ALBERT

Notes: Statistical thermodynamic theories have been developed in order to explore the consequences of two prominent models of length regulation in linear protein aggregates: the length-determining factor model and the cumulated strain model. In the former, the possibility that the final subunit is bound with extra stability has been incorporated. Calculations explore the dependence of the sharpness of the onset of extensive polymerization, and the breadth, shape, and most probable value of the distribution as functions of concentrations of template and polymerizing subunit, of subunit association constant, of the strain free energy, and of the maximum length and extra stability of the last subunit in the length-determining factor model. Numerical calculations are made with particular reference to the parameters of bacteriophage T4 tail-core polymerization. Both models predict suitably narrow length distributions with plausible values of the thermodynamic parameters. The length-determining factor model may exhibit highly cooperative behaviour, with either fully polymerized or very short aggregates being observed as the product K[C] of association constant and free monomer constant is varied. This cooperativity is accentuated as binding of the final subunit becomes more stable. Species of intermediate length will be observable only if the length-determining factor is shortened. In the cumulated strain model, if K[C] is lowered at constant strain free energy per subunit, shorter aggregates will be observed; but increasing K[C] will not yield appreciably longer polymers. As [C] is lowered, the distribution will remain narrow.

Notes: An electron microscopic investigation on the structure of fibrin is reported. Fibrin morphology was investigated in a wide variety of experimental conditions, and by carefully controlled staining procedures. Two main band patterns A (230-Å-spaced main dark bands) and B (230-Å-spaced main light bands) are observed for stained fibrin; it is shown that the former results from the superimposition of both positive and negative staining, and the latter is given by positive staining. By suitable denaturation experiments, it was found that the fiber is composed of a regular alternation of lose and dense regions along the axis.We have assumed that the monomer of fibrin is described by the three-nodular model of Hall and Slayter, as supported by recent investigations. The monomers are arrayed according to a head-to-tail sequence along the fiber, and to a staggered lateral association. This model accounts for all the experimental observations, and predicts well the high-resolution band pattern of fibrin. It further agrees with the results of a recent work on the early stages of the fibrinogen-to-fibrin conversion.

Notes: Oligopeptides having the general formula N-carbobenzoxy(γ-methyl-L-glutamyl)n-dimethyl-L-glutamate (with n = 1, 3, 5, 7, 11) were prepared, using both solid-phase synthesis and conventional peptide couplings in solution. The appearance of an ordered structure with increasing chain length was studied in organic solvents and at the air-water interface. The results obtained by thin-layer chromatography were interpreted. All the studies have demonstrated a very similar behaviour of this type of oligopeptide in solution and in monolayers.

Notes: Raman spectra are reported for crystalline nonactin, monactin, dinactin, trinactin, and tetranactin and their solutions in CCl4, CHCl3, CH3OH, and 4:1 (v/v) CH3OH:CHCl3. The macrotetrolide nactins selectively bind a wide variety of cations, and are important model compounds for the study of ion complexation. The conformations of nonactin, monactin, and dinactin in solution are similar. Their conformations are found to be sufficiently open to permit the ester carbonyl groups to form hydrogen bonds with CH3OH; this gives rise to characteristic changes in the vibration frequencies associated with the ester groups. Nonactin, which is the least soluble of the nactins in CH3OH, is also the least effective at forming hydrogen bonds with CH3OH. The greater ability of the higher nactins to form hydrogen bonds with CH3OH may be due to the increased inductive effect of ethyl over methyl side chains, which may increase the dipole moment of the ester carbonyl groups. Spectra of crystalline nonactin, monactin, and tetranactin are fairly similar, while the spectra of dinactin and trinactin comprise a second, distinct family. This is consistent with X-ray crystallographic studies, which show that nonactin and tetranactin form monoclinic crystals, while trinactin is triclinic.

