. It is concluded from the calorimetric data that the driving force for the poly (A + U) to poly (A + 2U) conversion reaction is the favorable entropy change.

Notes: The effect of salt type and concentration on the transformation of an oriented crystalline collagen tendon into a crosslinked network under conditions of equilibrium swelling was investigated. Our main observations are the following. The degree of swelling of crystalline tendons increases at low salt concentration Cs, and decreases at higher Cs for a wide variety of salts. The observation is not reconcilable with swelling taking place in interfibrillar spaces or structural voids. Within the tropocollagen units and at their ends, regions of reduced organization are postulated (as suggested by Bear and by Schmitt) which are able to interact with the diluent in the amorphous-like manner. At least four different factors should be considered in assessing the role of salt and salt concentration on the shrinkage temperature Ts under isoelectric conditions. They are: (1) specific effects, (2) diluent effects, (3) crosslinking effects, and (4) nonequilibrium effects. The diluent effects are correlated with the salting-in-salting-out power of the ions which was characterized in Part I of this series. Smaller amounts of diluents are generally available to the tendon when the salt has a higher salting-out power, and this corresponds to higher shrinkage temperatures, other conditions being the same. The crosslinking effect raises Ts due to a reduction of the diluent content and, probably for p-benzoquinone and formaldehyde, also to a reduction of the conformational entropy in the molten state. Nonequilibrium effects arise from the fact that shrinkage and recrystallization are kinetically hindered when the tendon is highly deswollen in strong salting-out solutions, or when the salt has a crosslinking power. The specific effect is the only effect which is not related to the amount of diluent present in the tendon. Its origin is less clear. For anions such as Cl- and SCN-, it is possibly related to an ability of the ion to prevent intersegmental hydrogen bonding and water carbonyl bridges. The competition of several of the above effects for a given salt solution makes possible various types of dependence of Ts upon Cs: Ts may either continuously decrease or continuously increase with increasing Cs, or it may go through a minimum. In absence of salt, the cooperative character of the transition at the pH at which maximum swelling occurs appears extremely reduced. The large swelling maintains the tendon in the elongated state and this simulates a continuous decrease of Ts on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and Ts is decreased with pH up to pH 2, when maximum swelling occurs, and then reincreases on further lowering of the pH. The dependence of Ts upon Cs is more complex than under isoelectric conditions. There is generally an increase of Ts with Cs which is equivalent to an increase of the denaturation temperature with Cs for helical polyelectrolytes in solution. At higher salt concentrations, however, Ts may decrease again, and possibly increase again at still higher salt concentrations, depending upon the effect of the salt solution in the isoelectric zone.

Notes: By using the Nemethy-Scheraga theory of water structure, a calculation was made of the energy changes for the rupture of a DH…A solute-solute hydrogen bond in water. A partition function was also obtained for the binding of water and other solutes to the DH and A groups in the special case where these are the NH and CO groups of a random coil polypeptide chain. In subsequent papers of this series, these calculated quantities will be used in a statistical mechanical treatment of the helix-coil transition for polypeptides in water.

Notes: A partition function is derived for a simple model of interacting helices in a short (20 residues) chain of poly-L-alanine. It is found that interhelical hydrophobic bonds effect a marked stabilization of helical forms, and give rise to a sharp transition of the type found in many proteins.

Notes: The electric birefringence of collagen solutions has been measured over a wide range of field strength with the pulse technique. The soluble collagen was from rat tail tendon. The solvent used was dilute acetic acid. Very pronounced saturation of the electric birefringence was observed, permitting calculation of the optical anisotropy factor. The Kerr constant was determined by extrapolation to zero field strength. From the dependence on field strength of the birefringence, the permanent dipole moment and the anisotropy of polarizability were separately determined. The contribution of the former to the Kerr constant was found to be twice as large as that of the latter. The same conclusion was obtained from the initial slope of the rise curves of the birefringence at low fields. The permanent dipole moment was 1.5 × 104 Debye, and the anisotropy of polarizability was about 3 × 10-15 cm.3. The magnitude of the latter indicates that the ion atmosphere polarization is important. Effects of added salt and thermal denaturation on the electric birefringence were explored.

Notes: The γ-benzyl-L-glutamate N-carboxyanhydride (NCA) polymerization initialed by diisopropylamine was studied in dimethylformamide (DMF)-dioxane mixtures of different compositions. It was found that the shape of the conversion versus time plots and the molecular weights of the polymers depend on the solvent composition. Auto-catalysis is present only when dioxane predominates in the solvent mixtures. Moreover, the molecular weight of the final polymer depends strongly on the precipitation conditions when the polymerization is carried out in DMF.

Notes: Crosslinked synthetic polypeptides of poly Glu51Lys33Tyr16 (mol. wt. 31,000) and of poly Glu52Lys33Tyr15 (No. 3) (mol. wt. 52,000) containing from one to six crosslinks per molecule have been prepared by use of FFDNB, WRK and ICD reagents.The monomeric fractions of these derivatives were isolated by Sephadex G-100 chromatography. The number of crosslinks per molecule of DNPene derivatives was determined by total hydrolysis of the derivatives, isolation of O, N∊-DNPene-Tyr-Lys bridges by paper chromatography and then spectrophotometric quantitation. The number of the amide-type crosslinks in Am derivatives was established by their deamination followed by total hydrolysis and quantitation of the remaining lysine residues. Crosslinked derivatives appear to have a more compact structure, as judged by their behavior on the Sephadex columns and by their intrinsic viscosities. They were further characterized in regard to their amino acid composition, average number of the crosslinks per molecule, nitrogen content, solubility, root-mean-square end-to-end distance, and their spectral properties. Their properties recommend them as useful models for the study of the tertiary structure of proteins in solution.

Notes: The introduction of intramolecular amide bonds into synthetic polypeptides produces molecules with organized spatial structure which are good models for the tertiary structure of proteins. Polarization of fluorescence measurements were used to study the internal structure and the overall rigidity of the intramolecularly crosslinked polypeptides. The graph of [(1/p) + (1/3)] against T/η changes from a straight line to a continuous curve: the temperature at which this change occurs and the slope of the straight line segment measure the stability of the internal structure of the molecule. The introduction of one to six crosslinks produces an organized internal structure that becomes more stable as the number of crosslinks increases. In contrast to the fluorescence measurements, the intrinsic viscosities, reflecting the overall hydrodynamic domain of the molecules, change (decrease) to the same extent whether one, four, or six intramolecular crosslinks are present. The overall rigidity of the polymers can be assessed by the rotational relaxation time ρh and the polarization at 10°C. p10. Both of these criteria show that the presence of six crosslinks significantly increases the rigidity, but one or four does not. The various hydrodynamic measurements may be fitted into a hierarchy of discrimination: intrinsic viscosity, sedimentation, and diffusion for size and shape; rotational relaxation time and polarization of fluorescence at a given temperature for overall molecular rigidity; and transition temperature and rate at which it is attained for internal molecular detail.

