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: 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: 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 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: 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: 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: 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: The molecular weight distribution of poly-γ-benzyl-L-glutamate prepared by N-carboxy anhydride (NCA) polymerization with the use of di-n-butylamine, diisopropylamine, and sodium methoxide as initiators was investigated. In every case, narrow distributions were found. Moreover the results allow us to conclude that the same polymerization mechanism is operative with each of the above initiators. The experimental distribution curves show fairly good agreement with the theoretical distributions expected for a polymerization process without termination.

Notes: The influence of counterion radius on the polyelectrolyte behavior of poly-L-cysteate in aqueous solution under different conditions was investigated. A linear relationship was found between a critical concentration in solution of the synthetic charged poly-peptide and the crystallographic radius of different counterions, indicating that the stability of this macromolecule is inversely proportional to the size of the cations.

Notes: X-ray diffraction patterns from dilute solutions of poly-3,5-dibromo-L-tyrosine in dimethylformamide and equivalent concentrations of 2,6-dibromophenol in the same solvent are compared. The intensity difference as a function of scattering angle is indicative of the arrangement of residues in the polymer. A novel averaging procedure is used to compute the intensity patterns arising from regular arrays of bromine atoms. Interbromine spacings measured on a Drieding model of a right-handed α-helix with side groups restricted to rigid positions of maximum stagger give moderate agreement with experiment. Very good agreement with observations is obtained by allowing for a small oscillation of phenyl rings which effectively cancels out the contribution of large distances to the intensity curve.

Notes: Aqueous solutions of ribonuclease and human serum albumin were subjected to periods of controlled heating and the nuclear magnetic spin-lattice relaxation times, T1, of the water protons measured. The heat treatment causes an initial increase in the relaxation times of the water protons. These T1 variations indicate that a configurational change, compatible with a disordering of the protein structure occurs, which involves an increase in various modes of internal mobility. The T1 measurements also indicate a close, ordered association of the water molecules and the protein.

Notes: The melting characteristics of unfractionated yeast s-RNA in neutral aqueous solution have been examined by high-resolution proton magnetic resonance spectroscopy. These studies indicate that the bases of s-RNA become rotationally activated at lower temperatures than their associated ribose units. These observations, as well as the recently determined base sequence of an alanine-specific s-RNA, suggest that base complementarity may not be the primary determinant in the stabilization of the secondary structure of s-RNA. In addition, evidence for exchange of C8-protons of purine bases of s-RNA with D2O was obtained at temperatures above 70°C.

Notes: 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.

Notes: Poly-N5-(3-hydroxypropyl)-L-glutamine (PHPG) samples of molecular weights from 20,000 to 140,000 were synthesized by the action of 3-amino-1-propanol on poly-γ-benzyl-L-glutamate. PHPG is freely soluble in water and in a variety of organic solvents and was shown to be devoid of functional groups ionizing in the pH range 2-12. From hydrodynamic data (viscosity, sedimentation, diffusion) and optical rotatory measurements (ORD in the range 289-650 mμ, circular dichroism and Cotton effects in the range 210-280 mμ) on samples of different molecular weight it was concluded that PHPG is largely helical in methanol, ethylene glycol, formamide, dimethylform-amide, n-butylamine, and acetic acid, and randomly coiled in dichloroacetic acid and formic acid. In water the polymer is partly helical, the degree of helicity increasing with decreasing temperature and increasing molecular weight. The following solutes were found to decrease the helicity in the order indicated: NaOH 〉 guanidinium chloride 〉 urea 〉 HCl 〉 LiBr 〉 NaBr 〉 NaCl 〉 H2O. Detergents do not destroy the helical conformation. Helix-coil transition curves were obtained for the solvent pairs: methanol-water, acetic acid-water, and chloroform-dichloroacetic acid. Thermal transitions were observed in water, water-methanol (70:30 v/v), and dichloroacetic acid-ethylene dichloride (22:78 v/v), the latter being of the “inverted” type. Heats of helix formation were calculated and their significance is discussed.

Notes: Polyadenylic acid is degraded into mononucleotides and low molecular weight oligonucleotides in a 20 hr. period at 64°C. by the action of Mn (II), Co (II), Ni (II), and Cu (II) ions, and in a 2 hr. period by Zn (II) ions. The latter also degrade poly C, poly U, and RNA at approximately the same rate, but poly I is degraded very much more slowly. No such difference in reaction rates can be observed in the alkaline degradation of polynucleotides. The sluggishness of the reaction with poly I is not due to any highly ordered structure of the zinc-poly I complex, but probably to the weakening of the zinc-phosphate bond through simultaneous binding of the zinc to the inosine base. Evidence for such a hypothesis is derived from the inhibition of the zinc degradation of poly A by inosinic acid or poly I. No drastic difference in the nucleotide composition of the undegraded residue can be observed in the degradation of RNA by zinc and by alkali.