Notes: The kinetics of the acid hydrolysis of Streptococcus salivarius levan were studied by examining the weight-average molecular weight. The molecular weights were obtained in a continuous manner from light scattering. Two first order reactions were observed: an initial rapid reaction in competition with a slower reaction.Activation energies, activation entropies, as well as the rate dependence upon substrate and hydrogen ion concentrations were determined. The data seem to indicate that the fast reaction is related to the breaking of branch-point bonds while the slower reaction is related to the breaking of main-chain bonds. Thus, levan hydrolysis seems to be fundamentally different from the completely random degradation of other branched polysaccharides, i.e., dextran, glycogen, and amylopectin.

Notes: The conformations accessible to the internucleotide phosphodiester group in deoxydinucleoside monophosphates, deoxydinucleoside triphosphates, and deoxypolynucleotides have been explored in detail by potential energy calculations. The two most predominant conformations for the nucleotide moiety (3E and 2E) and their possible combinations (3E-3E, 3E-2E, 2E-2E, 2E-3E) have been employed, similar to our earlier studies on polyribonucleotides. The internucleotide P-O bond torsions are very sensitive to the sugar pucker (3E and 2E) and sugar type (ribose and 2′-deoxyribose) on the 3′-residue of dinucleoside phosphates. The preferred phosphodiester conformations found for the deoxydinucleoside monophosphates and triphosphates, in general, follow the same pattern as those obtained for ribose sugars when the sugar on the 3′-side of the molecule has the 3E sugar-ring conformation. The internucleotide P-O bonds show a greater degree of conformational freedom when the 3′-sugar has the 2E pucker. The double gauche g-g- conformation for the phosphodiester, which leads to the overlap of the adjacent bases, is shown to be one of the energetically most favored conformations for all the sequence of sugar puckers. It is found that the 2E-2E sequence of sugar puckers shows a greater energetic preference for the stacked helical conformation (g-g-) than the (3E-3E) and the mixed sugar-pucker combinations. This effect becomes more pronounced in going from a dinucleoside monophosphate to a dinucleoside triphosphate suggesting that the 2′-deoxy sugars favor the 2E sugar pucker in di-, oligo-, and polydeoxyribonucleotide structures. In addition to g-g-, the conformations g+g-, tg-, g-t, tg+, and g+t are also found to be possible for the phosphodiester in a polydeoxyribonucleotide and their populations depend to some extent on the sugar-pucker sequence. It is shown that the short-range intramolecular interactions involving the sugar and the phosphate groups dictate to a large extent the backbone conformations of nucleic acids and polynucleotides.

Notes: Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature tm, corresponding to the helix-coil transition of free base pairs, and another band at a higher temperature t′m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the tm and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a tm about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dtm/dXG·C, where XG·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of Tm is similar to that observed for pure DNA in high ionic strength. Although the t′m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the tm0 extrapolated from the linear plot of tm against log Na+. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).

Notes: Electron paramagnetic resonance spectroscopy has been used to study the coupling of Mn2+ ions with the tRNAVal1 modified with a spin label at four pseudouridylic residues and with the valyl-tRNAVal1 modified with a spin label at the α-amine group of the valyl residue. A sharp increase of spin-label mobility has been found in these samples, due to the conformational transition induced by the first and second Mn2+ ions. Analysis of dipole-dipole couplings of spin labels with the coordinated ions revealed a definite order in the occupation of ion coordination sites in the tRNA. For some valyl-tRNAVal1 molecules, the second Mn2+ ions were shown to coordinate on the α-amine group of the valyl residue at a distance of 15-25 Å from a spin label. As a result of the conformational transition, a coordination site appeared in the tRNA at one of the pseudouridylic residues, its distance from the spin label being less than 10 Å. It has been suggested that the conformational transition induced by ions excluded some bases from the system of hydrogen bonds at the level of the tRNA tertiary structure. As a result, these bases acquired sufficient sterical freedom to participate in the Mn2+ ion coordination.

