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

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

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

Notes: Some experimental data are given on the infrared spectra between 3300 and 3500 cm-1 of dilute solutions in carbon tetrachloride of three types of model compounds: CH3-CONH-CH(R1)-CONH(R2), (I); CH3-CON(CH3)-CH(R1)-CONH(R2), (II) and CH3-CONH-CH(R1)-CON(R2)2, (III). In studying the N-H stretching bands, it was found that there are two types of intramolecular hydrogen bonds in these molecules; these result in two different cyclized conformations, C5 and C7, which contain respectively, five and seven atoms in the ring. By using model substances I, II, and III, in which the nitrogen atoms are unequally substituted, it is possible to identify the N-H stretching bands which are to be ascribed to the N-H oscillators included in the two different chelated conformations. It is found also that the stretching frequency of a free N-H oscillator depends upon the substituent on the nitrogen atom. Thus, it is possible to observe, with some of the model compounds I, four different absorption bands located at 3340, 3420, 3440, and 3460 cm-1. The first two are ascribed to the N-H oscillators included in the H—bonds which lock the C7 and C5 conformations; the last two correspond to free N-H which differ with the substituent on the nitrogen atom.

Notes: Molecular orbital calculations of the extended Hückel type have been used to study the conformations of glycyl and alanyl residues in ground and excited states. The ground-state surfaces show features similar to those obtained with the standard calculational methods in which the total energy is partitioned into components such as torsions, nonbonded and electrostatic interactions. The molecular orbital calculations provide the first independent theoretical check on such calculations. The excited-state surfaces, Uniquely available from the molecular orbital calculations, exhibit a better definition and sharpening of potential minima in the sterically allowed regions.

Notes: We derive equations for calculating the number-, weight-, and z-average lengths between single-strand breaks in DNA molecules. Provision is made for the existence of preformed breaks under genetic control: these are assumed to be fixed in number in each molecule, but may be variable in position. Breaks due to random degradation may be superimposed on the pre-existing breaks. An example is given for illustrative purposes.

Notes: The conformation in solution of fractionated 30 S and 19 S ribosomal RNA from rabbit reticulocytes has been studied by optical rotatory dispersion, analysis of thermal melting profiles and their derivatives, and spectrophotometric acid-base titration. From a consideration of the limitations of these methods, it has been possible to set limiting values on the degree of base-pairing and the lengths of the double helices: between 60 and 80% of the bases in 19 S and 30 S RNA are estimated to be paired. The paired segments are not shorter than 4 base pairs, and evidence from other sources is available which indicates that they are not longer than 8-16 base pairs. The spread of helix lengths is greater in the 30 S than in 19 S RNA; and other differences are noted. Several distinct populations of double helices, differing in their thermal stability, are present. Estimates are presented from spectrophotometric and titration data for the base compositions of the paired and unpaired regions.

Notes: A small perturbation analysis is carried out to determine the stability of a fluid containing two layers of diffusing solutes in a common solvent and acted upon by a uniform gravitational field. It is found that instability can arise even though the unperturbed diffusion does not lead to the formation of a density inversion within the fluid.

Notes: The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix-coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix-coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.

Notes: Sodium counterion association with partially neutralized poly(D-glutamic acid) or poly(DL-glutamic acid) was measured by use of Wall's transference method with radioactive sodium. In the region where both polyacids are in completely random coil form, fractions of association were considerably less than that with poly(acrylic acid) in the same region of degree of neutralization. Even in the region where poly (D-glutamic acid) is in the helical form, the fraction of association was less than that with poly(acrylic acid) in the same region. No pronounced characteristics attributable to counterion association corresponding to the helix-coil transition could be found. The association phenomena were discussed on the basis of a rodlike model of polyelectrolyte.

Notes: The isomerization of poly-L-proline in different solvents has been studied by NMR spectroscopy. Different resonance signals for the CHα protons have been obtained for the two different helical conformations of thus compound, namely form I and form II.

Notes: Poly-β-N-diphenylmethyl-L-asparagine and poly-γ-N-diphenylmethyl-L-glutamine were prepared from the corresponding N-carboxyanhydrides. Poly-L-aspuragine and poly-L-glutamine were obtained by removal of the diphenylmethyl protecting groups with liquid anhydrous hydrofluoric acid.

Notes: The Hydrogen-tritium exchange character of poly-D,L-alanine was studied in detail as a model for the hydrogen exchange behavior of the unhindered, polymeric peptide group. The random chain nature of poly-D,L-alanine was evident in the uniformity of exchange rate of all its hydrogens and in the similarity between this rate and that of random chain poly-D,L-lysine and other known, unhindered secondary amide groups. An equilibrium isotope effect favoring the binding of tritium over protium to the extent of 21% was measured. Specific acid and base catalysis of the exchange and the absence of detectable general catalysis were demonstrated. Apparent energy of activation is 17 kcal/mole for deprotonation, largely due to dependence of Kw on temperature, and 15 kcal/mole for protonation, which correlates with the extreme apparent pK. The hydrogen -tritium exchange half-time rate; of poly-D,L-alamine at any pH and temperature (T: °C) is given by the equation: \documentclass{article}\pagestyle{empty}\begin{document}$$ \[t_{\frac{1}{2}} \,(\min )\, = \,200\,\, \times \,10^{0.05{\rm T}} /\,[10^{{\rm pH} - 3} \, + \,10^{3 - {\rm pH}} ]\] $$\end{document}