Notes: A theory for the dependence of the net thermodynamic solvation of a macroion in an electrolyte solution is presented. The solvation in moles salt/mole macroion is shown to approach - |Zp|/v in the limit of infinite dilution of salt and macroion. The solvation in moles water/mole macroion is shown to approach zero at zero water activity. Isopiestic determinations of the hydration of sodium deoxyribonucleate in NaCl, Na2SO4, and NaClO4 solutions indicate that short-range interactions of the NaDNA with solvent account for more than half of the observed solvation. The net hydration appears to be predominantly influenced only by water activity.

Notes: 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.

Notes: 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.

Notes: It is shown how the technique of fluorescence may be used to assess conclusions about conformation which are based on a statistical mechanical treatment of simple protein models. Specifically, the state of bonding and distance between two chromophores in a molecule depend on the overall conformation, which can be computed for model systems. On the assumption that the intensity and polarization of fluorescence are affected by exciton transfer between the chromophores, it is possible to compute the effect of conformation on fluorescence. It is demonstrated that the conformational changes computed in the preceding paper will give rise to marked changes in the intensity and polarization of fluorescence.

Notes: Intrinsic viscosity-temperature studies for isoelectric gelatin in KCl and KSCN aqueous solutions and equilibrium degree of swelling, ν-1, measurements for amorphous crosslinked rat tail tendons in the same solutions were carried out. On increasing salt concentration Cs, both [η] and ν-1 increase, go through a maximum, and then decrease at high Cs, KCl being more effective than KSCN in bringing about this decrease. The trend observed is similar to the variation of solubility of polypeptides and soluble proteins with Cs. By regarding a water-salt solution of a given Cs as a single diluent interacting with the protein modified by solvation and binding of ions, usual polymer solution theories were applied to the experimental results. Thus, quantities related to the entropy and enthalpy components of the excess chemical potential of the diluents were obtained. The data indicate that the initial increase of [η] and ν-1 with Cs results from the balance of an enthalpy component which, on increasing Cs, becomes less favorable to dilution and an entropy component which, conversely, becomes more favorable. The subsequent decrease of [η] and ν-1 with Cs is due to the prevailing of the enthalpy component. The maximum is reached at higher Cs for KSCN than for KCl, primarily because of the large increase in the entropy component operated by the former salt. The increase of the entropy parameter with Cs is associated to a breaking down of the coordinated water structure and to an alteration of the conformation of the macro molecules due to ion-dipole interaction and to ion absorption. The decrease in exothermicity of dilution with Cs indicates a reduced thermodynamic affinity of the diluent toward the polymer which probably results from alteration of the nature of both polymer and diluent.

Notes: Nine samples of poly-γ-benzyl-L-glutamate (PBLG), ranging in M̄w from 19,000 to 410,000, were examined viscomctrically and by ultracentrifugation with dimethylforma-mide (DMF) at 25°C. as helicogenic solvent. The data for [η] and s0 (limiting sedimentation coefficient) as functions of M̄w were fitted well by the theories for a rigid prolate ellipsoid of revolution whose major axis increases linearly with M̄w, but whose minor axis is independent of M̄w. This implies that the overall shape of the PBLG molecule in DMF is represented by a straight cylinder whose cross section is independent of its length. The length per monomeric residue h evaluated from [η] is about 1.3 A., whereas that from s0 is about 1.6 A. No adequate explanation for this difference in h can be found at present. More serious is the fact that these hydrodynamically evaluated values of h are appreciably larger than, the value obtained from our light-scattering measurements reported previously. All these values of h from our studies are not consistent with the value characteristic of the α-helix, for which h is 1.5 A. The concentration dependence of s0 was found to agree well with the recent theoretical prediction of Peterson for cylindrical macromolecules.

Notes: Precipitation of soluble forms of collagen from solutions containing the soluble protein-polysaccharide (PP-L) of bovine nasal cartilage, followed by centrifugation at 100,000 g, resulted in the formation of coherent elastic pellets whose wet weights increased with the concentration of PP-L in the initial solution. Dry weights and uronic acid contents of these pellets showed that the amount of water held in the wet pellet was nearly constant for any one kind and concentration of collagen, and ranged from 20 to 100 mg./mg. PP-L in the pellet. Soluble collagens from four different sources and PP-L from three kinds of cartilage showed similar effects. Precipitation of soluble collagen in the presence of hyaluronate or dextran yielded pellets of much smaller size than those formed in the presence of PP-L. The presence of chondroitin sulfate had only a slight effect on wet pellet weights. Wet weights of pellets formed in the presence of PP-L decreased with increasing ionic strength. A model involving entanglement between insoluble collagen fibrils and the relatively stiff chondroitin sulfate chains of branched PP-L seems qualitatively capable of accounting for these results.

Notes: The conformations of oligopeptides derived from L-alanine and co-oligomers of L-alanine with γ-methyl-L-glutamate were studied in several solvents via optical rotation and far-ultraviolet spectroscopy. Calculated values for optical rotation based on model compounds were compared with experimental values for the oligomers. In trifluoroacetic and dichloroacetic acids, the oligomers and co-oligomers exhibit rotations in close agreement with predicted values based on model compounds. Thus, in these solvents only nonhelical conformations exist. In trifluoroethanol, the experimental points of molar rotation for the pentamer and larger oligomers no longer follow the predicted values. In addition, the benzyloxycarbonyl and acetyl cononamers show b0 values of about -150, which demonstrates the presence of stable helical forms for these peptides. We also examined the molar extinction coefficients of oligopeptides in the 190 mμ region and determined the values for nonhelical peptide groups. The molar extinction coefficients per amide bond for the benzyloxycarbonyl and acetyl cononamers show extensive hypo-chromism, once again indicating the presence of stable helices for these compounds in trifluoroethanol.

Notes: Studies were made of the influence of hydrostatic pressure on the helix-coil transitions of poly (A + U) and poly (A + 2U). The results were analyzed by a thermodynamic treatment which emphasized the cooperative aspect of the transitions. The helix-to-coil volume changes were found to be small and negative indicating pressure stabilization of the coil form. The significance of the results with respect to other denaturation measurements was discussed.

Notes: The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400-500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.

Notes: Investigations have been made on the optical rotatory dispersion properties of E. coli ribosomes and their constituent RNA and proteins. The results indicate that (1) no conformational changes are involved in the formation of a 70S particle from the 50S and 308 subunits, (2) the E. coli ribosomal proteins are similar to most globular proteins with little α-helix content, and (3) the conformation of RNA and proteins inside the ribosome is very similar to that in the free state.

