ALBERT

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

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

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

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

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

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

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

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

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

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

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

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

Notes: The cohesive energy densities (CED) of three amylose derivatives have been estimated from viscosity measurements in a range of solvents, by using methods proposed by Mangaraj and Bristow and Watson. The values assigned to the triacetate, (2.5) nitrate, and tricarbanilate are 92 ± 2, 90 ± 2, and 87 ± 2 cal./cc., respectively. The CED of unsubstituted amylose has been measured and found to be 154 ± 4 cal./cc. The results are discussed in relation to solvent power, structure, and possible correlation with the internal pressure of the polymer.

Notes: Viscosity measurements and calculated rotary diffusion constants show that collagen undergoes photopolymerization when irradiated with ultraviolet light of 2537 A. Fibril formation at the same time is inhibited. The results are correlated with the aromatic amino acid content of the dangling peptides protruding from the rigid portion of the macromolecule.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Notes: 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: A molecular theory of protein secondary structure is presented that takes account of both local interactions inside each chain region and long-range interactions between different regions, incorporating all these interactions in a single Ising-like model. Local interactions are evaluated from the stereochemical theory describing the relative stabilities of α- and β-structures for different residues in synthetic polypeptides, while long-range effects are approximated by the interaction of each chain region with the averaged hydrophobic template. Based on this theory, an algorithm of protein secondary structure prediction is proposed and examples are given of “blind” predictions made before the x-ray structural data became available.

Notes: To increase our understanding of peptide-water interactions, we are simulating the behavior of water molecules in the intermolecular channels of [Phe4Val6]antamanide dododecahydrate crystals. There is good overall agreement between the positions predicted using two alternative potential functions and those that have been observed by x-ray diffraction. Detailed differences between the predictions for the two potential functions are discussed.

Notes: Experimental measurements of disulfide bond stability at various stages of protein folding are considered in terms of the effective concentrations of the thiol groups relative to each other; values of up to 107M are observed, so that intramolecular interactions within the interior of a protein are much more stable, and provide greater stability to the folded conformation, than those on the surface or in a flexible segment. Intramolecular interactions can have substantially lower free energies than intermolecular, for solely entropic reasons; this implies that polar interactions, such as hydrogen bonds and salt bridges, can provide net stabilization to a folded conformation, in spite of the unfolded protein having intermolecular interactions with the solvent. These considerations can account for the lower free energy and enthalpy of the folded state and are useful for considering protein flexibility.

Notes: Near- and far-uv CD spectra of microtubule protein preparations have been examined to study the possible role of protein conformation in relation to the kinetics of the self-assembly of these proteins into microtubules in vitro. Although tubulin can form conformations with high helical content under apolar solution conditions, this transformation is apparently not involved in self-assembly. There is no major perturbation of tubulin near-uv CD by reagents and solution conditions favoring assembly. Thus, in these preparations, tubulin, as dimer and as oligomer with MAPs, is effectively in the conformation in which it undergoes self-assembly. This conclusion is consistent with a hybrid model of assembly of microtubule protein involving direct incorporation of oligomeric species as an alternative to the condensation polymerization of tubulin dimer as the exclusive assembly mechanism.

Notes: The ligand-modulated kinetics of the autoproteolysis of thermolysin and the high-molecular-weight products of the reaction provide evidence for the conclusion that separation of the two structural domains is most probably the first step on the unfolding pathway of the protein under native conditions.

Notes: The in vivo pathway of folding and subunit assembly of a trimeric bacteriophage protein has been studied by characterizing precursors to the native protein and by analyzing temperature-sensitive mutations that kinetically block the pathway. The native trimer is formed via an intermediate composed of three partially folded chains, the protrimer. At 39°C, temperature-sensitive mutations prevent the formation of both the native trimer and the protrimer, possibly by destabilizing earlier intermediates. However, the mutations do not affect the stability of the native protein, formed at 30°C. Thus, these mutations identify amino acid residues involved in interactions that determine the folding pathway.

Notes: We studied the kinetics of O2 release by oxyhemoglobin caused by sodium dithionite, in the presence and in the absence of organic cosolvents (monohydric alcohols and formamide) at 10°C. This study was performed by using standard stopped-flow techniques coupled with microprocessor-based data acquisition. We have fitted the experimental data to a mathematical expression obtained on the basis of a two-state model that takes into account the kinetic heterogeneity between α- and β-chains and the presence of αβ-dimers in oxyhemoglobin solutions. Results indicate that the cosolvents mainly affect the allosteric parameter L, i.e., the T ⇄ R conformational equilibrium of hemoglobin, leaving the intrinsic deoxygenation rates of both R and T states almost unaltered. The L values obtained in the present work are in excellent agreement with analogous values previously estimated from oxygen equilibrium measurements.