Notes: We have studied the electrical conductivity of NaDNA solutions under “saltfree” conditions at temperatures well below the melting point of DNA, using radio-frequency dielectric and noise measurements. A conductivity discontinuity is observed at a temperature well below that at which the usual denaturation processes and trans conformation may commence. The radio-frequency permittivity also exhibits a discontinuity at the same temperature. For the premelting phase, the conductivity versus temperature curves consist of two linear regions with a change in slope occurring at 23°C. This effect is related to the behavior of the ionic sheath covering the DNA macromolecule. The activation energy of the alternative current conductivity as well as that the equivalent noise conductivity results as 3.11 kcal/mole below and 4.08 kcal/mole.

Notes: The solution complexes of ethidium bromide with nine different deoxydinucleotides and the four self-complementary ribodinucleoside monophosphates as well as mixtures of complementary and noncomplementary deoxydinucleotides were studied as models for the binding of the drug to DNA and RNA. Ethidium bromide forms the strongest complexes with pdC-dG and CpG and shows a definite preference for interaction with pyrimidine-purine sequence isomers. Cooperativity is observed in the binding curves of the self-complementary deoxydinucleotides pdC-dG and pdG-dC as well as the ribodinucleoside monophosphates CpG and GpC, indicating the formation of a minihelix around ethidium bromide. The role of complementarity of the nucleotide bases was evident in the visible and circular dichroism spectra of mixtures of complementary and noncomplementary dinucleotides. Nuclear magnetic resonance measurements on an ethidium bromide complex with CpG provided evidence for the intercalation model for the binding of ethidium bromide to double-stranded nucleic acids. The results also suggest that ethidium bromide may bind to various sequences on DNA and RNA with significantly different binding constants.

Notes: The Miyazawa-Blout-Krimm (M-B-K) treatment of polypeptide absorption in the infrared is extended to the calculation of circular dichroism (CD), linear dichroism, and oriented CD for the amide I and amide II transitions. Matrix methods are applied to the α helix and β structures using measured values for the strengths and directions of the transition dipole moments and empirical values from M-B-K for the coupling constants.Relatively small aggregates, a 36-residue helix, and 8-chain × 4-residue β sheets, are large enough to show calculated absorption agreeing with M-B-K results, which are based on infinite lattices.In all cases the predicted CD is an approximately conservative couple. The strongest CD should appear in the α helix, Δε/ε ≃± 10-3 for both transitions. The amide II transition should show moderate CD couples in both β structures, Δε/ε ≃ (+2 to -1) × 10-4. The amide I transitions in β structures should show weak CD couples, Δε/ε = (+3 to -2) × 10-5, except that the negative branch in the antiparallel structure may be detectable (Δε/ε ≃ -2 × 10-4) because absorption is very low at its wavelength peak.CD on oriented samples should be enhanced over the unoriented cases, giving values as large as Δε/ε = 3 × 10-3 because particular directions of observation allow the light to avoid much of the absorption in the sample.If all three structures are considered as helices, then the larger distance of the transition dipoles from the axis in the α helix, and the orientations of the transitions in the different structures, are the factors that, in terms of our previous theoretical work [Snir and Schellman (1973) J. Phys. Chem. 77, 1653] satisfactorily explain the calculated results. Simple dipole-dipole interaction is calculated to make a substantial contribution to the coupling between groups.