Notes: Evidence is presented that polyriboadenylic acid (Poly A) is a single-strand flexible helix of stacked bases at neutral pH and room temperature. As the temperature is raised the stacking decreases, but there is still perceptible order at 80°C. The thermal transition from the double-strand helix (which exists at low pH) to the single-strand helix has been followed by optical rotatory dispersion, optical absorption, and viscosity measurements.

Notes: The binding curves of various aminoacridines on calf thymus DNA have been determined by a spectrophotometric method. The dominant role of electrostatic forces in the interaction has been confirmed by the effect of ionic strength. Side chain and ring substituents in the aminoacridines do not inhibit binding, since this decreases in the order: acranil 〉 neomonacrin 〉 atebrin 〉 9-aminoacridine ∼ proflavine 〉 9-amino-1,2,3,4-tetrahydroacridine (THA). The last named shows the effect of diminishing the flat area of the rings in the aminoacridines. The shape of the binding curves of proflavine, 9-aminoacridine, and THA on thermally denatured DNA show that about 30-50% more binding sites are available to these cations on denatured DNA than on native DNA, but that the binding constants are the same. These observations are discussed in relation to the intercalation and other models for the interaction. It is concluded that exact and complete intercalation is not a necessary condition of strong binding and that other, less regular, models, in which the positive ring nitrogens of the acridines are close to the DNA phosphates and the acridine rings partially interact with the DNA base rings, are probably more consistent with the effect of denaturation of DNA on the binding curves which are characteristic of the interaction in solution.

Notes: 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.

Notes: A mathematical method has been developed, together with a digital computer program for its evaluation, for the determination of possible conformations of a polypeptide chain for any given amino acid sequence. Allowed conformations were required to satisfy predetermined criteria regarding the locations of potential energy minima for rotation around single bonds, steric restrictions arising from van der Waals contacts between atoms, and the formation of a closed polypeptide loop by means of a peptide linkage or a disulfide bridge. Requirements for specific noncovalent interactions can also be considered. Various assumptions regarding the selection of the minima for rotational potentials and of the van der Waals contact distances are discussed. According to the assumptions adopted in the paper, 21 conformations are allowed for a glycine residue which is part of a dipeptide. These conformations are assumed to lie at minima of the rotational potential function, and satisfy simultaneously the steric requirements. Dipeptides containing alanine or other residues are restricted to 7 such conformations. As an example of the applicability of the method, the computation was performed for the smallest loop containing a disulfide bridge in the ribonuclease molecule, i.e., the cyclic octapeptide formed by residues 65 to 72. Only 15 allowed conformations were found for the loop as a whole. Parameters characterizing these allowed conformations are tabulated. Approximate calculations indicate the importance of steric restrictions and of the presence of side chains in reducing the conformational freedom of this peptide. Further potentialities of the method are discussed.

Notes: A combinatorial formulation of the partition function is developed for short polypeptide chains, where summations can be evaluated. From the complete combinatorial expression it is possible to assess the validity of any approximations which are made in evaluating the partition function. It is shown that it is an excellent approximation to consider the hydrogen-bonded portion of a short (20 residues) chain as consisting of a single helical sequence of any size, with all sizes of helical sequences being about equally probable at the transition temperature. Another approximation, involving a linear dependence of the energy and entropy on the number of hydrogen bonds, helps to clarify the nature of the intermediate states in the denaturation process. Expressions are developed which incorporate the effect of hydrophobic bonds on the stability of the α-helix, and the binding of water to the random coil. It is concluded that hydrophobic bonds in helical sequences can greatly stabilize the helix, but that similar bonds in the random coil can largely offset this stabilization.

Notes: The Lifson-Roig matrix theory of the helix-coil transition in polyglycine is extended to situations where side-chain interactions (hydrophobic bonds) are present both in the helix and in the random coil, as discussed for short chains in paper II of this series. It is shown that the conditional probabilities of occurrence of any number and size of hydrophobic pockets in the random coil can be adequately described by a 2 × 2 matrix. This is combined with the Lifson-Roig 3 × 3 matrix to produce a 4 × 4 matrix which represents all possible combinations of any amount and size sequence of α-helix with random coil containing all possible types of hydrophobic pockets in molecules of any given chain length.