Notes: The specific rotation of starch components, corrected for refractive index variation, exhibits a discontinuity in the region of the water-dimethyl sulfoxide (H2O-DMSO) system that corresponds to the composition of the complex 2H2O-DMSO. This discontinuity is a property dependent upon the presence of a number of consecutively linked α-1,4 glucose units and, therefore, must reflect a change in symmetry of a segment, of polymer chain. The optical rotation of amylose between 26.5 and 92.5°C. does not change in DMSO and is only slightly lowered in water at the higher temperature. The behavior of amylose in both DMSO and H2O is like that of a random coil, as indicated by viscosity and sedimentation measurements. These results may be interpreted either as being compatible with models of amylose in solution in which the polymer backbone has helical twist, or as indicating removal of strong interactions between polymer chain segments by a good solvent.

Notes: Computer simulation shows that the time required to attain near sedimentation equilbrium is dramatically reduced by a two-step initial loading in which a macromolecular solution at low or zero concentration is layered above one at a higher concentration. To achieve the minimum time requires a good estimate of the molecular weight, but at least a 50% reduction in time can be achieved if the molecular weight of the macromolecule is known only within a factor of 2. Numerical solutions to the differential equation of the ultracentrifuge are calculated using the finite element method. An efficient Gaussian elimination algorithm can be used to minimize calculation time and computer storage requirements.

Notes: The aldehydes present in acid-soluble type I collagen react with pyrenebutyrylhydrazine to form various types of complexes under different reaction conditions. These complexes exhibit one or more of three different pyrene fluorescence bands: monomer, excimer, and aggregate fluorescence. Collagen, whose aldehydes have been reduced with NaBH4, does not react with this fluorescent hydrazine, confirming that the hydrazine reacts specifically with aldehyde groups to form hydrazones. The absence of a reaction with pepsin-treated collagen also shows that the fluorescent labels are primarily in the nonhelical terminal telopeptides. Upon dialysis, the pyrene label bound to a saturated aldehyde in an α-chain is lost; whereas that bound to an unsaturated aldehyde remains on the protein. The pyrene monomer fluorescence in the β-chain of old collagen is stronger than that of young collagen. The formation of the pyrene excimer fluorescence implies the proximity of two pyrene molecules, probably attached to two adjacent aldehydes. Upon changing from acidic to neutral pH, both excimer and aggregate fluorescence bands disappear within a few seconds, revealing a very rapid alteration at the telopeptides.

Notes: We describe conditions which lead to complete helix formation of poly(I) in the presence of NH4+. Binding of NH4+ is shown to be specific in the presence of Li+, which does not by itself support helix formation under these conditions. The NH4+-poly(I) complex is characterized by uv, CD, and ir spectroscopy. The CD spectrum is strikingly different from those of the Na+ or K+ complexes, the first extremum being changed from negative for the metal ions to positive for NH4+. A stereospecific model is proposed for the NH4+-poly(I) helix in which the N of NH4+ is located on the axis of the four-stranded helix, midway between planar tetramers formed by the bases. The model is consistent with the tetrahedral symmetry of NH4+, the requirement for four acceptable hydrogen bonds, the observed stability of the helix, and the accepted geometry of the backbone.

Notes: It has recently been proven that the counterion condensate around an isolated line charge in an electrolyte, as characterized by nonlinear Poisson-Boltzmann theory, is an encapsulating δ-function. Here the identical result is shown to hold in the framework of the polyelectrolyte theory of Fuoss, Katchalsky, and Lifson. The proof fully exploits analytic solutions to the differential equation which are not available for the nonlinear, cylindrical Poisson-Boltzmann equation.

Notes: The extent and modes of binding of the divalent metal ions Mn2+ and Co2+ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA-bound Mn2+ and Co2+ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn2+ and Co2+ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn2+ and Co2+ upon binding to DNA. An average 2-3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn2+ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn2+ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.

Notes: The dc electrical conductivity of films of the polyelectrolyte complexes of glycol chitosan (GlChi) with the sodium salts of dextran sulfate (DS), carboxymethyl cellulose (CMC), polygalacturonic acid (GalUA)n, and alginic acid (AlgA) was measured at temperatures above and below room temperature. The maximum field strength in the thinnest film used amounted to 3 × 104 V/cm. A plot of normalized current against the reciprocal of the absolute temperature revealed two regions with different slopes, and activation energies in these two regions have been obtained for all the complexes. The activation energies in the high-temperature region vary from 0.85 to 1.18 eV and in the low-temperature region from 0 to 0.22 eV. Reasons are given to show that the conductivity is probably ionic. Near room temperature, the current-voltage relation is almost linear in the GlChi-DS complex, while in the other three complexes the current varies as a power n of the voltage with the value of n ranging from 1.7 to 2.5. A rise in temperatures causes an increase in the slope of the log I vs log V plot in GlChi-DS and GlChi-CMC complexes. The nonlinear current-voltage relation is ascribed to a space-charge-limited conductivity.