Notes: Porcine submaxillary mucin (PSM) is a glycoprotein composed of a protein core and frequent, short oligosaccharide side chains. We report static and dynamic light scattering experiments and intrinsic viscosities for PSM in aqueous solvent systems. In 0.1M NaCl solution, the data suggest PSM exists as large, internally branched, highly hydrated, polydisperse aggregates that slowly dissociate to give a stable species of weight-average molecular weight (Mw) 7.4 × 106. In 6M GdnHCl solution, the noncovalent bonds between PSM molecules are broken, giving a highly elongated molecule of Mw = 2.0 × 106. The irreversible nature of this dissociation suggests that the forces that stabilize the native aggregates of PSM in 0.1M NaCl are specific in nature. On reduction of PSM with mercaptoethanol, the polydispersity decreases and Mw also decreases to 9 × 105. A discrete change is observed in the solution properties of PSM in 0.1M NaCl at a concentration of 2mg/mL, manifested by a sudden decrease in the translational diffusion coefficient, an increase in viscosity number, and a decrease in slope of the osmotic compressibility. We tentatively propose that a weak and reversible secondary association process occurs at this concentration, although a purely hydrodynamic interaction cannot be ruled out.

Notes: The uv linear dichroism of calf thylus DNA has been studied at different degrees of orientation both in flow-oriented ethanol-water solutions and in a stretched aqueous host of poly(vinyl alcohol) (PVA). The reduced dichroism (LDR) curves in the region 250-290 nm for DNA in PVA films at 75 and 100% relative humidity (r.h.) are in fair agreement with the curves calculated for the A- and B-forms of DNA, based on the fiber structures and the π-π* transitions of the free bases. This suggests that DNA adopts its A and B conformations in PVA at 75 and 100% r.h. In ethanol, on the other hand, a deviation from the A-form spectrum shows that the conformation of DNA in the solution can differ from the fiber structure. At shorter wavelenghts, a positive contribution to LDR is explained in terms of an out-of-plane polarized n-π* transition.

Notes: A vibrational analysis has been performed for a double-helix-single-strand junction. A Green's function technique has been used in treating the junction as a defect on an otherwise perfect system of infinite chain homopolymers. We calculate that the hydrogen-bond stretching at the junction is amplified by a factor of two relative to the interior of the double helix, B poly(dG)-poly(dC). Breathing modes localized near the junction have also been predicted at 77 and 94 wave numbers. The calculated results are shown to be consistent with predictions from recent nmr studies.

Notes: Nmr studies of the protected and free tetrapeptide Gly-Pro-Gly-Gly were carried out in β-turn-supporting solvents, that is, in CDCl3 for Z-Gly-Pro-Gly-Gly-OMe and in Me2SO-d6 for H-Gly-Pro-Gly-Gly-OH. Comparisons with specifically α-deuterated analogs gave complete assignments of the NH and methylene regions. Analysis of chemical shifts, coupling constants, and the temperature dependence of chemical shifts show that the peptide adopts a type II β-turn conformation. This turn is stabilized for the protected tetrapeptide by two hydrogen bonds between (i) C=O (Gly1) and NH(Gly4), and (ii) urethane function NH and methyl ester C=O.

Notes: Ab initio minimal and split-valence basis set calculations have been performed on compounds that are involved in retro-inverso modifications, i.e., gem-diaminoalkyl and malonyl structures. These calculations are compared with empirical force field calculations and the minor differences discussed. All calculations agree that the preferred helical conformation of the isolated gem-diaminoalkyl and malonyl derivatives of residues found in the retro-inverso modified peptides is 5-8 kcal/mol lower than the Ceq7 conformation preferred by the isolated peptide residues. Population analysis and contour plots of the charge distribution are used to help explain the differences between the model compounds.

Notes: SCF-LCAO-MO ab initio calculations were carried out for the interaction between a charged amino acid and a water molecule. The results obtained were fitted by an analytical potential function of the atom-atom type, and the corresponding potential surfaces were examined by means of orientationally optimized isoenergy contour maps. Monte Carlo simulations were also carried out on a few selected solute-water clusters at T = 300 K, in order to obtain insight into the solvation structure.