Notes: The method of sequence-generating functions is applied to long polypeptide chains to describe various types of hydrophobic bonding in the random coil. These results are then combined with a similar treatment of the α-helix in order to discuss the helix-coil transition in single helices and in molecules whose helical segments interact by side-chain hydrophobic bonding. Numerical calculations, based on the equations derived in this and preceding papers, are presented to show the relative probabilities of occurrence in the random coil of neighbor-neighbor hydrophobic bonds and pockets of hydrophobic bonding, and the relative probabilities of occurrence of the various states in a system of interacting helices. A discussion is also presented of the dependence of the helix-coil nucleation and growth parameters on solvent and side chain.

Notes: The partition function of paper II for short polypetide chains is incorporated here into one for a simple model for side-chain hydrogen bonds between helices. It is thus possible to calculate the fraction of amide hydrogen bonds, the fraction of side-chain hydrogen bonds, and the number of bound protons as a function of temperature and pH. Illustrative data are presented for the probabilities of occurrence of intermediate structures and for the influence of the overall conformation on the observed pK's of ionizable groups.

Notes: The helix-coil transition temperature Tc of poly(γ-benzyl L-glutamate) in binary solvent mixtures of dichloroacetic acid and 1,4-dichlorobutane, 1-chlorooctane, or 1-chlorododecane have been measured. A treatment is presented with which the transition enthalpy can be calculated from the observed dependence of Tc on solvent composition. Results are compared with previously obtained calorimetric data. The underlying assumptions of the calculation are discussed.

Notes: A marked molecular weight fractionation accompanies the demixing or phase separation resulting from the complex coacervation of mixtures of aqueous solutions of salt-free isoionic unfractionated gelatins with pI's of 5 and 9. Viscosity studies show that the fractionation is such that the concentrated phase tends to maintain constant, homogeneous composition. A second feature, seen in phase volume and concentration measurements, is a marked self-suppression of coacervation intensity with increasing mixing concentration. These data were interpreted in terms of a dilute-phase aggregate model which assumes nearly equal electrostatic free energies of mixing in dilute aggregate and concentrated random phase. The driving force for phase separation is the entropy increase upon formation of the random phase but demixing also depends upon the polymer-solvent interaction parameter χ, in the same fashion as in simple coacervation. The dilute-phase aggregate model indicates that the sharp molecular weight selection takes place in the aggregate formation step and explains the self-suppression. Phase equilibria studies utilizing fractionated, paucidisperse high molecular weight gelatins, emphasize the requirement for concentrated phase homogeneity and indicate that aggregates of different molecular weight may act as different components, so that χP1Q1,PkQk 〉 0, bringing about a separation of the system into three or more coexisting phases. The formation of several coexisting phases from a homologous polyelectrolyte system and the very marked requirement for phase homogeneity suggest that the phenomenon of complex coacervation is a very good model for some of the essential steps in the pre-biologic organization of polymeric polyions.

Notes: Light scattering of multichain poly-α-amino acids was studied in dimethylformamide (DMF). The polymers consisted of a backbone of poly-L-lysine of degree of polymerization n with side chains of benzyl L-glutamate and benzyl L-aspartate of degree of polymerization, m, on each ε-amino group. The backbone length n is known and m is obtained by amino acid analysis. The results on a series of such materials confirm this structure and show that the molecules are dissolved in highly compacted conformations. It was found that DMF is a poor solvent for these polymers. In the case of the higher molecular weight polymers, the solutions initially were not molecularly disperse. The aggregates were resistant to dilution in the experimental range. Mild heat treatment, however, disaggregated the solutions irreversibly, and the light-scattering data indicated that a structural rearrangement of the molecules had occurred.

Notes: The intrinsic viscosity, sedimentation and diffusion of a series of branched, multichain poly-α-amino acids having a poly(L-lysine) backbone and poly(γ-benzyl L-glutamate) and poly (β-benzyl L-aspartate) side chains was studied at room temperature in dimethylformamide. The molecules were found to be extremely compact structures in which the molecular backbone is either lying along the major axis in a slightly twisted configuration (the longer the side chain the smaller the twist) or is coiled up in the form of a disk with backbone and side chains coplanar. Heat treatment (to 70°C.) introduces only small changes in the hydrodynamic parameters showing that the heat-labile aggregates detected by light scattering are reversibly broken up during the hydrodynamic measurements. The above structural information concerns the initial metastable conformation of the molecules which is irreversibly destroyed by heat treatment.