Notes: Five different glucomannan samples were recrystallized from dilute solution. Depending on the experimental conditions, the crystals obtained could be identified as corresponding to the mannan I (anhydrous precipitate of more or less regular lozenge-shaped crystals) or mannan II (hydrated gel-forming pseudo-fibrillar precipitate). High-molecular-weight material, low temperature of crystallization, or a polar crystallization medium favored the mannan II polymorph, whereas a low-molecular weight, a high temperature of crystallization, and a crystallization medium of low polarity yielded the mannan I polymorph. Since the base-plane unit-cell dimensions are fairly constant with respect to variation of glucose, it is likely that isomorphous replacement of mannose by glucose occurs in glucomannan crystallization; the data also indicate that perfection of the glucomannan crystals was reduced in specimens having a high glucose:mannose ratio. The oriented crystallization of glucomannan on cellulose microfibrils was also studied under conditions where the mannan I polymorph was obtained. This gave shish-kebab structures that were characterized.

Notes: The primary hydration process of native biopolymers is analyzed in a brief review of the literature, pertaining to various aspects of biopolymer-water systems. Based on this analysis, a hydration model is proposed that implies that the solution conformation of native biopolymers is stable at and above a critical degree of hydration (hp′ = 0.06-0.1 g H2O/g polymer). This water content corresponds to the fraction of strongly bound water, and amounts to ∼20% of the primary hydration sphere. In order to test this model, detailed sorption-desorption scanning experiments were performed on a globular protein (α-chymotrypsin). The results obtained are consistent with the proposed hydration model. They show that under certain experimental conditions, sorption isotherms can be obtained that do not exhibit hysteresis. These data represent equilibrium conditions and are thus accessible to thermodynamic treatment. Valid thermodynamic functions, pertinent to the interaction of water with biopolymers in their solution state, can be obtained from these sorption experiments.

Notes: The light scattering of bovine serum albumin (BSA) has been measured at protein concentration up to 90 g/L and at pH values between 4.4 and 7.6. The dependence of scattering on both protein concentration and pH may be quantitatively accounted for by a simple extension of the hard-sphere model for protein solutions [Ross, P. D. & Minton, A. P. (1977) J. Mol. Biol. 112, 437-452] allowing for electrostatic repulsions between molecules. According to the extended model, the radius of the effective hard spherical particle representing BSA varies with the net electrical charge of the BSA molecule in a manner which may be calculated from electrostatic theory.

Notes: A complete analysis of all possible conformations with correct hydrogen bonds of the collagen II type was performed on the basis of developed simultaneous equations. Using a unimodal search (by varying Ψ3), the energetically favorable structure was obtained. No other energetically satisfactory structural solutions are possible. The next aim was to obtain a precise model of the molecule. The program used includes a subroutine for continual deformation of the pyrrolidine rings. The set of parameters determining the structure consists of 14 independent variables (8 dihedral and 6 bond angles). As starting points for the energy optimization, conformations produced by scanning and some structures from previous work were used. The final structures (practically the same for both polymers) have helix parameters h = 0.285 nm and t = 52°, which are in excellent agreement with the 7/2 symmetry of diffraction data. The conformations of the pyrrolidine rings are of the B type, i.e., C2-Cβ-exo-Cγ-endo. For both polypeptides, the conformations of imino acids in position 3 of the triplet are the same; in position 2, however, they are slightly different. The difference in diffraction patterns for the 7/2 and 10/3 helices is discussed.

Notes: The CD spectrum of the enzyme adenylate kinase has been investigated. Theoretical calculations, based on the x-ray crystal structure, have been carried out by means of an origin independent matrix formalism. The entire molecule was included in the calculations in the sense that essentially all electronic transitions that occur at wavelengths longer than 185 nm were included in the basis set. A linear dielectric function was utilized to evaluate the intertransition coupling potentials. The results of the theoretical calculations were in reasonable agreement with experimental CD spectra of the molecule.

Notes: The mode of action of many antitumor agents entails the inhibition of nucleic acid synthesis. Because many of the drugs can intercalate, it is assumed that intercalation is an important step in the mechanism of biological activity. As intercalants contain a planar chromophore as an ingredient essential for intercalation, chromophores that should fit into DNA are desired. This is the main theme of this investigation. Binding to DNA of fundamental moieties, protonated pyridine, aniline, phenol, quinone, and 4H-thiopyran-4-one, is studied to determine their optimum placement in DNA. The optimum orientations for each moiety are superimposed to form polyaromatic systems that can intercalate in a manner in which functional groups on these chromophores are oriented as in the moieties themselves. Ideal intercalants proposed contain three and four fused ring system, have protonated ring nitrogen atoms located to maximize the electrostatic interactions with DNA, hydroxy and amino groups that can hydrogen bond to the OII and O5′ phosphate backbone atoms, and carbonyl and sulfur groups in the central position of the ring system to provide variations in the chromophore and to interact with the relatively positive region in the intercalation site. The optimum orientation occurs when the chromophore and the base pairs overlap to the maximum extent. The ideal intercalants are fundamentally of the type:

Notes: DNA reassociation kinetics using the phenol emulsion reassociation technique (PERT) [Kohne, D. E., Levison, S. A. & Byers, M. J. (1977) Biochemistry 16, 5329-5341] has been investigated at high DNA concentrations using an endonuclease S1 assay of reaction progress. Apparent second-order rate constants fall on two intersecting straight lines when presented as a function of DNA concentrations on a log-log plot. In the low DNA concentration range, the rate constants drop about 10-fold when concentration increases 1000-fold. In the high DNA concentration range, the rate constants drop more than 10-fold when concentration increases 10-fold. The slopes of these lines are the same in different solvents and at different temperatures. The intersection between the lines occurs when the available catalytic surface is saturated. At high DNA concentrations, high-complexity heterologous denatured DNA apparently competes 2-4 times better for the surface than homologous DNA because it does not participate in a reassociation reaction. Native and partially native DNA molecules cannot compete with single-stranded DNA for a saturated surface. At high DNA concentrations, reactions using PERT become dependent on the single-strand DNA length. Increasing length lowers reassociation rates.

Notes: Classical potential functions (CPF) calculations on 3′-mononucleotides, the building blocks of nucleic acids, predict a correlation between the sugar ring pucker and the torsion angle Φ′ around the C3′—O3′ bond. In ribonucleotides, the value of Φ′ depends on the sugar pucker, viz. the C2′-endo sugar pucker is associated with Φ′ = 210° and 270°, while the C3′-endo sugar pucker favors only Φ′ = 210°. On the other hand, in deoxyribonucleotides, both sugar puckers show a preference for Φ′ = 180°. These theoretical predictions are fully corroborated by the results obtained from x-ray and nmr studies on mono-, di-, and polynucleotides.

Notes: The effect of several surfactants on the secondary structure of bovine β-lactoglobulin B was determined from the circular dichroism spectra. The spectra were measured at several concentrations of surfactant ranging from 1 mg/mL to the critical micelle concentration. The surfactants studied were sodium dodecyl, decyl, and octyl sulfate, sodium dodecyl sarcosinate, dodecyltrimethylammonium bromide. The data were analyzed using the method of Chen et al. [Biochemistry (1974) 13, 3350-3359] to determine the percentage of α-helix, β-sheet, and unordered form at each surfactant concentration. In every case, an increase in structured form and a 20-25% decrease in the amount of unordered form was noted when the surfactant concentration reached the critical micelle concentration. However, the relative amounts of the two structured forms present depend on the surfactant used. The profile of the secondary structure of the protein also varied from surfactant to surfactant as the protein was titrated, probably reflecting the delicate balance between ionic and nonionic forces that governs the secondary structure of β-lactoglobulin and most other globular proteins in aqueous solution.

Notes: Amino acids are known to differ in their individual preferences for each of the four positions of the β-turn conformation formed by tetrapeptide segments. Proline and glycine show relatively high preferences for positions 2 and 3, respectively, of the β-turn. Using tripeptides of the type N-acetyl-Pro-Gly-X-OH, where X = Gly, Ala, Leu, Ile, and Phe, we have sought to study the influence of the 4th residue X on the stability of the β-turn conformation in these tripeptides. Our nmr and CD results show that the β-turn stability is quite significantly governed by the nature of the amino acid residue at this position in the following order: Leu 〉 Ala 〉 Ile, Gly 〉 Phe.

Notes: Thermodynamic studies of the binding of adamantanecarboxylate to cyclodextrins have been made as a function of temperature and added organic cosolvent (methanol) using flow microcalorimetry. The negative heat capacity change associated with the adamantanecar-boxylate/β-cyclodextrin interaction and the fact that the interaction is weakened by the addition of methanol implicate the binding process as being a hydrophobically driven one. The negative enthalpy change (ΔH0 = -5.5 kcal/mol) and near-zero entropy change (ΔS0 = 1.5 cal/mol deg) are quite different from the values normally expected for a hydrophobic bond, indicating that other bonding forces are important in addition to the hydrophobic effect. The relative contribution of the hydrophobic effect and other bonding forces (most likely van der Waals forces) to the overall binding was judged from an analysis of the dependence of the thermodynamics of the association process on the surface tension of the water-methanol mixtures following a model for “solvophobic” bonding described by Sinanoglu [Molecular Associations in Biology (1968) Academic Press, New York, pp. 427-445]. From this analysis, adamantane-carboxylate/cyclodextrin complex formation is found to be driven to the extent of -1.9 kcal/mol by the hydrophobic effect. Furthermore, the hydrophobic driving force is found to be characterized by a positive ΔS0 of 10 cal/mol deg. The remaining free energy of binding (and the ΔH0 of binding of ∼-6 kcal/mol) is then due to the intrinsic (surface-tension-independent) van der Waals interaction between the ligand and cyclodextrin cavity.