Notes: The iodine which is added to an aqueous amylose solution is bound only partly by the amylose while forming the blue complex and partly remains free. The equilibrium normality of the free and the bound iodine at half-saturation of amylose by iodine is designated as [If]v and [Ib]w, respectively. The stability of the poly iodine chain formed within the axis of amylose helices depends on its length, i.e., indirectly on the DP of the amylose helices: the greater this stability, the lower the [If]v value. The amylose molecule consists of helical segments. Such a molecule may behave as a random coil. The average length of the helical segments in freshly prepared amylose-iodine complexes depends on temperature, pH, iodide concentration, the presence of other complex-forming agents, and the DP of the amylose. This latter factor is investigated in the present paper. By the aid of an automatically recording photometrictitrating device the coherent values of [Ib] and [If] were determined. Plotting these values against DPn for mechanochemically degraded as well as for periodateo-xidized amyloses resulted in curves consisting of two linear sections. The break of the curves occurred between DPn 110 and 130. It was concluded that below DPn = 100 the DP of helical segments (= sDPn) is identical to the DPn of the total molecule, i.e., the molecule consists of only a single, relatively stiff helix. Above this limit the molecule contains several helical segments. The DP of these helical segments can be calculated as follows: sDPn = 141.1 - 10.2 × 105[If]v. This equation is considered to be valid for 0.5-0.6 mg. amylose in 100 ml. 0.1N HCl at 20°C., λ = 650 mμ, euuvet diameter 3.4 cm., the feed rate of the iodate-iodide titrating solution (in acid medium resulting in a 5 × 10-3N I2 solution with a molar iodide to iodine ratio of 1.5) is 0.4ml./min. Amylose molecules of, e.g., DPn = 1380 consist of an average of 11.4 segments having a DP of about 120 and consisting of an average of 15-18 helical turns.

Notes: The electrochemical possibilities for investigation of nucleic acids with high molecular weight are restricted to the determination of the adsorption behavior. According to our experience the alternating current polarography (Breyer-polarography) is mainly appropriate for the characterization of changes in the secondary structure of DNA. The Breyer-polarogram shows the alternating current of the dropping electrode in dependence on their potential which varied from 0-2 v. negative against the normal calomel electrode (NCE). By addition of native DNA to the supporting electrolyte (buffer solution) the current drops down in the range of adsorption between 0 and 1 v. At 1.16 v. against NCE the desorption takes place together with the formation of a rounded desorption peak. The investigation was carried out in phosphate buffer solution 0.1m with 0.075m NaCl or in a phosphate buffer 0.18m with 0.03m NaCl. In the pH range above pH 8 NaOH was added to realize the higher pH values. A calf thymus DNA sample having a mean molecular weight of about 18 million was used. The concentration of DNA was 5 × 10-3-1 × 10-1 wt.-%. The polarographic measurements were performed with an a.c./d.c.-polarograph “GWP 564” from Akademiewerkstätten für Forschungsbedarf der Deutschen Akademie der Wissenschaften zu Berlin (DAW). The denaturation of the double helix causes a sharp desorption peak at negative potentials of the alternating current polarogram. This new criterion for the helix-coil transition is due to formation of unpaired bases. These nearly free bases undergo a specific adsorption and the desorption takes place within a narrow potential range. Nevertheless, at present time an electron transfer to particular bases cannot be excluded at special conditions. The increase of the sharp peak permits to estimate: (a) the melting curve of the double helix in agreement with spectroscopic measurements; (b) the photolysis of the double helix; (c) the strand separation in acid and alkaline solution. In the alkaline range the sharp peak increases and reaches its maximum at pH 〉 12. In the acid range, however, no sharp peak is observed and the rounded desorption peak decreases. Therefore, the best way of following the conformation changes is to measure the current difference between the curves of the solutions with and without DNA at electrocapillary-zero-potential. On the classical d.c.-polarogram one can measure small current steps only, which may be caused mainly by capacity changes. Moreover, the scission of the molecule by ultrasonic action can be followed. In this case the rounded peak of DNA increases but the sharp peak does not appear. Similar alternating current polarograms are obtained with poly-A in the native state, because helical and unordered regions coexist in the same molecule. The very rapid indication of these structure changes allows one to carry out kinetic measurements at a fixed potential with this method.

Notes: The theory of Kirkwood for the translational frictional coefficients of structures composed of subunits has been generalized in two ways in order to consider aggregates of nonidentical subunits. One of these generalizations fails when the sizes of subunits are too disparate; the other, derived from a surface shell distribution of frictional elements, is effective over the whole range of relative sizes. It is shown that, in the limit of a continuous surface distribution, a shell model reproduces Stoke's law for a sphere. Comparison is made between the frictional coefficients of spheres, ellipsoids, and rods modeled by finite numbers of subunits and by continuous shells of frictional elements, and those calculated from other theories. Agreement is generally good, though the shell model for prolate ellipsoids of revolution deviates by a few per cent from the Perrin value.

Notes: The reaction, V amylose hydrate ⇄ H2O + V amylose dehydrate, where the amylose hydrate and dehydrate are pseudohexagonal, helical species with packing diameters of 13.7 and 13.0 A., respectively, has been studied. The V amyloses were exposed to water vapor pressures at various temperatures, with phase determination obtained by identifying solid reactant and product from their x-ray patterns. Reversibility of reaction was found to occur over a 50-96°C. range. A Clausius-Clapeyron plot yields a ΔH of reaction of 10.4 kcal./mole of water released, which value is close to analogous heats of reaction of many common hydrate systems.

Notes: The thermal helix-coil transition of DNA was studied by means of dielectric constant measurements. The dielectric dispersion of native helical DNA is characterized by a large dielectric increment and a large relaxation time, whereas that of denatured coil DNA is characterized by a small dielectric increment and a small relaxation time. The dielectric dispersion of partially denatured DNA is of particular interest. At the intermediate stage of the helix-coil transition, dispersion curves which are different from either that of helix DNA or that of coil DNA appear. This is particularly pronounced for large DNA. This indicates the presence of an intermediate form of DNA. Flow birefringence measurements were carried out simultaneously. The negative birefringence of helical DNA diminishes as the helix-coil transition proceeds. However, the extinction angle remains constant, as long as it can be measured. These results indicate the absence of intermediate forms during the helix-coil transition. The discrepancy between dielectric and birefringence measurements can be resolved by assuming that the intermediate forms are not birefringent. The size distribution of native DNA and of the indicated intermediate form of DNA was studied. It is found that a logarithmic normal distribution function explains the distribution of size of DNA reasonably well.

Notes: Poly-γ-berizyl-L-glutamate prepared by polymerization of γ-benzyl-L-glutamate NCA in dimethylformamide (DMF) with the use of diisopropylamine as the initiator was precipitated from the polymerization mixture under different conditions. A portion of the almost completely polymerized solution was treated with an excess of isopropylamine and then precipitated into diethyl ether (sample A). The remaining portion of the polymerization mixture was concentrated in a rotating evaporator, stored at room temperature for a few days, and then diluted with DMF and precipitated into diethyl ether (sample B). The molecular weight distributions of the two polymer samples were determined by the chromatographic procedure of Baker and Williams. The molecular weight of sample B is roughly three times that of sample A. However both samples have the “most probable” distribution of molecular weight. The results are interpreted according to Bamford's polymerization mechanism.

Notes: The sedimentation coefficient of T2 phage DNA has been studied by zone centrifugation in sector-shaped preparative centrifuge tubes over a concentration range of 0.02-2.0 μg./ml. DNA. These results have been compared to a similar study in the analytical centrifuge of T4 DNA over the range of 0.50-5.75 μg./ml. DNA. A value for the sedimentation coefficient of 60.7 ± 1.8 S. was obtained by the first method and a value of 61.3 ± 1.5 S. by the second.