Notes: The hexanucleotide Gm-A-A-Y-A-ψp excised from the anticodon loop of yeast tRNAPhe and its constituent oligonucleotides have been studied by ultraviolet absorption spectroscopy, static fluorescence, and circular dichroism. Gm-Ap has a melting point of 45°C and a high melting enthalpy when compared with G-Ap; hence 2′-O-methylation seems to stabilize stacking interactions. The nucleobase Y adjacent to the 3′-side of the anticodon triplet interacts stronger with its 3′-neighboring A than with its 5′-neighboring A. It is concluded that the base Y disconnects the stack of the anticodon itself from the stack of the anticodon stem, thereby setting a reading frame for the mRNA in the course of protein biosynthesis. From the opposite signs of the short-wavelength Cotton effects in the spectra of Gm-A-A-Y-Ap and Gm-A-A-Y, it is concluded that Y after removal of its 3′ neighbor undergoes a dramatic change in its conformation. The fluorescence of the nucleobase Y upon addition of Mg2+ is enhanced in oligonucleotides longer than two. An identical enhancement is observed for tRNAPhe, indicating that this Mg2+ effect is a property of an oligonucleotide segment and does not reflect conformational changes of the whole tRNA. The data presented here reveal that the basic structural features of the anticodon loop are already present in the hexanucleotide Gm-A-A-Y-A-ψp and are not determined by the overall structure of tRNA.

Notes: Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10-4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε220 and Δε278 than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε278 and Δε220 induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε220 of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.

Notes: The structural features of calcium guanosine-3′,5′-cytidine monophosphate (GpC) have been elucidated by X-ray diffraction analysis. The molecule was crystallized in space group P21 with cell constants of a = 21.224 Å, b = 34.207 Å, c = 9.327 Å, and β = 90.527°, Z = 8. The hydration of the crystal is 21% by weight with 72 water molecules in the unit cell. The four GpC molecules in the asymmetric unit occur as two Watson-Crick hydrogen-bonded dimers related by a pseudo-C face centering. Each dimer consists of two independent GpC molecules whose bases are hydrogen bonded to each other in the traditional Watson-Crick fashion. Each dimer possesses a pseudo twofold axis broken by a calcium ion and associated solvent.The four molecules are conformationally similar to helical RNA, but are not identical to it or to each other. Instead, values of conformational angles reflect the intrinsic flexibility of the molecule within the range of basic helical conformations. All eight bases are anti, sugars are all C3′-endo, and the C4′-C5′ bond rotations are gauche-gauche. The R factor is 12.6% for 2918 observed reflections at 1.2-Å resolution.

Notes: The growth of the weight-average double-strand length of renaturing sonicated DNA was investigated as a function of the degree of renaturation. Endonuclease S1 was used to digest single-strand ends and gaps. The ratio of the average renatured length to the initial length was found to be 0.6 ± 0.1 early in the reaction and to approach 1.0 at infinite time, while the ratio of the average single-strand length of native regions increased from 0.6 ± 0.1 to 0.8 ± 0.1 at infinite time. The ratio increases are nonlinear, with the greatest growth occuring around 50% renaturation. Relaxation times of double-strand DNA were measured in low salt using alternating field electric birefringence and were found to increase to only 70% of the relaxation time of the initial DNA at infinite time. In the molecular-weight range examined (150-320,000 daltons), relaxation times vary with the 2.3 ± 0.3 power of molecular weight.

Notes: Poly-β-1-naphthylmethyl-L-aspartate and copolymers of β-1-naphthylmethyl-L-aspartate and γ-benzyl-L-glutamate were prepared. From the results obtained by a study of infrared and circular dichroism spectra, poly-β-1-naphthylmethyl-L-aspartate was found to be a left-handed α-helix both in the solid state and in solution. The fluorescence spectra of these polymers showed excimer emission of the naphthyl chromophores and gave some information about the arrangement of the side-chain chromophores. By optical titration experiments, it was found that an increasing amount of β-1-naphthylmethyl-L-aspartate residues in the copolymers induces a progressive instability of the helical structure.