Notes: Pivaloyl-L-Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in (CD3)2SO solution, by 1H-nmr methods, suggesting the existence of β-turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II β-turn conformations are about 2 kcal mol-1 more stable than Type III structures. A crystallographic study has established the Type II β-turn in the solid state. The molecule crystallizes in the space group P21 with a = 5.865 Å, b = 11.421 Å, c = 12.966 Å, β = 97.55°, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II β-turn conformation is stabilized by an intramolecular 4 → 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are φPro = -57.8°, ψPro = 139.3°, φAib = 61.4°, and ψAib = 25.1°. The Type II β-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.

Notes: The folding-unfolding process of reduced bovine pancreatic trypsin inhibitor was investigated with an idealized model employing approximate free energies. The protein is regarded to consist of only Cα and Cβ atoms. The backbone dihedral angles are the only conformational variables and are permitted to take discrete values at every 10°. Intraresidue energies consist of two terms: an empirical part taken from the observed frequency distributions of (φ,ψ) and an additional favorable energy assigned to the native conformation of each residue. Interresidue interactions are simplified by assuming that there is an attractive energy operative only between residue pairs in close contact in the native structure. A total of 230,000 molecular conformations, with no atomic overlaps, ranging from the native state to the denatured state, are randomly generated by changing the sampling bias. Each conformation is classified according to its conformational energy, F; a conformational entropy, S(F) is estimated for each value of F from the number of samples. The dependence of S(F) on energy reveals that the folding-unfolding transition for this idealized model is an “all-or-none” type; this is attributable to the specific long-range interactions. Interresidue contact probabilities, averaged over samples representing various stages of folding, serve to characterize folding intermediates. Most probable equilibrium pathways for the folding-unfolding transition are constructed by connecting conformationally similar intermediates. The specific details obtained for bovine pancreatic trypsin inhibitor are as follows: (1) Folding begins with the appearance of nativelike medium-range contacts at a β-turn and at the α-helix. (2) These grow to include the native pair of interacting β-strands. This state includes intact regular secondary conformations, as well as the interstrand sheet contacts, and corresponds to an activated state with the highest free energy on the pathway. (3) Additional native long-range contacts are completely formed either toward the amino terminus or toward the carboxyl terminus. (4) In a final step, the missing contacts appear. Although these folding pathways for this model are not consistent with experimental reports, it does indicate multiple folding pathways. The method is general and can be applied to any set of calculated conformational energies and furthermore permits investigation of gross folding features.

Notes: Peptide NH chemical shifts and their temperature dependences have been monitored as a function of concentration for the decapeptide, Boc-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-OMe in CDCl3 (0.001-0.06M) and (CD3)2SO (0.001-0.03M). The chemical shifts and temperature coefficients for all nine NH groups show no significant concentration dependence in (CD3)2SO. Seven NH groups yield low values of temperature coefficients over the entire range, while one yields an intermediate value. In CDCl3, the Aib(1) NH group shows a large concentration dependence of both chemical shift and temperature coefficient, in contrast to the other eight NH groups. The data suggest that in (CD3)2SO, the peptide adopts a 310 helical conformation and is monomeric over the entire concentration range. In CDCl3, the 310 helical peptide associates at a concentration of 0.01M, with the Aib(1) NH involved in an intermolecular hydrogen bond. Association does not disrupt the intramolecular hydrogen-bonding pattern in the decapeptide.

Notes: A formalism for extracting the conformations of a proline ring based on the bistable jump model of R. E. London [(1978) J. Am. Chem. Soc. 100, 2678-2685] from 13C spin-lattice relaxation times (T1) is given. The method is such that the relaxation data are only partially used to generate the conformations; these conformations are constrained to satisfy the rest of the relaxation data and to yield acceptable ring geometry. An alternate equation for T1 of 13C nuclei to that of London is given. The formalism is illustrated through an example.

Notes: We have used broadline proton magnetic resonance to study molecular motion in cellulose, a sodium pectate solution, a calcium pectate gel, and isolated bean cell walls. All samples were prepared in D2O to minimize the contribution of water to the observed signals. For each sample, a free induction decay was obtained, and the second moment, spin-lattice relaxation, and dipolar relaxation were measured. Our results show that the large majority of protons in cellulose are immobile. Rigid and mobile domains were also observed in the pectate samples. We have shown that gelation induces large-scale changes in the free induction decay, the second moment, and the relaxation behavior of the pectate. As with the other samples, rigid and more mobile domains were found in bean cell walls. The fraction in the rigid domains is much larger than the fraction of cellulose in the sample, suggesting that the noncellulosic wall components are also organized into rigid and mobile domains.

Notes: Conformational energy computations were carried out on collagenlike triple-stranded conformations of several poly(tripeptide)s with the general structure CH3CO—(Gly—X—Y)3—NHCH3. The sequences considered had various amino acid residues in position X or Y of the central tripeptide, with either Pro or Ala as a neighbor, i.e., Gly-X-Pro, Gly-X-Ala, Gly-Pro-Y, and Gly-Ala-Y. Minimum-energy conformations were computed for the side chains, and their distributions were compared for the four sequences. The residues used were Abu (= α-aminobutyric acid), Leu, Phe, Ser, Asp, Asn, Val, Ile, and Thr. The conformational energy of a —Ch2—CH3 side chain in Abu was mapped as a function of the dihedral angle χ1. Intrastrand interactions with neighboring residues do not affect the conformations of a side chain in position Y, and they have a minor effect on it in the X-Ala sequence, but they strongly restrict the conformational freedom of the side chain in the X-Pro sequence. Conversely, interstrand interactions do not affect side chains in position X, but they strongly restrict the conformational freedom of a side chain in position Y if there is a nearby Pro residue in a neighboring strand. Hydrogen bonds with the backbone can be formed in some conformations of long polar side chains, such as Asp, Asn, or Gln. All amino acid residues can be accommodated in collagen. Because of the interactions mentioned above, steric and energetic constraints can be correlated with observed preferences of certain amino acids for positions X or Y in collagen. Hence, these preferences may be explained, in part, in terms of differences in the conformational freedom of the side chains in the triple-stranded structure.

