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: 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: 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: The matrix method of statistical mechanics is used to calculate equilibria for the binding of small molecules to polymers. When there is only one kind of binding site the problem is simple; some examples are given for illustrative purposes. If, however, the binding sites are not all equivalent and the bound molecules interact or interfere with each other, the problem is no longer trivial, being formally analogous with calculation of the helix-coil transition equilibrium in a heterogeneous polypeptide. Particular difficulties arise when the sequence of binding sites is aperiodic; most naturally occurring materials fall in this class. The purpose of this paper is to point out that problems of this type are readily solved with good accuracy by use of random-number methods on a high-speed digital computer. One such calculation is presented for illustration. The methods developed are applicable to such systems as the binding of actinomycin, Hg-, and acridine dyes to DNA.

Notes: The aggregated form of poly(L-glutamic acid) can be isolated by gel permeation chromatography, whereby it was found that in a polydispersed sample the lower molecular weight fractions precipitate preferentially and the higher fractions tend instead to aggregate. In addition, aggregation has the effect of retarding or inhibiting precipitation.

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

Notes: The interaction of hapten (ε-DNP lys) with native and S-sulfonated antibodies specific towards the 2,4-dinitrophenyl group, as well as the interaction with isolated chains and a complex obtained by mixing light, (L) and heavy (H) chains of these antibodies, were followed both by polarography and by equilibrium dialysis. With the S-sulfonated antibodies and with the mixture of H and L chains the binding heterogeneity observed in the original antibodies was much lowered or entirely removed. At the same time, the amount of active proteins in the sample decreased approximately by half. The association constants of modified antibodies were of the same order as the average association constants of the original antibodies. A slow increase of the amounts of hapten bound with proteins was observed on mixing the H and L chains and adding hapten. This slow reactivation was not obtained with the original or S-sulfonated antibodies and with isolated chains. It was shown that the reaction determining the kinetics of this reactivation (the slowest reaction) was not the association of H and L chains but the interaction of complexes of the H and L chains with hapten. It was reported previously that H chains were nonspecifically reactivated by binding L chains. The amount of hapten bound by the complex of H and L chains increased with increasing excess of L chains following a curve resembling the Langmuir isotherm. The limiting value of the amount of hapten bound when using antibody L chains was higher than in the case of nonspecific L chains.

Notes: The rate of breakage by hydrodynamic shear of the cohered ends of λ-DNA molecules has been observed for the circular monomers, joined half molecules, and joined quarter molecules, in a capillary apparatus with known flow parameters. The rate constant for breakage has been measured as a function of shear stress, temperature, ionic strength, and molecular length. There is a large temperature coefficient, with an activation energy of 120 ± 20 kcal./mole. The values of d ln k/dG, where k is the rate constant for breaking and G is shear gradient, in aqueous solution at 25°C. are about 3.8 ± 0.3 × 10-4 see. The shear stresses needed for breakage of joined quarter molecules and of circular monomers, respectively, are about equal, and about half that needed for breakage of joined half molecules. The rate of breakage at a given shear stress increases with decreasing ionic strength, approximately as [Na+]-1.6. Self-protection effects are not observed for opening of circular monomers at a DNA concentration of 5 μg./ml. but are observed for breakage of joined half molecules at concentrations down to 0.5 μg./ml. The large temperature coefficient which is approximately equal to that of the thermal dissociation of the cohered ends is interpreted to mean that shear breakage is a mechanically assisted thermal reaction in which the thermal fluctuations provide most of the free energy of activation for breakage. A detailed model for this interpretation is presented. The self-protection effect implies that those molecules which break are not average molecules but exceptional ones which, due to some fluctuation, are more fully extended in the flow field.

Notes: The dynamic viscoelastic properties of hyaluronic acid solutions have been measured over the frequency range 0.02-1.67 cps. The effects of varying temperature, hyaluronic acid concentration, pH, and ionic strength on the dynamic shear moduli were studied. The solutions exhibited a sharp transition from viscous to elastic behavior as the strain frequency increased. No entanglement coupling of the hyaluronic acid molecules was evident over the concentration range 2.0-4.0 mg./ml. Solutions at pH 2.5 showed a pronounced elastic behavior relative to both higher and lower pH's. This effect was attributed to a stiffening of the hyaluronic acid molecule at this pH.