Notes: The method of matrix rank analysis has been applied to a large body of experimental data to determine and identify the minimum number of independent spectral components that, were contained within it. The optical rotatory dispersion of TMV RNA was measured in the wavelength region 230-350 mμ, for a wide range of temperatures and ionic strengths. Over the whole range of conditions considered, the observed optical rotatory dispersion spectra were found to be a superposition of two basic spectra only. Evidence is put forward relating these spectra to the single-strand and double-strand helical forms of the molecule. A model is proposed to explain the observed spectral changes in terms of an equilibrium between these two conformations, which leads to a direct calculation of the percentage composition of the double strand at any of the conditions considered. This equilibrium, while simple in general description, appears multi-state and complex in detail. This method of analysis is both powerful and of wide applicability, since it is independent of the source of the data.

Notes: An infinite series solution to the Mason-Weaver equation is presented for the case in which a synthetic boundary is formed originally between solution and solvent. Digital computations based on this series, and confirmed independently, have been made for a range of parameters. For given conditions, the maximum rate of change of concentration at the meniscus and the time at which it occurs can be easily estimated by means of the curves presented. In equilibrium experiments which commence with formation of a sharp boundary, this enables the fringes to be identified with certainty.

Notes: The course of the reversible helix formation of poly(γ-benzyl L-glutamate) (PBG) dissolved in a mixture of dichloroacetic acid (DCA) and 1,2-dichloroethane (EDC) was followed by measuring the heat capacity and the optical rotation of the system through the transition region. The results of these measurements indicate that the transition enthalpy ΔH the transition temperature Tc, and the Zimm-Bragg parameter σ depend considerably on the PBG concentration as well as on the composition of the solvent. For the standard state of infinite dilution, however, a linear extrapolation of the measured ΔH if values results in a standard value ΔH° = 950 cal./mole, independent of the solvent composition. The results of the calorimetric measurements are discussed in relationship to changes in optical rotation. Some peculiarities in the measured thermodynamic and optical properties in solutions with relatively high content of dichloroacetic acid are reported.

Notes: Helical conformations of infinite polymer chains may be described by the helical parameters, d and θ (the translation along the helix axis and the angle of rotation about the axis per repeat unit), pi (the distance of the ith atom from the axis), dij, and dij (the translation along the axis and the angle of rotation, respectively, on passing from the ith atom to the jth). A general method has been worked out for calculating all those helical parameters from the bond lengths, bond angles, and internal-rotation angles. The positions of the main chain and side chain atoms with respect to the axis may also be calculated. All the equations are applicable to any helical polymer chain and are readily programmed for electronic computers. A method is also presented for calculating the partial derivatives of helical parameters with respect to molecular parameters.

Notes: A dilatometric technique is employed to measure the volume changes occurring on denaturation of collagen solutions and tendons. Partial specific volumes of tropocollagen are calculated to be 0.686 ml./g. in water and 0.689 ml/g in 0.1M citrate buffer. From data on non-aqueous solutions, it is estimated that most of the volume change arises from changes in polymer configuration rather than from changes in solvation.

Notes: We have generated solutions to the Lamm equation to examine the effects of concentration dependence on velocity experiments. Two forms of c dependence are considered: s/s0 = 1 - kc and s/s0 = (1 + kc)-1. Features of these solutions are discussed. The magnitude of the errors resulting from the usual procedure of measuring the rate of movement of schlieren maxima or of the position at which the concentration is one half the plateau value have been examined. These errors are usually negligible after sufficient centrifugation time. The errors in using the half-plateau concentration are less than those using the movement of the peak. We have also examined a technique due to Fujita for determining D from boundary spreading when s/s0 = (1+kc)-1. This method is satisfactory when s/s0 is actually of this form, or under certain limitations when s/s0 = (1 + kc)-1. Creeth has shown that under certain conditions the concentration gradient, curve remains virtually unchanged in shape after separating from the meniscus. When this occurs it is possible to estimate s/D from the data. The condition for such a steady state is that kc0 be sufficiently large. Numerical confirmation of this method is presented in the final section.

Notes: Preliminary experiments have been carried out which measure the variation in the intensity of the light scattered by DNA solutions under the influence of an electric field. Data have been collected on the length of DNA molecules, their electric polarizability, and their dispersion behavior.

Notes: Information concerning the structures of rA-rU, rA-rU2 rI-rC, rA-rI2, and acid rA helices in solutions is reported. Through the use of diquaternary ammonium salts of the general structure, \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}_1 {\rm R}_2 {\rm R}_3 \mathop {\rm N}\limits^ + ({\rm CH}_2 )n\mathop {\rm N}\limits^ + {\rm R}_1 {\rm R}_2 {\rm R}_3 \cdot 2{\rm Br}^ - $\end{document} (I), it is shown that (1) the distances between adjacent negatively charged oxygen atoms on the helix increases in the following order rA-rI2 〈 rI-rC 〈 rA-rU ≤ rA-rU2; (2) the density of the helices increases in the order. rA-rI2 〈 rA-rU 〈 rA-rU2 〈 rI-rC; (3) there is a large hydrophobia site in rA-rI2 and possibly also in rA-rU, rA-rU2, and rI-rC helices; (4) the results of the interactions between the salts of type I and the helices may be formulated in semi-quantitative terms by the use of two parameters, α, and β which are shown to be related to the charge separation and the density of the helices, respectively; (5) the studies in solutions compare favorably with the x-ray studies on the fibers; and (6) the acid rA helix differs significantly from the other helices by the fact that the electrostatic interstrand interactions between the negatively charged oxygen atom of a phosphate group and the positively charged 10-amino group of adenine contribute significantly to the stabilization of the helix, and thus it is found that the presence of the salts, I, leads to a significant destabilization of the acid rA helix.

Notes: The half molecules of 186 DNA have been isolated by the Hg(II)-Cs2SO4 density gradient centrifugal ion technique. The buoyant densities of the two halves in CsCI at 25°C. are 1.713 and 1.709 g./cm.3, corresponding to GC contents of 54% and 50%, respectively. Similarly, 5-bromouracil labeled λ DNA halves were separated. The isolation of the four DNA halves made it possible to test for homology in base sequences between the cohesive ends of λ and those of 186. There was no indication of any significant homology in base sequences between the cohesive ends of the two DNA's, as indicated by the absence of a band with intermediate buoyant density in CsCI when either half of 186 DNA was annealed with either half of 5-bromouracil labeled λ DNA and then centrifuged. The lack of cohesion between the two DNA's made it possible to demonstrate unequivocally the formation of interlocked rings (catenanes) between the two DNA's. The existence of a dimeric catenane is evidenced by the formation of a species of intermediate buoyant density when 5-bromouracil labeled λ DNA is cyclized in the presence of cyclic 186 DNA of a relatively high concentration. The molecular weight of one DNA relative to the other can be calculated from the position of the dimeric catenane in a density gradient by using the method of Baldwin. The result was in complete agreement with our previous measurements from the sedimentation coefficients and by electron microscopy. The probability of dimeric catenane formation when one DNA is cyclized in the presence of another DNA is discussed. The experimental results agree with the theoretical expectation.