Notes: The ionization constants of the tyrosyl groups of chymotrypsinogen and of nitrated-chymotrypsinogen (two tyrosyl residues nitrated) have been determined by difference spectrophotometry. In chymotrypsinogen, two of the four tyrosyl groups ionize without any time dependence. Above pH greater than ca. 12.5, time-dependent spectral changes are seen for 0.7 group equivalent. The data can be fitted to the values of pK′1 9.75 ± 0.07, pK′2 11.55 ± 0.05, pK′3 13.30 ± 0.05. In nitrated-chymotrypsinogen, the two nitrated tyrosyl residues have pK′1 6.44 and pK′2 8.30. For both proteins, these pK′ values are in agreement with those evaluated from potentiometric titration and calorimetric data using computer-assisted curve-fitting analysis.

Notes: The conformation of several samples of poly(α,β-L-Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L-Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm-1 was shifted to 620 cm-1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.

Notes: The use of 1H-nmr spectroscopy is demonstrated to be a useful analytical method to characterize the structure of synthetic peptides attached to soluble, macromolecular polyoxyethylene (POE) supports in the liquid-phase method (LPM) of peptide synthesis. We report an extensive 360-MHz 1H-nmr study of POE-bound homo-oligo-L-methionine peptides. A combination of high field and selective saturation or Redfield pulse methods allows resolution of individual backbone NH and α-CH resonances of dilute peptides in the presence of strong resonances from macromolecular POE and/or protonated solvents. The nmr spectra for the POE-bound peptides in CDCl3 are qualitatively similar to those of the low-molecular-weight Boc-L-Metn-OMe peptide esters. This corroborates other observations that POE has little effect on peptide stucture. The backbone α-CH region of peptides is overlapped by signals from the terminal oxyethylene group of POE, but the peptide side-chain and low-field backbone NH resonances are well resolved. In trifluoroethanol the Boc-(L-Met)n-NH-POE heptamer and octamer adopt the right-handed α-helical structure, and the present nmr studies provide evidence for two strong intramolecular hydrogen bonds to stabilize the helices. In water, the N-deblocked derivatives, (L-Met)n-NH-POE oligomers adopt β-sheet structure and manifest well-resolved nonequivalent NH resonances with 6-7 Hz 3JNH-CH coupling constants.

Notes: The concerted model of Monod, Wyman, and Changeux is generalized so that all effects of interactions for an enzyme operating at a nonequilibrium stationary state are considered. In contrast to the original model, which is based on an analogy to equilibrium ligand binding, the generalization may show both “positive” and “negative cooperativity” in both catalytic binding and conformational processes. Furthermore, in contrast to any equilibrium binding model, the Hill coefficients may be greater than the number of sites n. For catalysis, the maximum value is 2n, and for conformational changes, n + 1. These points are illustrated by two cases that yield simpler analytic expressions. The first obtains when catalysis occurs on a much faster time scale than the conformational changes, and the second, when this situation is reversed.

Notes: By combining gel permeation chromatography (GPC) and light-scattering spectroscopy, including photon correlation and angular distribution of absolute scattered intensity, we were able to characterize immunologically active Haemophilus influenzae type b polysaccharide (HIB Ps) bovine serum albumin (BSA) conjugates in terms of equivalent hydrodynamic radius rh ∼ (6.2 ± 0.6) × 102 Å, apparent radius of gyration rg ∼ (5.4 ± 0.3) × 102 Å, apparent molecular weight Mw ∼ (3.5 ± 0.4) × 106 g/mol, and a second virial coefficient A2 ∼ (1.9 ± 0.3) × 10-4 cm3 mol/g2. We could study the effects of each of the processes in the conjugate formation according to the following procedure: BSA (dialysis, modification, fractionation) + HIB Ps → HIB Ps/BSA conjugate (conjugate formation, fractionation). Narrow distributions of HIB Ps BSA conjugate formation can be achieved using fractionated BSA.

Notes: We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ∼25 to ∼500 cm-1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm-1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm-1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm-1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.