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: Some general rules governing hydrogen bonding at the ring oxygens of furanosides, pyranosides, and bridge oxygens of glycosides have been formulated from existing data on crystal structures of carbohydrates. Ring oxygens of the majority of the glycopyranosides in the hemiacetal or acetal form are involved in hydrogen bonding such that the hydrogen bond direction is usually equatorial to the ring plane and not axial. In contrast, there are no known examples of ring oxygens of glycofuranosides and methyl-glycopyranosides displaying hydrogen bonding in the crystal. Also, the bridge oxygens of glycosides are not involved in hydrogen bonding. The observed shortening in the exocyclic and endocyclic anomeric C(1)—O bonds and the geminal C—O bonds indicate that compounds with two oxygen atoms attached to the same saturated carbon atom may participate in double-bond-no-bond resonance interaction in the same manner as difluoromethane. It is also possible that under these circumstances the carbon atom exhibits greater than tetracovalency. The “anomeric effect” may also be related to (a) the differences in the “double bonding” or bond shortening in the anomeric C—O bonds of the anomeric glycopyranosides, (b) the shorter intramolecular O(1)…O(5) non-bonded interaction, and (c) the smaller O(1)C(1)O(5) valence angle in the equatorial anomer compared to the axial anomer. An analysis has been made of the energetically preferred conformations about the glycosyl and glycosidic bonds of 1,4- and 1,3-polysuc-charides. In the 1a, 4e-glycopyranosides the projected angle φ1 [O(5)C(1)OR, where R = C or H] is positive, while it is negative in the 1e, 4e-glycopyranosides. Angle φ2 [C(1)OC(4′)C(3′)] is positive in both the 1,4-anomeric polyglycosides. 1e, 4e- and 1a, 4e -polysaccharides are stabilized by intramolecular O(5)…H—O(3′) and O(2′)…O(3′) hydrogen bonding, respectively, and generate linear and helical (cyclic) structures, respectively. 1e, 3e- and 1a, 3e-polysaccharides may be stablized by one of two possible intramolecular hydrogen-bonding schemes such that the 1a, 3e -polysaccharides generate helical structures while the 1a, 3e-polysaccharides generate nonhelical structures. The conformation about the C(5)—C(6) bond in the pyranosides falls into two groups where the angle φ00 [O(5)C(5)C(6)O(6)] is either positive, ∼+60 ± 30°, or negative, ∼-60 ± 30°, the former conformation being found more frequently. In the furanosides the latter conformation is preferred.

Notes: The synthesis of spermine derivatives (II), \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}_1 {\rm R}_{\rm 2} {\rm R}_{\rm 3} \mathop {\rm N}\limits^ + \left( {{\rm CH}_2 } \right)_3 \mathop {\rm N}\limits^ + {\rm R}_{\rm 1} {\rm R}_{\rm 2} \left( {{\rm CH}_2 } \right)_2 ]_2 \cdot 4{\rm X}^ - $\end{document}, and spermidine derivatives (III), \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}_1 {\rm R}_{\rm 2} {\rm R}_{\rm 3} \mathop {\rm N}\limits^ + \left( {{\rm CH}_2 } \right)_4 \mathop {\rm N}\limits^ + {\rm R}_{\rm 1} {\rm R}_{\rm 2} \left( {{\rm CH}_2 } \right)_3 \mathop {\rm N}\limits^ + {\rm R}_{\rm 1} {\rm R}_{\rm 2} {\rm R}_3 \cdot 3{\rm X}^ - $\end{document}, are reported. The effects of these salts on the helix-coil transition of rA-rU and rI-rC helices were examined. Increasing the size of the hydrophobic substituents, R1, R2, and R3 lowers the degree of stabilization of the helical structure. The disproportionation reaction, 2rA-rU→rA-rU2 + rA occurs readily with salts II and III, especially when the substituents, R1, R2, and R3 are small, i.e., H or Me. Spermine is found to stabilize the rA-rU2 and rI-rC helices to approximately the same extent; however, large differences between the degree of stabilization of rA-rU2 and rI-rC helices are observed when the substituents R1, R2, and R3 are large hydrophobic groups. Similar results are also obtained for the spermidine series. Finally, differences in the interactions of the salts II and III with rA-rU2 and rI-rC helices suggest that the latter helix is denser.