Notes: The optical rotatory dispersion and ultraviolet absorption spectra of the 64 trinucleoside diphosphates containing the bases A, U, C, and G have been calculated by using a simple semiempirical approach. These calculations accurately predict the optical properties of the nine trimers for which extensive experimental results are available. The computed optical data should be useful in the identification of oligonucleotides obtained in the course of sequence determination of ribonucleic acids and should simplify the determination of the concentration of oligonucleotides in aqueous solution. Additional calculations indicate that it should be possible to analyze most, mixtures of sequence isomers of trinucleoside diphosphates by direct, measurement of the ORD of the mixture at neutral pH.

Notes: The equilibrium binding constants of Cd++ and Cu++ to native and denatured calf thymus DNA were determined polarographically. The binding constants are an exponential function of the potential at the binding site and as such they vary with ionic strength and with the charge on the DNA molecule. The correlation between the fraction of sites occupied by heavy metal ions and between the thermal stability of DNA in solution is discussed.

Notes: Complex shear viscosity η* = η′ - iη″ of poly (L-glutarmic acid) solution was measured by the torsional crystal method at 50 kc./sec. as a function of pH. A sharp peak was found at the midpoint of the helix-coil transition region in both η′ and η″. The relaxation time is calculated from η′ and η″ assuming a single relaxation process and the peak value at the midpoint of transition is estimated at 10-6 sec. Such behavior agrees well with the prediction from the theory of Schwarz. The attenuation of longitudinal sound waves at,3 Me./sec. was measured as a function of pH for solutions of poly(glutamic acid), glutamic acid, and gelatin. A small attenuation peak was observed for the three solutions, the peak height being almost, the same for them. The peak is interpreted in terms of the dissociation reaction of side chains.

Notes: We report the circular dichroism (CD) spectrum of poly-Lacetoxyproline in trifluoroethanol, a solvent in which the form I to form II conformational isomerization occurs slowly enough to permit observation of the spectral changes. A comparison is made to poly-Lproline. As judged by the similarity of the CD spectra, the conformations of the corresponding forms of the two polymers must be nearly the same. Transition assignments are proposed; these are shown to agree with the theoretical calculations of Pysh. There is a serious unexplained discrepancy between our solution data and those of Fasman for poly-Lacetoxyproline.

Notes: The dielectric relaxation of DNA solutions has been investigated with and without extraneous ions covering a wide frequency range. The effect of monovalent ions such as Na, K, and Li as well as divalent ions such as Mg, Ca, and Hg have been included in the study. These ions are found to have a profound effect on the dielectric increment and the relaxation time without affecting the molecular dimension drastically. This dielectric effect is interpreted as indicating the importance of counterion fluctuation on the low frequency dielectric constant of DNA in solution. The effect of an organic ion, tetra-methylammoniun bromide, has also been studied. This ion has no noticeable effect. A simple theory is derived on the basis of a microscopic model to account for the effect of external ions on the dielectric behavior of solutions of DNA.

Notes: Block and random copolymers of γ-benzyl L-glutamate and glycine were studied by optical rotatory dispersion methods in order to ascertain the extent and stability of the α-helix formed by these polymers in appropriate solvent mixtures. Results indicate that when in solution such polymers do contain sections of α-helix. However, it appears that glycine does not readily fit into the α-helix which is largely due to the γ-benzyl L-glutamate segments. Further, there are indications that the helical sections in the random copolymers are due to the nonrandomness of the polymerization.

Notes: It is demonstrated that, poly(A + U) and poly(I + C) are both formed under low ionic strength conditions. Continuous variation studies indicate the formation of copper(II) complexes of poly A, poly C, and poly I, but not of poly U. Copper(II) in a 1:1 ratio to polynucleotide prevents the formation of poly(A + U) and brings about the dissociation of the poly (A + U) complex produced in the absence of the metal. Poly (I + C) is similarly dissociated by copper(II) ions. The addition of sufficient electrolyte reverses the copper(II) induced dissociation of poly(I + C). The effect of copper(II) on ordered synthetic polynucleotides is thus very similar to its effect on DNA.