Notes: The Raman spectrum of poly-DL-alanine (PDLA) in the solid state is interpreted in terms of the disordered chain conformation, in analogy with the spectrum of mechanically deformed poly-L-alanine. The polymer is largely disordered with only a small α-helical content in the solid state. When PDLA is dissolved in water, the spectra suggest that short α-helical segments are formed upon dissolution. These helical regions might be stabilized by hydrophobic bonds between side-chain methyl groups. Addition of methanol to the aqueous PDLA solutions results in a Raman spectrum resembling that of solid PDLA. This result suggests that the methanol disrupts the helical regions by breaking the hydrophobic bonds.The Raman spectra of poly-DL-leucine (PDLL) and poly-L-leucine (PLL) are compared and only slight differences are observed in the amide I and III regions, indicating that PDLL does not have an appreciable disordered chain content. Significant differences are observed in the skeletal regions. The 931-cm-1 lines in the PLL and PDLL spectra are assigned to residues in α-helical segments of the preferred screw sense, i.e., L-residues in right-handed segments and D-residues in left-handed segments (in PDLL). On the other hand, the 890-cm-1 line in the spectrum of PDLL is assigned to residues not in the preferred helical sence, i.e., L-residues in left-handed segments and D-residues in right-handed ones. The Raman spectra of poly-DL-lysine and poly-L-lysine in salt-free water at pH 7.0 are compared. The Raman spectra of the two polymers are very similar. However, this does not negate the hypothesis of local order in poly-L-lysine because the distribution of the residues in poly-DL-lysine probably tends towards blocks, and the individual blocks may take up the 31 helix.

Notes: The Raman spectra of collagen, gelatin, and elastin are presented. The Raman lines in the latter two spectra are assigned by deuterating the amide N-H groups in gelatin and by studying the superposition spectra of the constituent amino acids. Two lines appear at 1271 and 1248 cm-1 in the spectra of collagen and gelatin that can be assigned to the amide III mode. Possibly, the appearance of two amide III lines is related to the biphasic nature of the tropocollagen molecule, i.e., proline-rich (nonpolar) and proline-poor (polar) regions distributed along the chain. The melting, or collagen-to-gelatin transition, in water-soluble calf skin collagen is studied and the 1248-cm-1 amide III line is assigned to the 31 helical regions of the tropocollagen molecule.Elastin is thought to be mostly random and the Raman spectrum confirms this assertion. Strong amide I and III lines appear at 1668 and 1254 cm-1, respectively, and only weak scattering is observed at 938 cm-1. These features have been shown to be characteristic of the disordered conformation in proteins.

Notes: Native calf thymus DNA was sheared by sonication in a viscous solvent to the molecular-weight range from 3 × 104 to 3 × 105 daltons, and fractionated by gel chromatography. Number and weight average molecular weights (M̄n and M̄w) were determined for individual fractions by electron microscopy; the ratio M̄w/M̄n for the peak fraction is approximately 1.1. Sedimentation coefficients (s020,w) of these fractionated samples show an approximately linear dependence on the logarithm of the molecular weight M̄w. This behavior is that expected for rodlike molecules, and is in quantitative agreement with the theory of Yamakawa and Fujii [(1973) Macromolecules 6, 407-415] for the sedimentation coefficient of a wormlike chain with a persistence length of 625 Å, a diameter of 25 Å, and a mass per unit length of 195 daltons/Å. It appears that the wormlike coil model, without excluded volume, can represent the sedimentation behavior of DNA over the entire conformational range from rigid rod to flexible coil, using the above parameters.Equilibrium melting curves were determined for various fractions in aqueous 2.4 M tetraethylammonium bromide. A substantial broadening of the transition and decrease of the melting temperature were observed with decreasing molecular weight. Empirical expressions have been obtained relating both the transition temperature and breadth in this solvent to molecular weight.