Notes: An improved cube method has been developed for calculating the intensity of diffuse x-ray scattering of macromolecules in solution using a certain set of their atomic coordinates. The technique is based on the ideas of B. Lee and F. M. Richards [(1971) J. Mol. Biol. 55, 374-400] and Richards [(1977) Annu. Rev. Biophys. Bioeng. 6, 151-176] on the possibility of estimating the molecular and accessible surface of a particle by “rolling” a sphere, simulating a water molecule, on its molecular surface. It is shown that this technique is more advantageous than earlier versions of the cube methods. The improved technique for calculating scattering curves was utilized for several globular proteins, and for the first time, reliable scattering curves were obtained for protein-“bound” water complexes. In the case of globular proteins and tRNA, this technique has permitted a strict evaluation of their accessible surfaces, their volumes, and, apparently for the first time, their complete molecular surfaces.

Notes: An extended retro-inverso modification was introduced at the central six residues of the somatostatin molecule, the region of internal enzymatic degradations. The synthesis of the analog [Ala4,g-Phe6-r-D-Phe7-r-D-Trp8-r-D-Lys9-r-D-Thr10-m-R,S-Phe11]-somatostatin required a unique strategy accommodating the unusual structure. Side-chain protection based on the t-butyl group in combination with Fmoc and Nps α-amino protection was employed. The key component containing the gem-diaminoalkyl residue was generated by an iodobenzene bistrifluoroacetate-mediated reaction. The separation of diastereomers of the cyclic tetradecapeptide in highly pure form was accomplished by high-performance liguid chromatography on a semipreparative scale. The analogs exhibited very low potency in the growth hormone inhibition test in vitro. This is interpreted as the consequence of the complex structural changes created by the extended retro-inverso modification.

Notes: The nature of internal and overall motions in native (double-stranded) and denatured (single-stranded) DNA fragments 120-160 base pairs (bp) long is examined by molecular-dynamics modeling using 13C-nmr spin-relaxation data obtained over the frequency range of 37-125 MHz. The broad range of 13C frequencies is required to differentiate among various models. Relatively narrow linewidths, large nuclear Overhauser enhancements (NOEs), and short T1 values all vary significantly with frequency and indicate the presence of rapid, restricted internal motions on the nanosecond time scale. For double-stranded DNA monomer fragments (147 bp, 24 Å diam at 32°C), the overall motion is that of an axially symmetric cylinder (τx = ∼10-6 s;τZ = ∼1.8 × 10-8s), which is in good agreement with values calculated from hydrodynamic theory (τx = ∼1.8 × 10-6 s; τZ = ∼2.7 × 10-8 s). The DNA internal motion can be modeled as restricted amplitude internal diffusion of individual C—H vectors of deoxyribose methine carbons C1′, C3′, and C4′, either with conic boundary conditions (τw = ∼4 × 10-9 s, θcone = ∼21°) or as a bistable jump (τA = τB = ∼2 × 10-9 s, θ = ∼15°). We discuss the critical role in molecular-dynamics modeling played by the angle (β) that individual C—H vectors make with the long axis of the DNA helix. Heat denaturation brings about increases in both the rate and amplitude of the internal motion (described by the wobble model with τW = ∼0.2 × 10-9 s, θcone = ∼50°), and overall motion is affected by becoming essentially isotropic (τx = τZ = ∼5 × 10-8 s) for the single-stranded molecules. Since 13C-nmr data obtained at various DNA concentrations for C2′ of the deoxyribose ring is not described well by the above models, a new model incorporating an additional internal motion is proposed to take into account the rapid, extensive, and weakly coupled motion of C2′.

Notes: The interaction of methyl orange with poly(L-lysine) was studied kinetically by the stopped-flow technique with CD detection, as well as by static CD titration experiments. In the static experiments, the differences observed in the polymer-to-dye ratio dependences of the CD spectra and absorption spectra suggested at least two kinds of bound states of the methyl orange attached to the polymer. The kinetic experiments using the stopped-flow apparatus, however, revealed four distinct reaction processes. The reaction mechanism was elucidated from the concentration dependence of the time constant for each process as follows: the first process was attributed to the bimolecular binding step of methyl orange to the side chain of poly(L-lysine), the second and third process were ascribed to the intramolecular reaction of the polymer-dye complex, and the fourth process was found to be the intermolecular aggregation of the polymer-dye complex. The origin of the stacking of methyl orange on poly(L-lysine) is discussed on the basis of the characteristics of signal amplitudes obtained from the kinetic experiments for these processes.