Notes: The synthesis and some of the physical-chemical properties of tricopolymers of L-glutamic acid, L-lysine, and L-alanine are reported here. The molar ratios of the glutamyl: lysyl: alanyl residues were 1:1:X or 3:2:X, where the alanyl content X was increased in regular steps. The α-helix content calculated from the optical rotatory dispersion of the polypeptides is compared with a predicted helix content estimated from the composition of the polymers and the known behavior of the homopolypeptides at pH 3, 8, and 12. At pH 3 copolypeptides containing 20 mole-% or more alanine exhibit a helix content equal to the sum of their alanyl and glutamyl residue contents. At pH 8 the helix content equals the alanyl content when the latter was 40 mole-% or higher; at lower alanyl contents the electrostatic interaction between charged glutamyl and lysyl residues makes some contribution. At pH 12 the amount of helix observed is proportional to the mole ratio of alanine residues present in the polymer. The helix content of a tricopolymer containing 1:1:3 mole ratios of glutamyl: lysyl: alanyl residues was determined in solutions of lithium bromide and in urea solutions. Both reagents led to a decrease in helix content at pH 3 and 8 to a minimum of approximately 20% helix in 8M urea or 5.5M LiBr. The helix-random chain transition curves at pH 3 and 8 are parallel when the urea concentration is varied, but differ in shape when the lithium bromide concentration is varied at pH 3 and 8. The mode of action of these two “denaturing” reagents may thus be different. Heating the same tricopolypeptide at pH 3 or 8 from 5 to 80°C. also led to a helix-random chain transition centered at approximately 45°C.

Notes: Potentiometric titration curves of the silver(I) complexes of cytidine, adenosine, and uridine show little uptake of base below pH 7, unlike the curve for silver(I)-guanosine, which shows extensive base uptake at neutral pH. This observation is correlated with spectrophotometric data showing little difference between the silver complex spectra of adenosine, cytidine, and uridine and the spectra of uncomplexed nucleosides, except at high pH, but showing a great difference between the silver complex spectra of guanosine and inosine and the corresponding uncomplexed nucleosides even at pH 6. Similar comparisons of the silver complexes of poly A, poly C, poly I, and poly U, both by potentiometric titration and by spectrophotometry, show that poly I behaves like guanosine and inosine as expected, differing from poly A and poly C. However, poly U behaves like poly I and thus does not resemble uridine in its complexing behavior. There is thus a dichotomy between poly A and poly C on the one hand in silver complexing phenomena, compared with poly U and poly I on the other. When silver(I) is added to systems containing zinc(II) and various polynucleotides, the same dichotomy is noted. Silver(I) inhibits the degradation by zinc(II) of all four polynucleotides, but the degradation of poly I and poly U is prevented virtually completely. Silver(I) alone has no degradative effect on RNA and inhibits, the zinc(II) degradation of RNA. Polynucleotide complexes in which silver(I) and zinc(II) are simultaneously bound to different positions on the macromolecules are postulated as intermediates in the inhibited degradation reactions.

Notes: The relation of the high-frequency elastic moduli of semicrystalline polymers to volume fraction crystallinity is correctly described by the Hashin-Shtrikman theory, without any disposable constants, as a function of the ratio of the modulus of the amorphous to that of the crystalline phase. Hence the (high-frequency) reduced modulus of semicrystalline polymers is largely a function of the temperature T/Tg. The importance of T/Tm for the modulus of the crystalline phase precludes the existence of a single universal reduced modulus versus temperature curve.

Notes: The crystallization kinetics and morphology of poly(tetramethyl-p-silphenylene)siloxane spherulites have been investigated over a temperature range of 25-130°C. The effect of molecular weight on the spherulitic growth rates, ranging from the monomer to molecular weights about 106, is discussed in terms of conventional rate theory. Surface free energies of crystal growth are computed on the basis of a spherulitic model in which the polymer chains are presumed to be incorporated within the lamellar crystallites which are comprised in the spherulites. Mention is made of the change in mechanical properties with molecular weight.

Notes: The depolarized light intensity (DLI) technique detec's transitions in birefringent materials by simultaneously measuring sample temperature and intensity of polarized light roatated by the sample. Most polyolefins in the solid state rotate 30-40% of the polarized light. At any temperature at which the sample exhibits an ordered state, some rotation occurs. Temperatures of transitions and changes in refractive index can be determined in this way. Many of these transitions can also be followed by the well-known method of differential thermal analysis (DTA). A comparison of DLI and DTA curves of polyethylene, polypropylene, mixtures of these polymers, and block copolymers of ethylene and propylene shows the two techniques to be complementary and of use in determining the physical behavior of these polymers. Instrumentation and system variables of the DLI technique are discussed.