Notes: Based on the four electrodes technique, an apparatus is described which measures the Very Low Frequency (VLF) conductivity of ionic solutions, all electrode effects being completely eliminated. It is thus possible to measure the conductivity frequency dependence between 0.8 and 500 cps, with a relative error of 10-4. Applying this method to DNA solutions, one always finds a conductivity dispersion in the VLF range, which disappears when the biopolymer is heat-denatured. The relaxation time is different from one solution to another, but is always greater than 10 msee. approximately, sometimes even greater than 0.1 sec., the upper limit which one can estimate with our apparatus. The different, explanations of the DNA very low frequency polarization, assuming that its relaxation is connected with the rotational diffusion of the biopolymer long axis, is discussed.

Notes: Studies of the properties of copolymers of N-carboxy sarcosine anhydride with other N-carboxy α-amino acid anhydrides have shown that the secondary structure may be a function of the type of initiator used. In particular, when polysarcosine is the initiator, and the “chain effect” becomes possible, blocklike character appears in the copolymer. This is the result of selective and rapid polymerization of N-unsubstituted N-carboxy α-amino acid anhydrides by the chain-effect mechanism. In suitable circumstances, therefore, the latter may be used to induce order into copolymerizations of this type.

Notes: Sterically allowed forms of the poly-N-methyl-L-alanine chain were found by calculation of conformational energies as a function of the rotation angles of its chain bonds. The lowest energy form seems to be a right-handed, approximately threefold helix.

Notes: Oriented amylose fibers in the V form were prepared and subjected to x-ray analysis. Unit cells and the probable space group of P212121 were determined for the V anhydrous and V hydrate forms of amylose; the analysis confirms previous predictions of these structures based on x-ray powder patterns. Chain folding in V amyloses is discussed in view of crystallographic evidence and folding experiments conducted with space-filling models. Reported also is evidence for amylose helices having diameters intermediate between 13.0 and 13.7 A.

Notes: Conformational energies of the poly-N-methyl-L-alanine chain have been calculated as a function of the rotational angles of its skeletal bonds, taking into account torsional potentials, van der Waals repulsions, and London attractions. Four pronounced minima in the energy were found; the relative magnitudes of the energies at these minima suggest that the preferred conformation of this chain is either a right-handed, approximately threefold helix, or a slightly distorted, left-handed α-helix. Inclusion of an estimate of the dipolar contribution to the total conformational energy does not significantly affect this conclusion.

Notes: The equilibrium normality [If] of free iodine in amylose-iodine complex formation is a function of the length of the polyiodine chains. This length depends on the DP of helical segments of amylose (sDPn). Values of [If] and of the concentration of the bound iodine [Ib] were determined by the continuous photometric titration with automatic recording. Plots of [Ib] versus [If] give an integral distribution curve. Since the relation between [If] and sDPn is known, the graphic differentiation of the [Ib] versus [lf] curve furnishes the differential distribution curve, representing the mass distribution of the helical segments according to their DP. The peak of this curve is characteristic of the percentage and DP of those helical segments, which occur in the largest amount. On the basis of the differential distribution curve the polymolecularity of the investigated sample may be judged. The titration of amylose samples degraded by various methods gives different distribution curves. Titrating mixtures of samples with widely differing average DP values results in differential curves having more than one maximum.

Notes: The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (∼3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.

Notes: The degree of helicity θ of a series of homologous polypeptides as a function of solvent composition was investigated. The polypeptides studied were: poly-N5-(3-hydroxypropyl)-L-glutamine (PHPG) as well as the corresponding 2-hydroxyethyl and 4-hydoxybutyl derivatives (PHKG and PHBG, respectively). PHPG, which is nonhelical in formic acid, attains helicity on addition of relatively small amounts of formates, formamide, and urea to its solution in formic acid. This demonstrates that the high acidity of pure formic acid is largely responsible for its helix-breaking power-probably through protonation of the peptide bonds. In formic acid-water mixtures all three polymers show a maximum in degree of helicity at a mole fraction of about 0.3 formic acid. This is interpreted as being due to interaction between the two helix-breaking solvents, which results in the formation of an inactive molecular species. It is shown that solvent-induced transitions with helicity maxima are predicted by the Bixon-Lifson treatment when applied to this system.

Notes: In aqueous solutions of the amylase-iodine complex the concentration of free iodine [If]v after reaching equilibrium (or closely approximating it) is determined by the following factors: temperature, pH, concentration of iodide ions and amylose, and DP of amylose. In the present paper the role of temperature, amylose concentration, and DP has been investigated. At half-saturation of amylose by iodine, the reciprocal value of free iodine defines the equilibrium constant: 1/[If]v = K. The relation between [If]v, in normality and temperature is the following: 5 + log [If]v = -(2.132/T) + 8.52, for DPn = 1290, 0.4 mg. amylose in 100 ml. 0.1N HCl. The value of the energy of activation Ea between 2 and 52°C. is 9.72 kcal./mole. The influence of amylose concentration [Am] on photometrically determined [If]v, at 20°C, in the range of 0.1-1.2 mg./100 ml. 0.1 N HCl for DPn = 1290 is: 5 + log [If]v = 0.209 - 0.047 log [Am]. At [Am] = 0.6 mg. amylose/ 100 ml. 0.1 N HCl and 20°C, the value of [If]v depends on DPn as follows: 5 + log [If]v = 0.085 = + 0.222 log (104/DPn). These above equations are summarized by the relation: [If]v = exp {16.865 - (Ea/RT)}[Am]0.047(104/DPn)0.222 ×10-5 Considering that the determination of [If]v by automatic photometric titration can be performed quickly and with appropriate reproducibility, this method is convenient for a rapid empirical and approximate determination of DP of amylose on a microscale. The iodine-binding capacity [IBC] as well as the value of λmax, have been also investigated as functions of DPn, by photometric and by amperometric titration.

Notes: Dielectric properties of human and horse hemoglobin were studied at frequencies ranging from 20 kc./sec. to 7 Mc./sec. The relative errors in the measurements were usually less than 10-3 even for mildly conducting solutions (10-3M KCl). The experimental setup allowed us variation and measurement of the degree of oxygenation of the protein and to determine its dielectric parameters. Our main conclusion is that it was not possible to find any variation of the dielectric increment for hemoglobin oxygenation levels of 25, 50, 75, and 100%, approximately. This result is at variance with some previous reports. We cannot give the reason for this discrepancy but discuss some possible explanations. The specific dielectric increment, Δεs/c, of human hemoglobin was shown to be significantly smaller than that of horse hemoglobin (0.28 against 0.32). This physical property is lowered with increasing ionic strength I: Δεs/c = 0.28 and 0.20 for I = 10-4 and 10-3, respectively (human protein).