Notes: Light scattered from a macromolecular solution in a capillary tube is used to determine both the sedimentation and translational diffusion coefficients. The capillary tube is spun in a preparative centrifuge, removed, and placed in a light-scattering photometer equipped with a scanning mechanism. The intensity distribution of scattered light along the tube represents the concentration profile in the tube and provides the measure of boundary migration. The sedimentation coefficient is determined from this measure and the applied centrifugal field. The diffusion coefficient is obtained from a time-autocorrelation analysis of fluctuations in intensity of light scattered from any fixed point of the profile. These coefficients were obtained for two monodisperse systems, R17 bacteriophage and 28s ribosomal rat liver RNA. The molecular weights obtained from ratios of these coefficients are in good agreement with literature values. In the sedimentation analysis, deviations from linearity between boundary displacement and applied field were found to be less than 1%. This precision confirms that the boundary is stable for the capillary geometry even in the absence of a preformed density gradient. The sedimentation coefficients of identical samples were also measured with the Spinco Model E analytical ultracentrifuge; results of the two methods agree to within 4%.As a consequence of the capillary tube geometry and light-scattering detection, sedimentation coefficients can be obtained from sample volumes of less than 100 μl. This detection techniques is thus far demonstrated to be at least an order of magnitude more sensitive than Schlieren optics, thereby useful when uv absorption is not applicable. For diffusion measurements there are also several inherent advantages. The diffusion coefficient is obtained from the identical sample, and scanning provides the capability to measure D from various parts of the sedimentation profiles and thereby directly explore concentration dependence, homogeneity, and integrity of the sample. The capillary tube with a layer of silicone oil over the sample and centrifugation provides an effective method to cleanse the solution and trap all dust.

Notes: The Raman spectra of human IgG, IgM, and rabbit IgG in lyophilized form and solution are reported. The spectral results indicate that the predominant structure in these immunoglobulin proteins is the antiparallel β-sheet. The Raman spectra have also been obtained of rabbit anti-ovalbumin, and this antibody molecule precipitated with its respective antigen. The spectra reflect a conformational change on binding of antibody with antigen. The conformational change occurs in the direction of disordering.

Notes: 1H, 2H, 13C, and 81Br nmr measurements of mixtures of poly-L-lysine hydrobromide with water have been carried out over a range of temperatures and water contents. When n (number of molecules of water per residue) ∼13 at room temperature, a transition occurs from a gel to a liquid phase. The liquid phase contains polymer molecules that are flexible, but contain more intramolecular structure than the same molecules in trifluoracetic acid solution. The gel phase contains junction zones of hexagonally packed α-helices, linked by flexible regions of polypeptide chain. The α-helical residues impart to their associated water molecules a slight anisotropy of motion, which is dectable by 2H nmr. These residues bind up to about seven molecules of water each; the other six required to complete the gel-liquid transition space out the polymer molecules, allowing increased segmental motion of the residues in the flexible regions. This increased motion reduces the energy of the flexible regions and thus increases the proportion of residues in them (increasing the temperature has the same effect); the transition occurs when insufficient residues remain in the α-helical junction zones.

Notes: Copolymers of benzylaspartate with the corresponding para-nitro and ortho-nitro derivatives are known to undergo, at increasing nitro content, a transition from a left-handed α-helical conformation to a right-handed α-helical conformation. The circular dichroism associated with the n, π* transition of the amide group, as well as the extrinsic bands associated with the nitrobenzyl chromophore, have been studied for these copolymers as a function of the nitro content. The circular dichroism associated with the 330-nm extrinsic band is shown to present an abrupt change, which parallels the change in the 222-nm band characteristic of the left- to right-handed helix transition. The intensity of the 222-nm CD band seems unaffected by the interaction with the side-band chromophore. The circular dichroism associated with the 330-nm band is much stronger in the right-handed conformation and is unaffected by increasing interactions between side chains. Interpretation in the light of the possible mechanism for optical activity of helical molecules seems to indicate that the 330-nm extrinsic effect is essentially due to the Condon, Altar, and Eyring (CAE) mechanism and μ-m perturbation (in Schellmann terminology) on the side-chain chromophore n, π* transition.