Notes: The weight-average molecular weights of polymers of acrylonitrile prepared by a free-radical initiator and an organometallic catalyst have been determined by lightscattering measurements in N,N-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide at 25°C. and in dimethyl sulfoxide at 140°C. The apparent molecular weights of the polymers prepared with the NaAlEt3S(i-Pr) catalyst in DMF at -78°C. (referred to as high-melting polymers) changed from 54,800, 82,700, and 480,000 when measured in DMF at 25°C. to 36,000, 41,600, and 225,000 when measured in DMSO at 140°C., whereas the molecular weights of the free-radical polymers remained unchanged. Furthermore, from results obtained in DMSO at 140°C., The intrinsic viscosity-molecular-weight relationships were found to be identical for the high-melting and the free-radical polymer and in substantial agreement with an equation reported by Cleland and Stockmayer. The apparent decrease in molecular weight of the high-melting polymer from 25 to 140°C. indicates rather clearly that the high-melting polymers are associated in DMF at 25°C. The “aggregates,” even though present only at low concentrations, raised the weight-average molecular weight markedly but affected the number-average molecular weight only slightly, thus giving a high M̄w/M̄n ratio. It appears likely that when temperature and solvent are such that association does not occur, linear PAN's will have approximately the same intrinsic viscosity-molecular weight relationship (subject of course to slight change by polydispersity). The often reported abnormal molecular weight of samples prepared by solution polymerization especially at low temperatures, may be attributed to branching, or to an association, as reported here. The nature of association of PAN in dilute solution is also discussed.

Notes: Thermal expansion and dielectric relaxation of polyacrylonitrile were measured in the temperature range from -75 to 152°C. at frequencies from 30 cps to 3 Mc./sec. The thermal expansion curve and the temperature dependence of logarithmic relaxation time both show an inflection at 85°C. An x-ray study by Bohn reveals that this inflection comes from the thermal expansion of the paracrystalline phase of this polymer, and consequently the transition at 85°C. and the associated relaxation are ascribed to molecular motion in the paracrystalline phase. The relaxation strength increases with increasing temperature above this point. The transition is caused by freezing of the bending vibration of chains whereas the relaxation results from rotational vibration. The length of segments in this phase is roughly estimated to be ca. 100 A. by comparing the observed relaxation strength with the theory developed on the basis of the above considerations.

Notes: The adsorption of iodine by rayon tire yarn samples from aqueous solutions in which the concentration of iodide and triiodide ions is suppressed was studied. Fowler and Guggenheim's model of adsorption on regular localized monolayers appeared to be applicable to the adsorption phenomena considered. It is concluded that adsorption takes place on patches of sites in the cellulose-water gel.

Notes: Aromatic poly(amic acids) derived from pyromellitic dianhydride and 4,4′,-diaminodiphenyl ether were characterized by dilute solution techniques. Number-average molecular weights M̄n of 13 samples ranged from 13,000 to 55,000 (DP 31-131). Weight-average molecular weights M̄w of 21 samples ranged from 9,900 to 266,000. The ratio M̄w/M̄n was between 2.2 and 4.8. Heterogeneous polymerization yielded higher molecular weight polymer than homogeneous polymerization. The molecular weight could be varied systematically by control of stoichiometric imbalance. Use of very pure monomers and solvent gave polymers of relatively high number-average molecular weight (∼50, 000) and the most probable molecular weight distribution M̄w/M̄n = 2. Impure monomers and/or solvent resulted in lower number-average molecular weight (M̄n ≅ 20,000-30,000) and wider distributions (M̄w/M̄n = 3-5). The Mark-Houwink relation obtained was [η] = 1.85 × 10-4M̄w0.80 The exponent is characteristic of moderately extended solvated coils. The unperturbed chain dimensions (r02/M)1/2 were 0.848 A., and the steric factor σ was 1.24 which is close to the limiting value of unity for an equivalent chain with free internal rotations. The sedimentation constant-molecular weight relation was S0 = 2.70 × 10-2M̄w0.39. This exponent is consistent with the Mark-Houwink exponent.

Notes: Current network theory exhibits inconsistencies which show up particularly clearly in deformation of networks prepared by crosslinking a polymer in solution. A check of theory can be obtained if one knows precisely the number of crosslinks in the network and if a range of deformations is applied to the network. In an effort to explore this problem we have examined the relation of shear modulus to crosslink density, primary molecular weight, and polymer concentration for a series of poly(vinyl alcohol) gels at low to intermediate concentrations. Aqueous poly(vinyl alcohol) solutions were crosslinked to form infinite networks using terephthalaldehyde. We find a large discrepancy with these poly(vinyl alcohol) gels between measured shear modulus and that calculated from classical elasticity theory assuming quantitative reaction of crosslinking. The ratio of measured to calculated modulus is independent of crosslink density for a given primary molecular weight and concentration. It shows linear dependence on polymer concentration prior to crosslinking and extrapolates to a critical concentration which is consistent with the effective sizes of the polymer molecules.