Notes: Studies have been made of the d.c. semiconductive behavior of fibers and solid gels of DNA. The semiconductive behavior under high vacuum is similar in solid gels of DNA and stretched fibers of DNA measured along and across the fiber axis. Photoelectric effects have been studied, and an action spectrum is reported which agrees with the absorption spectrum of DNA. No photoelectric effects are observed at wavelengths corresponding to the thermal energy gap. The temperature dependence of the photocurrent has been determined, and the effects of air, oxygen, and nitrogen at different pressures on the photocurrent have been measured.

Notes: The predominant role of van der Waal interactions in determining the helical conformations of a simple synthetic linear polymer, as well as helical polypeptides, was pointed out in systematic studies by Liquori et al.1,2. In the case of homopolypeptides the conformational analysis carried out on the basis of a simple semiempirical function describing the van der Waal pairwise interactions between the non directly bonded atoms lead to the conclusion that only five helices are allowed (Rα, β, γ, δ, Lα).2,3In view of the close similarities with poly-L-alanine, we have investigated by x-ray and conformational analysis the molecular conformation of poly(S-lactic acid) which has recently been described by Kleine and Kleine4 and Schuls and Schwaab5 and studied in solution by Goodman and D'Alagni.6 In fact, this polymer may be related to the polypeptide by the interchange of a peptide bond with ester bond along the main chain. This operation is expected to involve only a relatively small change in the steric interaction within the possible helical conformation, but obviously rules out any possibility of hydrogen bonding.

Notes: Poly-L-prolines I and II were differentiated by the characteristic bands in the far infrared region. Form I showed two broad bands at about 280 and 160 cm-1 and form II two bands at, 400 and 670 cm.-1. Furthermore, three broad bands at about 250, 200, and 100 cm.-1 were observed in the spectrum for form II. Infrared absorption bands of the pentamer, hexamer, and octamer of tert-amyloxycarbonyl-L-proline were almost similar to those of poly-L-proline II in the 1800-75 cm.-1 region. In the far-infrared region, especially, the absorption bands of these three oligopeptides were in good agreement with that of poly-L-proline II. Accordingly we concluded that the molecules of pentamer, hexamer, and octamer had a helical structure of a left-handed threefold screw axis. The tetrapeptide of tert-amyloxycarbonyl-L-proline might also have a left-handed helix, probably one turn, since the tetramer clearly showed an absorption band at about 400 cm.--1 characteristic of poly-L-proline II.

Notes: The conformational properties of poly-L-alanine have been examined in aqueous solutions in order to investigate the influence of hydrophobic interactions on the helix-random coil transition. Since water is a poor solvent for poly-L-alanine, water-soluble copolymers of the type (D, L-lysine)m-(Lalanine)n-(D, L-lysine)m, having 10, 160, 450, and 1000 alanyl residues, respectively, in the central block, were synthezised. The optical rotatory dispersion of the samples was investigated in the range 190-500 mμ, and the rotation at 231 mμ was related to the α-helix content, θH, of the alanine section. In salt-free solutions, at neutral pH, the three large polymers show high θH values, which are greatly reduced when the temperature is increased from 5 to 80°C. No helicity was observed for the small (n = 10) polymer. By applying the Lifson-Roig theory, the following parameters were obtained for the transition of a residue from a coil to a helical state: ν = 0.012; ΔH = -190 ± 40 cal./mole; ΔS = -0.55 ± 0.12 e.u. Since ΔH and ΔS differ from the values expected for a process involving only the formation of a hydrogen bond, and in a manner predicted by theories for the influence of hydrophobic bonding on helix stability, it is concluded that a hydrophobic interaction is also involved. In the presence of salt (0.2M NaCl), or when the ε-amino groups of the lysyl residues are not protonated (pH = 12), the helical form of the two large polymers (n = 450 and n = 1000) is more stable than in water. Since the electrostatic repulsion between the lysine end blocks is greatly reduced under these conditions, the alanine helical sections fold back on themselves, and this conformation is stabilized by interchain hydrophobia bonds. This structure was predicted by the theory for the equilibrium between such interacting helices, non-interacting helices, and the random coil.

Notes: Infrared spectra of polyglycines I and II obtained at -170°C. have been compared with those obtained at room temperature. The changes in frequency of some of the C - H stretching bands are consistent with the earlier suggestion that C - H … O=C hydrogen bonds are present in the structure of polyglycine II.