Notes: A study of the oxygen replacement reaction of carbon monoxide-saturated hemoglobin (HbA0) was carried out using spectroscopic, calorimetric, and pH titration methods. Under fully saturated conditions the replacement reaction can be defined by a single partition constant over all ratios of bound oxygen to carbon monoxide. This indicates that under saturating conditions Haldane's first law for the ligand binding of gas mixtures holds for any CO/O2 ratio. It further shows that there is no appreciable difference in relative CO-O2 affinity between the α- and β-chains. The same partition coefficient was found to hold for different pH, buffer, and allosteric effector conditions. The lack of any pH dependence of the partition coefficient was confirmed by the absence of proton changes for the replacement reaction. The temperature dependence of the partition coefficient and calorimetric results yield a value for the enthalpy of the reaction of -3.65 ± 0.29 kcal/mol/heme.

Notes: We demonstrate that the isotropic absorption and linear dichroism in an unknown flow field can be used to determine base tilt in polynucleotides if three transitions are measured and the directions of the corresponding dipoles are known. The method is applied here to reach conclusions about the base tilt in poly(rA), poly(rA)+·poly(rA), and poly(rC). The respective values are: 28° tilt about the axis + 50° toward C8 from the C1′ → N9, and 25° tilt about the axis + 118° toward C5 from C1′ → N1. The results for poly(rA)+·poly(rA) are consistent with the accepted model. Spectra were measured for poly(rC)+·poly(rC), but definite conclusions must await reliable directions for transition dipoles. The dipole direction for the 218-nm transition in rC is found to be +13° or +43° toward C5 from C1′ → N1. The CD spectra to about 168 nm are presented and discussed.

Notes: Measurements of stress relaxation in uniaxial extension and associated time-dependent birefringence have been made on bovine fibrin film, prepared by gentle compaction of coarse fibrin clots, containing 13-22% fibrin plasticized with either aqueous buffer or glycerol. Both unligated and ligated (i.e., with α-α and γ-γ ligation by fibrinoligase, factor XIIIa) films were studied. Both types showed two stages of stress relaxation, with time scales of approximately 10 and 103-104 s, respectively, with a plateau region between. In the plateau, the nominal (engineering) stress for ligated glycerol-plasticized film is proportional to In λ, where λ is the stretch ratio, up to λ ≅ 2, and it decreases with increasing temperature. For unligated glycerol-plasticized film, the stresses are smaller by a factor of one-half to one-third. For ligated film, the second stage of relaxation is relatively slight, and recovery after release of stress is often nearly complete. For unligated film, the second stage involves a substantial drop in stress, and after recovery there is a significant permanent set. A second relaxation for ligated film reproduces the first, but for unligated film it reproduces the first only if the initial relaxation is terminated before the second stage; otherwise, the second relaxation shows a weaker structure. The behavior of water-plasticized film is similar to that of glycerol-plasticized except that the second stage of relaxation occurs at shorter times. During the first stage of stress relaxation, up to about 100 s, the birefringence and the stress-optical coefficient increase; during the plateau zone of stress relaxation, the birefringence of ligated films is approximately constant and is proportional to 2λ2/(λ2 + 1) - 1, where λ is the stretch ratio. This dependence is predicted by a two-dimensional model in which rodlike elements in the plane of the film are oriented with independent alignment. During the final stage of stress relaxation, the birefringence of ligated films decreases slightly; that of unligated films decreases substantially, but less rapidly than the stress, corresponding to a further increase in the stress-optical coefficient. With additional information from small-angle x-ray scattering reported in an accompanying paper, the first stage of relaxation is attributed to partial release of bending forces in the fibers by orientation, accompanied by increased birefringence. The second stage is attributed, for ligated films, to an internal transition in the fibrin units accompanied by elongation of some of the fibers; and in the unligated films, to a combination of the latter transition with slippage of protofibrils lengthwise within the fiber bundles that causes some loss of orientation, which diminishes the birefringence.

Notes: A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.

Notes: The distribution of fibers in agarose gels has been studied by electron-microscopic examination of replicas formed from freeze-fracture surfaces. For gels set in water, the results obtained support the model proposed for the gel structure by Arnott et al. (1974) of a random array of long, straight, connected fibers, with each fiber having a diameter equivalent to that of an aggregate of approximately 10-30 agarose helixes, depending on the initial agarose concentration. The density of these fibers, their water content, and the total length of fibers per unit volume have been derived from the measured distribution of intersections per unit area of freeze-fracture surfaces. For gels set in the presence of salt, the distribution of fibers becomes distinctly non-Poissonian, leading to larger interfiber spaces and a gel of greater effective pore size. The larger pore size of gels set in the presence of salt also has been revealed by electrophoretic measurements in which the relative migration rates of plasmid DNA molecules of varying conformations have been determined.

Notes: Energy embedding has been shown recently to be a useful extension of the distance geometry approach to conformational calculations in the case of very small molecules and simple energy functions. This paper tests the ability of energy embedding to locate low energy conformations satisfying both weak and strong geometric constraints when the molecule is the small protein, bovine pancreatic trypsin inhibitor, and the energy function is the complicated Oobatake-Crippen residue-residue potential. Using the potential function alone, the algorithm reaches a structure with energy lower than that of the native conformation, but with little resemblance to it. Aided by numerous geometric constraints, such as preformed secondary structure segments, the algorithm again finds a local minimum with energy better than that of the native, and with only 3.3 Å rms deviation from it. This is significantly closer to the native value than can be obtained using standard distance geometry and the geometric constraints alone. Thus, energy embedding using the Oobatake-Crippen potential function is a significant help in finding native conformations of proteins. However, additional trials on a hairpin bend fragment of trypsin inhibitor demonstrate the potential's shortcomings in encouraging proper secondary structure.