Notes: The transient electric birefringence behavior of bacteriophage T2 and the T2 ghost or protein coal was studied. The field free relaxation measurements show both the intact virus and its ghost to have two rotary diffusion coefficients. These coefficients have values of 555 ± 54 and 111 ± 22 sec.-1 for the intact virus and 688 ± 89 and 161 ± 29 sec.-l for the ghost. The equivalent ellipsoids for the fast and slow relaxation coefficients were obtained by use of Perrin's equation and were related to the bacteriophage structure in terms of a possible extension of the tail fibers or an enlargement of the head structure. The saturation of the specific birefringence of the phage and the ghost when compared with the specific birefringence of the free nucleic acid gave an average optical orientation of 10 to 18% of the nucleic acid parallel to the main axis of the phage. The analysis of the birefringence versus applied field strength in the Kerr region gave the following values for the anisotropy of the polarixability. αe,33 - αe,11 and intrinsic dipole, μ, of both phage and ghost : for T2 phage αe,33 - αe,11 = 5.0 × 10-14 cm.3 and μ = 64,400 Debyes; for T2 ghost αe,33 - αe,11 = 7.9 × 10-14cm.3 and μ = 57,200 Debyes. The high intrinsic dipole for phage and ghost is interpreted as to be associated with the mechanisms of the virus for attachment, to the host cell wall.

Notes: The hydration of several polyacids has been investigated with special attention to the effects of neighboring charged groups and hydrophobic groups on the hydration regions. The molar volume change due to the structural change of water by the hydration was obtained by the method of refractivity measurement. The polyacids employed were poly(acrylic acid), poly(methacrylic acid), poly(L-glutamic acid), and a copolymer of maleic acid and vinyl acetate. The measurement of the refractive index was performed for the solutions of these polymers neutralized to varying degrees by tetrabutyl-ammonium hydroxide and sodium hydroxide. The results confirm the characteristics of the previous model of the hydration of polyelectrolytes, that is, the cooperative action of neighboring charged groups induce the second hydration region in addition to the intrinsic hydration region. The stiff structure of water in these regions restricts the mobility of counterions forced to enter into these regions by the strong electrostatic potential of polyions. The results indicate also that hydrophobic groups induce an additional hydration region around their neighboring charged groups. Small but negative volume changes were observed for conformational changes of poly(L-glutamic acid) and poly(methacrylic acid) induced by the neutralization of carboxyl groups.

Notes: The heat of solution of a series of three polyglutamates as a function of solvent composition was measured. The abrupt increase in heat of solution at the solvent composition of the helix-coil transition (as evidenced by optical rotation data) allows the estimation of the transition enthalpy change. The difference of side chain in the three polyglutamates has no appreciable effect on the transition enthalpy, although it affects the helix stability, as judged from the solvent composition at the transition points. These facts are discussed on the basis of existing models of the transition.

Notes: Single crystals of poly(γ-benzyl L-glutamate) were formed by epitaxial crystallization from solution in mesitylene on NaCl, KI, and KCl (001) cleavage faces. From electron microscopy and diffraction studies, the structure of these overgrowths was determined to be that of lamellae containing chain-folded α-helical macromolecules. The usual type of crystal perfection, that of ordered helix axes and disordered side groups, was exhibited by this synthetic polypeptide. Unique orientation regimes were observed with each substrate.

Notes: As a model for a variety of reaction processes on long chain molecules, for example, helix formation, a kinetic theory on a linear lattice is presented. Each reaction site can undergo reversible transitions between two states (0 and 1) with rates depending on the slate of its nearest neighbors. The system of coupled rate equations for the frequencies of specified runs of 0's and 1's is infinite for an infinite chain. In contrast to the case of irreversible processes, the system cannot, be written down by inspection. A procedure for the systematic derivation of the rate equations is developed which can be programmed on a computer. Explicit expressions for runs up to length four, involving runs up to length five are obtained without recourse to the computer. For the solution of the rate equations a closure must necessarily be imposed, and a possible procedure is pointed out. Furthermore the equilibrium relations following from the model are considered. The well-known equilibrium results for nearest-neighbor interactions represent a special case of these equations.

Notes: Measurements of specific heat of wool-water systems were made at approximately 5°C intervals over the temperature range -70 to 100°C. Ten different, samples were used, each with a different amount of absorbed water in the range from dry ness to saturation at 0°C. The graph of specific heat against temperature for dry wool is precisely linear over the complete temperature range, suggesting that thermal motion is entirely vibrational. When absorbed water is present the data can be conveniently discussed in terms of behavior below and above an amount of absorbed water of 22.7 g in 100 g of wool (22.7% of absorbed water). Below 22.7% there is only one temperature range in which the results indicate an appreciable transition in heat absorbing properties. The temperature of transition depends on water content but is higher than 0°C. Above 22.7% a second transition appears in the range -30 to 0°C and grows rapidly larger with increase of water content. The first transition is tentatively ascribed to a slightly cooperative breakdown of polar bonds in wool, and the second to a process analogous to melting in the absorbed water. The results are discussed in these terms as well as with reference to specific heat theories, the heat absorption of the wool component and the water component, and enthalpy differences between the various samples.

Notes: Dielectric relaxation of poly(γ-benzyl L-glutamate) in solution has been studied in the 5 kcps-10 Mcps range for various values of the helix content. The results give first experimental evidence for three effects of major significance. (1) The system exhibits dielectric relaxation due to a chemical rate process (namely helix formation). This confirms recent theoretical predictions. (2) The mean relaxation time τ* of the helix-coil transition could be evaluated as a function of the degree of transition. The results are in excellent agreement with a previously developed theory. At the midpoint of transition it is found τ*max = 5 × 10-7 sec. The elementary process of helical growth turns out to be practically diffusion-controlled (with a rate constant of hydrogen bond formation of 1.3 × 1010 sec-1). (3) There is a considerable electric field effect of the helix-coil transition. This indicates that conformation changes in biological systems could be potentially caused by direct action of an electric field.

Notes: The interaction of unsubstituted purine with polyuridylic acid in D2O solution at neutral pD has been studied by high resolution proton magnetic resonance spectroscopy. The poly U proton resonances were shifted to higher fields by the added purine, indicating that purine binds to the uracil bases of the polymer by base stacking. Severe broadening of the purine proton resonances was also observed, providing strong evidence for the intercalation of purine between adjacent uracil bases of the polymer. The line widths of the poly U proton resonances were not noticeably broadened in the presence of purine; thus, the binding of purine to poly U does not result in a more rigid or ordered structure for the polymer.

Notes: Polyelectrolyte expansion effects on high molecular weight bacteriophage DNA have been studied by examining the influence of simple salt concentration upon the intrinsic viscosity, [η]. The viscosity-molecular weight exponent a in the expression [η] = KMa diminishes from 0.8 in 0.005M simple salt to a limiting value of 0.6 for salt concentrations greater than 0.6M at 25°C. The ε parameter of the N1+ε hydrodynamic representation thus varies from approximately 0.2-0.07 over this range of salt concentration. The intrinsic, viscosity of DNA decreases slightly with increasing temperature at low and moderate salt concentrations but becomes independent of temperature at high salt concentrations. The expansion of the DNA molecular domain is linear in the reciprocal square root of the simple salt concentration. Viscosity differences among DNA's isolated from several bacteriophage T5 mutants reflect small differences in molecular weight which are in agreement, with sine determination by other techniques. The DNA's isolated from various rII mutants of T4 bacteriophage including some very large deletion mutations were found to be identically the same size in accord with current genetic ideas. Details of the representation and extrapolation of viscosity data are discussed and the sensitivity of the technique is evaluated.