Notes: Novel devices for the spectroscopic and chromatographic analysis of the denaturation curves of the protein are described. A multidimensional spectroscopic measuring system makes it possible to carry out simultaneous and continuous acquisition of a set of data of different spectroscopic dimensions at several wavelengths in the course of increasing or decreasing denaturational perturbation. GuHCl-gradient chromatography can provide information about the progressive change of the protein volume in the course of increasing GuHCl concentration. Thus, denaturation curves with a high data-point density can be obtained. The data-storing function by a magnetic disk memory provides enough precision for a rigorous investigation of the correlation among the curves that probe different aspects of denaturation. The GuHCl denaturation of RNase A, cytochrome c, and pepsinogen are studied to demonstrate the high performance of these devices. Three types of transitions are found in these three proteins and the multiphasic nature of the transitions is clearly detected in the last two proteins.

Notes: A sufficiently long semiflexible filamentous macromolecule is theroretically expected to exhibit three different domains of behavior of its apparent diffusion coefficient Dapp(K) as a function of scattering vector K: (1) the small wave vector limit, where Dapp(K) = D0 is the translational diffusion coefficient of the center-of-mass; (2) the universal K3 region, where Dapp(K) = (kBT/6πη)K is a universal function of K independent of any property of the molecule itself; (c) the plateau region at large K2, where Dapp(K) approaches either a plateau, or gradually sloping quasiplateau, characteristic of local (elastic) rigid-body motions of the filament. The existence of each of these different domains has now been established experimentally for at least some polymers. The boundaries of the universal K3 region and the plateau region are determined theoretically here using precise quantitative criteria for universal or plateau behavior of Dapp(K) for a Rouse-Zimm model containing N + 1 subchains with rms subchain extension b. Allowing a maximum of 13% nonuniversal behavior, the domain of the universal K3 region is given by K2R2G = K2Nb2/6 ≥ 7 and K2b2 ≤ 0.54. Allowing as much as 10% nonplateau behavior, the boundary for onset of plateau behavior is K2b2 = 18.3. Dapp(K) is at least 50% nonuniversal when K2b2/6 = 6 ln 3. Extension of these results to DNA is examined theoretically, and good agreement of the pertinent predictions with published experimental data is demonstrated.It is concluded that no truly universal K3 region exists for DNA with Mr ≤ 107 and persistence length a ≥ 450 Å, although marginally (≤17% nonuniversal) universal behavior, is exhibited in a very narrow domain 0.64 × 1010 ≤ K2 ≤ 0.84 × 1010 cm-2 for φ29 DNA (Mr = 11.5 × 106). More than 50% of Dapp(K) is governed by local (elastic) rigid-body motions when K2 = 5.23 × 1010 cm-2. The existence of a very wide region of nonuniversal apparent K3 behavior extending up to very large K2, far into the plateau region, is demonstrated in a plot of Dapp(K)/K vs K2 for the Rouse-Zimm model. This is shown to stem in part from visual artifacts of plotting Dapp(K)/K vs K2, even for rigid species.

Notes: The rotational relaxation tiem τ3 of DNA molecules (Mw ≃ 5 × 106) in solution has been determined by the transient electric birefringence method. The analysis of the birefringence decay makes it possible to study only the higher-molecular-weight fraction, the molecules being considered as rigid elongated particles in a short time scale. A marked concentration dependence of the relaxation time has been observed for DNA in low ionic strengths. Above a critical concentration c*, τ3 increases with the DNA concentration, c. The value of c* increases with the ionic strength. For 10-3 ionic strength (with NaCl), c* is about 10 μg/mL; then we observe the same strong concentration dependence of rotational relaxation times as recently reported for rodlike M-13 viruses [Maguire, J. F., McTague, J. P. & Rondelez, F. (1980) Phys. Rev. Lett. 45, 1891-1894]. These results may be discussed in terms of the Doi-Edwards theory for rotational relaxation time of rigid macromolecules [Doi, M. (1975) J. Phys. 36, 607-611; Doi, M. & Edwards, S. F. (1978) J. Chem. Soc. Faraday Trans. 74, 918-932] and the critical concentration above which the interactions between the molecules begin to appear allows determining the corresponding molecular length. We observe a very good agreement between the DNA lengths obtained from the c* values and by using the infinite dilution value of τ3 and Broersma's equation. Therefore, only highly diluted solutions can be used if intrinsic molecular properties based on the rotational diffusion of high-molecular-weight elongated molecules are studied.

Notes: A thermodynamic treatment of the helix-coil transition of synthetic polypeptides in binary organic solvent mixtures is extended to describe isobaric heat-capacity increments associated with the phenomenon. This development resolves such increments into three components: two associated respectively with intrinsic differences between the ordered and disordered states of the macromolecule and between the coil-solvent complex and its components, and a third term derived from the temperature dependence in the fraction of coil residues bound to active solvent. Insights derived from this analysis are also applied to the discussion of some heat capacity increments associated with the denaturation of globular proteins.

Notes: The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.

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.