Notes: The rate of water sorption at 25°C. has been determined for a number of polyacetal films differing in structure, density, and orientation induced by extrusion. The equilibrium water uptake was found to be a linear function of density only; no other effect of structure or orientation was detectable. The extrapolated density for zero sorption was 1.51 g./cc., not far from the theoretical crystalline density. The diffusivity of water in unoriented films rose with decreasing density; for linear copolymer, the trend was parallel to that of the area under the dynamic mechanical loss peak associated with long-range chain motions in the disordered regions (β-transition). Less pronounced effects of molecular weight and long chain branching on diffusivity were also noted. Films crystallized while an extruded melt was still oriented showed considerable increases in water diffusivity, but no significant changes in the apparent activation energies of permeation (about 6.6 kcal./mole) or diffusion (about 11.5 kcal./mole). On annealing these films, the diffusivity remained almost constant while the sorption coefficient and retraction on remelting decreased.

Notes: The cavity model used in the theory of dielectrics was applied to hydrodynamics to calculate the force exerted by a system of soft dumbbells on a reference dumbbell in a hydrodynamic field. The influence of this force on the viscosity and flow birefringence and its dependence on both the concentration and velocity gradient were calculated. The system of equations has a real solution only for values of β = M[η]η0γ/RT which are smaller than a critical value rapidly decreasing with increasing concentration. At zero concentration the results obtained agree with the theory of a single isolated dumbbell model. The calculated Huggins constant is k′ = 0.4. The extinction angle is connected with the relative viscosity very nearly as derived from experiments. However, the theory fails at higher concentrations and gradients yielding an increase in viscosity with the gradient and infinite zero-shear viscosity for the concentration c = 2.5/[η].

Notes: The present study is concerned with some network properties of polyurethane elastomers in which stability is acheived via physical crosslinkages such as microcrystallites or secondary bonding. The techniques of isothermal, thermo-, and photoelasticity have been used to gain better insight into the mechanisms which occur during the deformation of these materials. It was found that stable networks are obtained only after mechanical and thermal conditioning. The well-known stress lowering which is observed during the second deformation is manifested primarily in the entropy component of the retractive force. This implies that the stress lowering results from a reduction in the number of effective network chains and not from time-dependent effects or crystallinity changes. Depending upon the chemical structure of the material, both positive and negative energy components of the force have been found.

Notes: An extension of the results of a previous to higher molecular weights is made. The viscoelastic parameters ηt, Je, τm, and Em are found experimentally through stress relaxation tests. The predictions of the Rouse-Bueche (RB) theory and its modified Ferry, Landel, Williams (FLW) form concerning the molecular weight dependencies of these perameters are compared with the data. It is found that the RB and FLW predictions are not rigorously obeyed.

Notes: Low-angle light-scattering patterns were obtained on ionotropic gels formed by the diffusion of Ca++ into a polygalacturonate sol. Near the sol-gel boundary, diffraction patterns were obtained which indicated organized structures with repeating units of 1100, 250, and 12.5 μ. Microscopic investigations confirmed that at this gel boundary capillaries run parallel to each other and perpendicular to the direction of diffusion. The diameter of capillaries was 12 μ while their length was in the 1000 μ range. Short interconnecting capillaries were about 200-300 μ in length. Inside the gel, in the more highly crosslinked parts, a disorientation occurs during the aging process with the partial disappearance of capillaries. However, at the gel-sol and gel-semipermeable membrane boundaries, the highly organized structures remain even during prolonged aging. The kinetics of the gel formation was also investigated.

Notes: Data on wettability of elastomers should be considered basic to the understanding of all phases of elastomer adhesion. However, no such data in the form of critical surface tensions were available for elastomers other than polydimethylsiloxane. For this study, 18 elastomers were selected to determine the effects of functional groups, of geometrical and structural isomerisms, of copolymerization, and of the induced orientation upon wettability. Most results support the constitutive law of wettability established by Shafrin and Zisman. The effect of structural isomerisms in the form of a vinyl side group and cyclization is discussed. An equation for the calculation of critical surface tension of a copolymer or of a mixture of isomers is proposed as follows: \documentclass{article}\pagestyle{empty}\begin{document}$ \gamma _c {\rm (copolymer or mixture of isomers)} \simeq \sum\limits_1^\infty {N_i \gamma _{c_i } } $\end{document} where Ni is the mole-fraction of the individual monomer in the copolymer and \documentclass{article}\pagestyle{empty}\begin{document}$ \gamma _{c_i } $\end{document} is the critical surface tension of each homopolymer. Most elastomer adhesion studies conducted in the past were concerned with the diffusion theory of adhesion. This study further supports the conclusion on the role of diffusion and adsorption in adhesion advanced in Part I, especially with respect to the physical state of polymer at the time of application. The wettability data in this study could shed some light upon major basic mechanisms involved in elastomer reinforcement.