Notes: Infrared spectra in the region 4000-60 cm-1 have been measured for acetylglycine N-methylamide and its deuterium homologs, CD3CONHCH2CONHCH3, CH3-CONHCD2CONHCH3, CH2CONHCH2CONHCD3, and CH3CONDCH2CONDCH3. Normal frequencies have also been calculated for these molecules with various conformations. The spectra show that this compound has two crystalline modifications, form A and form B. The frequencies and their isotope shifts show that the molecular conformation (Ψ, φ) of form B is near (0, 120) and that of form A near (180, 120). The short-range factors determining the conformation of peptide backbone having glycine residues are discussed.

Notes: The objective have been to establish if those ions which are known to change the stability of the structure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L-lysine (PLL) in the presence of KSCN, KCl, and KF, and for poly(L-glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCl. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pK0), slope of pKapp versus degree of ionization (α) curves, the degree of ionization at which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (δG°hel). The effects of nonspecific salts (KCl and LiCl for PLL and KSCN and KCl for PLGA) are small, and about, as expected from general electrostatic considerations. In line with the observations made with isoelectric and cat ionic collagen, specific, effects were noted with KSCN-PLL and with LiCl-PLGA. In the presence of KSCN, the poly-L-lysine helix becomes stabilized at much lower degree of ionization than in the presence of KCl, and the slope of the pKapp versus α plots is greatly reduced. However, ΔG°hel (for the uncharged molecule) is not affected, and pK0 is only slightly higher. We interpret these data in terms of binding of SCN- primarily to the side-chain amino groups (both to R—NH3+ and to R—NH2) solutions. (L-glutamic acid) in LiCl solution has its transition at the same α value as in KCl solution. However, both the slopes of the pKapp versus α plots and the absolute values of ΔG°hel are lower than in KCl solution. We interpret these results in terms of binding of Li+ to side chains as well as to the peptide bond.

Notes: Density increments (∂ρ/∂c2)°μ in solutions of NaDNA in NaCl and CsDNA in CsCl were determined over a wide range of salt concentrations; calf thymus DNA, fragmented by sonic irradiation to a molecular weight of 4-6 × 105 was used. The partial specific volume v̄2° of NaDNA at 25°C was found to ho 0.500 ml/g in water, and that of CsDNA 0.440 ml/g. Both values increase with increasing NaCl and CsCl concentration. Refractive index increments under various experimental conditions were also determined. The relevance of the density increments (at constant, chemical potential of diffusible solutes) to equilibrium sedimentation in a density gradient and the evaluation of molecular weights is discussed. Distribution coefficients of diffusible components, sometimes referred to as preferential solvation or net hydration, were derived from the density increments and partial volumes and compared with direct experimental results, whenever available, from membrane distribution and isopiestic distillation. The thermo-dynamic significance of the distribution coefficients as well as possible interpretations in terms of specific molecular mechanisms are considered.

Notes: A model for hemoglobin is proposed and its application to allosteric enzymes is discussed with particular reference to asparate transcarbamylase. The main assumptions made are that the molecule is composed of subunits and that occupation of a sub-unit produces a conformational change which affects the occupational probability of neighboring subunits. The results compare favorably with experiment and a number of specific predictions are made for aspartate transcarbamylase.

Notes: Complexes of amylose with n-butanol were prepared both as crystalline precipitates and as oriented fibers. These complexes were subjected to x-ray analysis, their unit cells were calculated, and the space group of P212121 was confirmed. n-Butanol complexes exist in both hydrated and anhydrous forms. There is no evidence for methanol, ethanol, or n-propanol structures similar to those shown by the n-butanol complex. The Complexes are unstable in the open air and revert to V-amylose hydrate on standing.

Notes: Collagen fibers were contracted “chemically,” i.e., by transferring them from water into KCNS solutions either isometrically or isotonically. Both changes in force and fiber length and in salt and water contents were measured as functions of time. The mechanical changes were found to follow the diffusional processes. The diffusion of water exhibited a plasmolytie effect. The role of water in the melting process is discussed.

Notes: The correlation between mechanical and chemical Processes in the contractile system collagen fibers-aqueous KCNS sulutions was investigated. Melting and contraction of the fibers were induced by applying a force sufficiently high as to prevent melting in a KCNS solution and then decreasing it either suddenly, or continuously at a constant rate. The kinetics of both processes are characterized by an initial rapid elastic response of the crystalline collagen, followed by a stationary region. The force-velocity relationship in this region was found to be the same under different types of mechanical deformations. It is probable that under the prevailing conditions, the behavior in the stationary state is determined by the melting process and is not markedly influenced by diffusional changes. Part of the experimental data could be explained by assuming a linear, rigid model or, better, by taking into account the highly elastic properties of the amorphous collagen. The kinetic, unit seems to be composed of several hundred amino acid residues.

Notes: The dynamic elastic behavior of collagen fibers treated by LiBr solutions was studied by the method of free longitudinal vibrations. The frequency response functions and the stress-strain relationship were evaluated for fibers denatured to different extents by various concentrations of the salt solution. The James and Guth model for rubber elasticity was applied to the experimental data. The elastometric parameter β, which is a measure of the degree of folding of the macromolecular chains, was found to decrease on increasing the salt concentration. It might thus serve as a characteristic of the degree of denaturation of fibrillar proteins.

Notes: Sodium hyaluronate (NaHy) and sodium carboxymethyl cellulose (NaCMC) behave similarly with respect to concentration.N 3 of an added 1 : 1 electrolyte. The second virial coefficient A2 (light scattering) is identical within experimental error at a given.N 3. The limiting viscosity number [η] also varies with N3-1/2in similar fashion for samples of similar [η] of the two polymers. Differences in Na+ activity in salt-free solutions are interpreted on the basis of weaker Na+ binding in NaHy, presumably due to the greater charge separation along its chain backbone. Added electrolyte is excluded in dialysis more strongly by NaHy (or its acid form) than by NaCMC. The Flory parameter Φ is smaller in good solvents for NaHy, as for many other polyelectrolytes, than for nonionic polymers.

Notes: In order to test theoretical predictions that poly-L-valine can exist in an α-helical conformation, water-soluble block copolymers of L-valine and D, L-lysine were prepared. By carrying out the synthesis on a resin support (with the use of N-carboxyanhydrides) contamination of the individual blocks by any unreacted monomer from the previous block was avoided. A single glycine residue was incorporated at the C-terminus of the chain for use in amino acid analyses. Using optical rotatory dispersion and circular dichroism criteria, about 50% of the short valine block of (D, L-lysine HCl)18-(L-valine)15-(D, L-lysine-HCl)16-glycine was found to be in the right-handed α-helical conformation in 98% aqueous methanol, in water, the polymer appears to be a dimer, with the valine block being involved in the formation of an intermolecular β-structure.