Notes: Methods for calculating the rate of cooperative transitions on a linear lattice, for which the helix-coil transition of polypeptides is an example, are reported. The problem is to determine the kinetic characteristics of the transition given the rate constants for a set of elementary steps: in this case, the transformations of individual segments between the helix and coil states. The most straightforward method is to store the state of a long lattice (in which helix and coil segments are represented by 1′s and 0′s) in a computer and to use random-number techniques to generate its behavior as a function of time. This is, in principle, a solution to the problem, but it requires very large amounts of computer time. We have devised a matrix iteration procedure which allows much faster computation while reproducing the results of the random-number method accurately. In this procedure the computer operates repeatedly with a transition probability matrix on a vector which represents the time-dependent state of a finite group of units. The choice of a finite group neglects kinetic correlations between the state changes of units inside and outside the group, but comparison with the random-number method indicates that these correlations are not important. Thus it is possible to generate the kinetic behavior of the model under essentially any conditions, for either relaxation or large perturbations. Examination of these calculated curves suggests a simple and quite generally applicable solution to the inverse problem - that of evaluating the rate constants from kinetic curves. The initial slope is well defined in almost every case, and since an analytic equation can be written relating this to the rate constants, these can be obtained directly from the initial rate. This latter is therefore the most convenient single measure of transition rate.

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

Notes: The absorption spectrum of the aqueous solution of acridine orange (AO)-poly-(D-glutamic acid) (PDGA) complex at pH 4.5 (helix form) did not show any wavelength shift, but at pH 7.5 (coil form) changed to the absorption curve of the helix form by compression up to 4500 atm. The ionization degree of PDGA estimated from the electric conductivity of the aqueous solution of PDGA at 4500 atm. was a value of about 5.3%. The entropy of the helix formation of PDGA from the titration data at 1 atm. and 30°C. was negative -2.98 e.u. It will be concluded in this report that the volume change for coil to helix could be positive for PDGA and negative for AO-PDGA complexes.

Notes: The optical rotatory dispersion, hypochromism, and proton magnetic resonance were measured for a number of dinucleoside phosphates in both dilute salt solutions and in 25.2% LiCl (which freezes below -70°C.) as a function of temperature. Two models for the “disordering” of dinucleoside phosphates: the two-state model and the oscillating dimer model, are used to analyze the data. Both models fit some of the data, but neither are completely satisfactory. Evidence is given for a dynamic structure of dinucleoside phosphates, in which the bases oscillate with respect to one another, remaining parallel to one another with no solvent in between, even at high temperatures. Implications of this model to the structures of single-strand sections of RNA's are discussed.

Notes: If the collagen triple helix is so built as to have one set of NH ⃛ O hydrogen bonds of the type N3H3(A) ⃛ O2(B), then it is possible to have a linkage between N1H1(B) and O1(A) through the intermediary of a water molecule with an oxygen O1W leading to the formation of the hydrogen bonds N1(B) ⃛ O1W and O1W (A). In the same configuration, another water molecule with an oxygen O1W can link two earbonyl oxygens of chains A and B forming the hydrogen bonds O2W O1(A) and O2W O0 (B). The two water oxygens also become receptors at the same time for CH ⃛ O hydrogen bonds. Thus, the neighboring chains in the triple helix are held together by secondary valence bond linkages occurring regularly sit intervals of about 3 Å along the length of the protofibril. The additional water molecules occur on the periphery of the proto-fibril and will contribute their full share towards stabilizing the structure in the solid state. In solution, they will be disturbed by the medium unless they are protected by long side groups. It appears that this type of two-bonded structure, in which one NH ⃛ O bond is to a water molecule, can explain several observations on the stability and hydrogen exchange properties of collagen itself and related synthetic polypeptides. The nature of the water bonds and their strength are found to be better in the one-bonded structure proposed from Madras than in the one having the coordinates of Rich and Crick.

Notes: The study of the structure of KBr-amylose begun by Senti and Witnauer has been extended by a three-dimensional crystallographic analysis and by stereochemical considerations. Location of Br- at (0.200, 0.200, 0.000) and K+ at (0.540, 0.540, 0.000) was obtained from the three-dimensional map of vector interactions. By using known parameters for the D-glucose residue and accurate space-filling models, an amylose helix was constructed to meet the fiber repeat spacing of 16.1 Å. The helix was determined to be left-handed, and the correct space group for KBr-amylose is P43 212. Placement of the helix in the unit cell resulted from structure factor calculations; minima in the grid of R values were checked with space-filling models to establish the final structure. Both ions are located in a waterlike environment. The oxygen atoms O(2), O(3), and O(4) from glucose residues on adjacent chains coordinate around K+.