Notes: Ultraviolet circular dichroism spectra have been obtained in aqueous solutions in the presence and absence of ethanol for a synthetic DNA, poly[d(AC):d(GT)], a synthetic RNA, poly[r(AC):r(GU)], and two DNA:RNA hybrids, poly[d(AC):r(GU)] and poly[r(AC):d(GT)]. In the absence of ethanol, we find that the RNA and DNA spectra are dissimilar, while the spectra of the hybrids show differing degrees of similarity to that of the RNA. In the presence of 60-80% ethanol by weight, the spectra of the DNA and both hybrids become much closer to the spectrum of the RNA, which remains relatively unchanged. We interpret the results as indicating that DNA can undergo a change to an A-type conformation in the presence of ethanol and that the DNA:RNA hybrids are not wholly restricted to an RNA-like conformation in the absence of ethanol.

Notes: The information that may be obtained from a fluorescence correlation spectroscopic study of a nonideal solution is considered. If all of the macromolecules in a two-component solution are fluorescently labeled, the mutual diffusion coefficient will be measured. If only a few of the macromolecules in a solution are fluorescently labeled, the tracer diffusion coefficient will be obtained. Two nonideal systems that probably may usefully be studied with fluorescence correlation spectroscopy are proposed. The application of fluorescence correlation spectroscopy to studies of lateral diffusion in biological membranes is discussed; the form of the contribution to the fluorescence correlation spectrum of bulk motion within a membrane is noted.

Notes: The circular dichroism (CD) and absorption spectra of uridine, thymidine, purine ribonucleoside, and the four adenine derivatives 2′-deoxyadenosine, adenosine, adenosine-3′,5′-cyclic phosphate, and arabinosyl adenine were measured in water at pH 7 and pH 2. The absorption and CD spectra of the pyrimidines were simultaneously fitted to four Gaussian bands, and the dipole and rotational strengths of the electronic transitions determined. Adenine-derivative CD spectra were determined by computer averaging six runs. The spectra showed CD bands at 268, 226, 209, and 195 nm. The band at 226 nm probably is an n-π* transition; the band at 209 nm cannot be detected without a computer. The CD and absorption spectra of purine ribonucleoside indicate three transitions in the 230-310-nm region.

Notes: Studies have been made of the precipitation of poly-L-glutamic acid from dilute aqueous solutions, over the pH range where the α-helical conformation is maintained. The major purpose is to establish the molecular conformation in the precipitate and to outline the mechanism of the precipitation. Utilizing molecular-weight fractions, it is shown that there are two distinctly different precipitation regions, which depend on temperature, concentration, and pH. In one of these regions, the α-region, a variety of physical properties demonstrate that precipitation occurs without any conformational change. The temperature coefficient of the precipitation process in this region indicates that it is nucleation-controlled.

Notes: Infrared and X-ray diffraction studies have established that in the β-precipitation region of poly-L-glutamic acid the chains are in the β-conformation. Therefore, a major molecular conformational change has taken place upon precipitation. It is shown that the size of the α-helical aggregates remains constant with time in the β-region. Strong evidence can be offered to indicate that the transformation involves a transitory random-coil intermediate. Reasons are advanced, in view of the stability of the β-form, as to why two distinct precipitation regions exist.

Notes: We have synthesized (Pro-Pro-βAla)n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)n followed a single-term Drude equation and the λc was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)n to (Pro-Pro-Gly)n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.

Notes: Water-soluble polypeptides of L-valyl and L-isoleucyl residues flanked with DL-lysyl blocks [poly(DLLys · HCl)x-poly(LVal)y-poly(DLLys · HCl)x, poly(DLLys · HCl)x-poly-(LIle)y-poly(DLLys · HCl)x] and homopoly(L-threonine) were prepared. The β conformation of these polymers in water, as well as in aqueous methanol, was confirmed by infrared spectroscopy and circular dichroism studies. The optical properties of these valyl and isoleucyl polypeptides were quite different from those of previously reported synthetic homopolypeptides in the β structure. Their differences could be explained by the presence of a “single extended β chain” without either intra- or interchain association.