Notes: Calculations of the dependence of the conformational energy and the rotational strength of the amide n-π* electronic transition (in a series of α-helical polyhel-α- amino acids with different side chains) on conformation have been carried out. The conformational energies were computed by procedures developed in this laboratory; the computation of rotational strengths was carried out by the method of Schellman and Oriel, with a slight modification. Polyamino acids with both nonpolar and polar side chains were considered; in the latter case, it was assumed that the only influence of the polar side chain was on the backbone conformation and on the electrostatic field which perturbs the amide chromophore of the backbone. Only conformations in the range of backbone dihedral angles of the right- and left-handed a-helices were considered, and the assumption of regularity (i.e., uniformity of dihedral angles in every residue) was made. The rotational strength per residue was found to vary markedly with chain length (in oligomers of up to 40 residues long); both the conformational energy per residue and the rotational strength per residue were found to vary significantly with the backbone conformation, which in turn depends on the nature of the side chain. The geometry of the hydrogen bond in the α-helical backbone is the most important factor which influences the dependence of the rotational strength on conformation. The implications of these results, for the interpretation of experimental circular dichroism and optical rotatory dispersion data, are discussed.

Notes: Heat-denatured DNA from HeLa cells interacts with natural as well as synthetic polysaccharides. Glucose does not inhibit the interaction nor will it produce it. Polysaccharides with a molecular weight of 10000 or greater are required before the interaction takes place.

Notes: A series of copolymers of β-p-nitrobenzyl L-aspartate with β-benzyl L-aspartate and with β-mcthyl L-aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.

Notes: The dichroic ratios of T5st-O and T2H bacteriophage DNA molecules were measured throughout the ultraviolet region of the spectrum. Two methods of DNA orientation were studied: (1) orientation in solution in a Shimadzu flow dichroism instrument attached to a Beckman DU spectrophotometer, and (2) alcohol precipitation of the DNA from solution and orientation in a thin film on the quartz face of a humidity chamber. Spectra in the latter case were recorded using a Gary Model 14 spectrophotomcter fitted with Glan prisms. The lower wavelength limit was 215 mμ in both systems. The DNA preparations were carefully characterized as to spectral purity, sedimentation coefficient, hyperchromicity, protein content, and DNA content. In addition, the structure of the DNA oriented in films was inferred from x-ray diffraction patterns of fibers of the precipitated DXA. The A and B configurations of DNA in films could not be distinguished by the dichroic ratio measuiements. The dichroic ratio obtained for the film-oriented DNA at high relative humidity shows the same wavelength dependence as for the flow-oriented DNA. The same wavelength dependence for DNA in the fibrous state and in solution, when considered together with the x-ray diffract ion results, indicates that DNA in solution maintains an orientation of bases which is similar to that in fibers. I1Or both solutions and films of DNA, the dichroic ratio is constant from 290mμ to 240 mμ and increases at wavelengths below 240 mμ. The increased parallel absorption below 240 mμ is consistent with the existence of an n→π* transition. The inherent molecular dichroic ratio is found to be the same for T5st-O DNA and T2H DNA in solution, and is a maximum of 0.09 ± 0.02 at 260 mμ.

Notes: This paper gives a very simple method based on the characteristic property of the electric free energy to calculate the repulsive force between parallel rodlike macroions in a solution as a function of the charge density on rods. The total extensive force (∂f/∂X) of an assembly of m rods of length l and charge number n (charge density - neo /l) at small extension X in the absence of low molecular sals is given by where z is the valency of counterions and Q (= neO2/εkTl) is a dimensionless quantity representing the charge density. The repulsion between two parallel rods is given by putting m = 2. At large charge densities the repulsion is very much smaller than the direct coulomb force between charged rods, even at small distances. The addition of low molecular salts does not depress the repulsion appreciably, as long as the average concentration of salt ions is much smaller than the concentration of counterions accumulated in the space between rods. The effect of fluctuation of the coumerion distribution is also analyzed, and it is found that the attractive force due to the ion fluctuation may predominate over the above repulsive force in the case of polyvalent counterions and rods of high charge densities at small distances.

Notes: An expression is derived for the melting point of a polymer when in equilibrium with a solution in which binding of low molecular weight compounds to the polymer takes place. Allowance is made for the possibility that the crystalline polymer itself is a complex. The argument is a purely thermodynamic one and is based on a consideration of the change in free energy as a result of a change in binding. Allowance is made also for non-specific polymer-solvent interactions, in which the mixture of low molecular weight solvents is treated as a single solvent. Special attention is paid to “inverted” melting transitions, i.e., cases in which the melting point increases with increasing dilution of the polymer. It is shown that as a rule this is accompanied by a corresponding, inverted effect of the solvent composition on the melting point. It is further shown that-in the absence of binding, “normal” behavior at the critical point (i.e., phase separation is induced by lowering the temperature) is always accompanied by “normal” melting behavior (i.e., a decrease in melting point when the polymer is diluted). Also, “inverted” melting always implies that phase separation at the critical point is induced by heating, but the reverse is not necessarily true.

Notes: An increase in temperature causes an increase in the amplitudes of intramolecular and intermolecular motions and mainfests itself as an increase in the effective radii of the various atoms, especially hydrogen atoms. If one uses a Lennard-Jones 6-12 potential function to account for nonbonded interactions, this increase in the radius of the hydrogen atom is equivalent to a modification of the coefficient of the repulsive part of the Lennard-Jones potential. Accordingly, this effect of increasing temperature on the helix sense of several polyamino acids is computed by allowing the effective radius of the hydrogen atom to increase. It is found that the preferred sense of several polyamino acid helices changes from right- to left-handed as the temperature is increased. This accounts for recent experimental observations of changes in helix sense with temperature.

Notes: Potentiometric titrations and some complementary optical rotation data are presented for solutions of poly(L- glutamic acid) (PGA) in several H2O-ethanol mixtures. The data allow the determination of the intrinsic pK (pK0), slope of the apparent. pK (pKapp), versus degree of ionization curves and of the enthalpy of ionization as a function of ethanol concentration. The variation of the degree of ionization at which the helix-coil transformation occurs with ethanol and temperature is also determined. Finally free energy, enthalpy, and intropy changes associated with the helix-coil transformation for the uncharged conformers are determined from the titration curves. The effect of the ethanol is to increase the stability of the helical conformation of PGA for both the charged and the uncharged forms of the polymer. The stabilization of the uncharged helix is essentially an entropic effect.

Notes: Infrared spectra of polypeptides were measured in the region of 1800-400 cm-1. For the α-helical form, disordered form, and antiparallel-chain β-form, amide V band- arising from N-H out-of-plane bending models were observed at 610-620, around 650, and 700-705 cm-1, respectively, and amide V′ bands arising from N-D out-of-plane bending modes were observed at 455-465, around 510, and a 515-530 cm-1, respectively. These correlations are useful for conformation diagnoses, particularly for copolyamino-acids or proteins which are not oriented. The nature of low-frequency amide bands are discussed with reference to potential energy distributions calculated for the α-helical form and β form.