Notes: Molecular-mechanics calculations have been carried out on the base-paired deoxy dodecanucleoside undecaphosphates d(CGCGAATTCGCG)2 and d(A12)·d(T12). These refinements were carried out using the model-built Arnott B-DNA geometry as initial coordinates (with a helix repaeat of 10.0 residues/turn), as well as helix repeats ranging from 9 to 12 residues/turn. There was some variation in the optimum calculated helix repeat, depending on the dielectric model, the presence or absence of counterions, and the method used for inclusion for nonbonded interactions; the most interesting general result of these calculations was the coupling between furanose sugar puckering and twist. This coupling was observed for all models. With a helix repeat of 9.0 residues/turn, all sugars remain C(2′)endo after refinement; as the helix repear increases to 12.0 residues/turn, the number of sugars repuckering to O(1′)endo and C(3′)endo increases also. With our most rigorous model (i.e., a model with no cutoff distance for nonbonded interactions) and a helix repeat of 10.0 residues/turn, we find a greater tendency for pyrimidine than purine repuckering in d(CGCGAATTCGCG)2, in agreement with the x-ray structural data of Drew et al. [(1981) Proc. Natl. Acad. Sci. USA 78, 2179-2185].We also carried out a number of calculations in which we “forced” one of two deoxy sugars to repucker or one of the C3′-O3′-P-O5′ (ω) torsion angles to change from gauche- to trans using dihedral angle constraints. After the constraints were removed, some of these structures “reverted” to the sugar pucker of the initial structures, while others remained repuckered. In all cases, the energies for repuckered structures after refinement were very similar to energies of the initial structure. Experiments and theory suggest that local conformational fluctuations play an essential role in nmr relaxation of 31P and 13C atoms in double-helical DNA. The results of our previous calculations on hexanucleoside phosphates and the calculations presented there are consistent with an important contribution to nmr relaxation processes of conformational changes in the torsion angle ω′ from gauche- to trans and deoxy sugar repuckering from C(2′)endo to C(3′)endo. Specifically, the calculations presented here indicate a very flexible phosphate backbone in helixes having an intermediate helix repeat of 10 to 11 residues/turn. These helixes may accommodate sugars of variable pucker without significantly disrupting base-base hydrogen-bonding and stacking interactions. All of the variant structures are similar in energy, suggesting that conversion between them can occur on a nanosecond time scale, as observed in nmr relaxation experiments.

Notes: The microwave absorption of aqueous solutions of DNA extracted from E. coli has been studied between 8 and 12 GHz by the use of an optical heterodyne technique. By measuring optically the temperature rise produced in an absorbing sample by pulsed microwave radiation, unambiguous, direct measurement of the microwave absorption is possible. Our results show that E. coli DNA absorbs microwaves in the 8-12-GHz region substantially more efficiently than water, which is itself an extremely efficient absorber. The observed absorption is featureless and decreases slightly with increasing frequency. These observations are consistent with an explanation involving direct absorption by longitudinal acoustic modes of the double helix.

Notes: The formation of polyelectrolyte complexes of glycol chitosan (GlChi) with sodium salts of dextran sulfate (DS), carboxymethyl cellulose (CMC), polygalacturonic acid (GalUA)n, and alginic acid (AlgA) has been studied. The number of ionizable groups per pyranose ring and the degree of dissociation as a function of pH in the polycation and polyanions have been determined using conductometric and potentiometric titrations. The formation of the complexes at different pH values has been followed by turbidity measurements. It is found that stoichiometric complexes of the polycation with CMC, (GalUA)n, and AlgA are formed at a mixing ratio of 0.5, indicating that the conformation may correspond to chains of equal lengths. In the case of the complex of GlChi with DS, the stoichiometric composition corresponds to a mixing ratio of 0.62. Thin transparent films of the complexes have been obtained by dehydration under reduced pressure on a layer of mercury. The dielectric constant ε′ and loss ε″ of thin films of these complexes have been measured in the range of frequencies of 1-100 kHz at different temperatures above and below room temperature. The GlChi-DS complex shows very little change in the values of ε′ and ε″ with frequency or temperature. On the other hand, films of GlChi-CMC and GlChi-(GalUA)n complexes show a significant increase in ε′ and ε″ as the temperature is increased above room temperature. The increase is more prominent at low frequencies. This behavior is attributed to the Maxwell-Wagner interfacial polarization. In the case of GlChi-AlgA film, the values of ε′ and ε″ increase enormously as the temperature increases. This behavior is similar to the increase in ε′ and ε″ observed by Michels et al. [(1965) J. Phys. Chem. 69, 1456-1465] as the salt concentration increases in another polyelectrolyte complex studied by them. The electric double-layer mechanism proposed by Schwarz [(1962) J. Phys. Chem. 66, 2636-2642] to account for the dielectric properties of biocolloids is shown to account quantitatively for the observations on the GlChi-AlgA film.

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.