Notes: Experimental data obtained from stress-strain curves of five different textile fibers, at a series of different, constant strain rates covering a range of 61/2 decades have been used to study two methods of nonlinear viscoelastic analysis proposed elsewhere. According to the first of these, time and strain effects are factorizable so that stress σ, strain ε and time t are related by the equation σf1(ε)/ε = f(t),. This is shown to be unsatisfactory with the present materials, but an empirical modification to σf1(ε)/ε = f2(ε) + f(t) is satisfactory. According to the second, general nonlinear viscoelastic behavior can be described by an equation which reduces to the form σ/ε = F1(t) + εF2(t) + ε2F3(t) + when applied to extension at a constant strain rate. This series is shown to be strongly divergent except at fairly small stains. In fact, if it is truncated after about three terms, which are as many as can be estimated with any significance in the present experiments, it is applicable only to strains of about 3-4% and less. Numerical techniques which enable standard statistical procedures to be used have been devised to perform the above analyses and are described in detail.

Notes: The theory of rate processes has been applied by Eyring to the plastic flow of solids, but he considers only the case of simple shear. Some assumptions concerning a generalization of this theory for an arbitrary strain tensor are here proposed and discussed. The components of the deformation and the work done by the deviator of the stress during the elementary flow process are evaluated. According to these results, it can be proved that the yield condition of Huber, Hencky, and von Mises is valid when there is no hydrostatic stress. The discrepancy between the yield stresses corresponding to tensile and compressive tests is explained by the influence of the hydrostatic component and evaluated by taking account of the amount of free volume required for an elementary jump. The calculated value agrees with the experimental data. Measurements of the inclination of Lüder's bands and deformation components on poly(vinyl chloride) flat bars strained by tensile tests are in good agreement with the proposed theory.

Notes: Heterogeneous olefin polymerization catalysts generally consist of a transition metal compound from groups IVB or VB in combination with an organometallic compound from groups I, II, and III of the periodic table of the elements, but in some instances may contain a third component possessing at least one strongly complexing ligand atom. The composition of a coordination catalyst of this type determines both the rate of polymerization of the α-olefin and the stereoregularity of the polymer produced. In order to predict activity and stereospecificity of catalysts for the polymerization of α-olefins, data from the polymerization of propylene using catalysts of the following types were correlated: (1) titanium(III) chloride catalysts in which the organometallic component was varied, (2) coordination catalysts containinig a ligand third component in which the ligand was varied, and (3) coordination catalysts in which the transition metal component was varied.The electronegativity of an orgariometallic compound is known to be important in determining its reactivity with ethers, hydrogen, and other reagents. Both the ionic size and the electronegativity of the central atom appear to be important in determining the utility of both the organometallic and the transition metal compounds as components for coordination catalysts. In general, organometallic and transition metal compounds having small central atoms with similar electronegativities favor the formation of stereospecific catalysts.The present correlation has made it possible to predict the activity and stereospecificity of coordination catalysts for olefin polymerization.

Notes: Degradation of poly(ethylene oxide) (PEO) by violent stirring was studied in various solvents. The chain scission or the limit of the degradation was measured, and the effects of solvents, polymer concentration, stirring speed and degree of polymerization (DP) were investigated. It was found that the number of bonds broken per polymer chain was independent of the concentration but increased with the stirring speed and with decrease in the DP. The rate was much affected by the solvent used, being larger in a poor solvent. It was also found that the rate could be represented either by Jellinek's or Ovenall's rate equations, which have been applied to the ultrasonic degradation of polymers in solution.

Notes: The small-strain mechanical behavior of crystalline polyethylene has been studied by using a microstrain technique with strain resolution on the order of 10-6. The strain rate was varied from 10-6 to 10-4 sec.-1, and a temperature range of 17-28°C. was investigated. A strong dependence on strain rate and temperature has been observed for the following parameters which characterize the mechanical response of polyethylene in the microstrain region: the initial modulus of the stress-strain curve, the deviation in strain from ideal linear elastic behavior at a given stress amplitude, and the energy dissipated in a deformation cycle. The Young's moduli that were observed by means of tensile tests in the microstrain region were only about 20% lower than the values reported in other investigations at kilocycle and megacycle frequencies. The experimental method made it possible to isolate a deformation process which was attributed to a crystallographic shear mechanism corresponding to a yield point of 27 psi. This shear mechanism is discussed in terms of the various shear processes, such as slip, twinning, and the orthorhombic-monoclinic phase change.