Notes: Two kinds of errors are found in protein models made with the tool of Rubin and Richardson. Global errors result from the accumulation of many errors too small to localize, while local errors are assignable to particular bends in the model. We here locate the sources of local errors, and show how to minimize both kinds of errors.

Notes: The thermal helix-coil transition of four samples of poly(γ-benzyl-L-glutamate) in the dioxane-dichloroacetic acid (DCA) mixture was studied by optical rotatory dispersion method. The transition occurred at very high DCA content (97.5% by weight) in this system. The transition parameters have been evaluated by two methods based on the theories of Zimm and Bragg and of Nagai. The values of the helix initiation parameter and of the enthalpy of transition were found to be (4.4 ± 0.4) × 10-5 and 340 cal/mole, respectively. The range of validity and equivalence of the two methods is discussed.

Notes: The Raman spectra of β-lactoglobulin in the crystalline, freeze-dried, and solution states are compared. The spectra of the freeze-dried and crystalline proteins were practically identical. The conformationally sensitive amide III line appearing at 1242 cm-1 increased in intensity 30% upon dissolution of the protein in water which is interpreted as a conformational change in the disordered chains of the protein. This result appears to be a phenomenon for globular proteins containing a large disordered chain fraction.The alkaline denaturation of β-lactoglobulin was studied. When the pH was increased from 6.0 to 11.0, the amide III line shifted from 1242 to 1246 cm-1, broadened, and decreased in intensity. This is consistent with the conversion of β-sheet regions in β-lactoglobulin to the disordered conformation, as has been proposed by other investigators. At pH 13.5 the amide III shifts to 1257 cm-1 characteristic of a completely disordered protein, indicating that any remaining “core” of β-sheet has been randomized.Several changes in the intensities of the tyrosine and tryptophan vibrations accompany the denaturation. As the pH is increased from 6.0 (native state) to 11.0 (denatured state) the intensity ratio of two tyrosine ring vibrations, I855 cm-1/I830 cm-1, decreases from 1.0:0.9 to 1.0:1.3. The same ratio for a copolymer consisting of 95% glutamic acid and 5% tyrosine at pH 7.0, where the polymer forms a random coil exposing the tyrosine to the aqueous environment, is 1.0:0.62. This ratio more closely resembles that corresponding to β-lactoglobulin at pH 6.0 (native state) than pH 11.0 (denatured state) suggesting that the average tyrosine in the denatured state may be in a more hydrophobic environment than in the native state. A time-dependent polymerization of the denatured protein reported by other investigators and observed by us may account for the change in the tyrosine environment.A tryptophan vibration appearing at 833 cm-1 in the spectrum of the native state becomes weak as the pH is increased to 11.0. The intensity of this line may also reflect the local environment of the tryptophan residue.

Notes: Circular dichroism spectroscopy has been used to investigate the influence of DNA molecular size, base composition, and the presence of intercalating agents upon the Ψ transition of DNA brought about by high concentrations of poly(ethylene oxide) and salt (Lerman (1971) Proc. Natl. Acad. Sci. (U.S.) 68, 1886-1890). A molecular weight of 0.15-3.0 × 106 daltons yields maximum formation of Ψ-DNA. Both the amplitude of the large negative CD band at 265 nm - a chief characteristic of the Ψ state - and the thermal stability of Ψ-DNA increase linearly with increasing mole fraction of guanine plus cytosine in the DNA sample. Either ethidium or proflavine, at concentrations where approximately one dye is bound per 5-10 nucleotide residues, can prevent the transition completely. Striking similarities between the Ψ-DNA produced by poly(ethylene oxide) + salt and the complexes formed between DNA and lysine-rich histone f1 suggest the presence of similar nucleic acid-nucleic acid interactions in both types of condensed phase.

Notes: The values of maximum frequencies, intensities, and other spectral parameters of the main absorption bands of amino acid residue side-chain groups have been obtained in the 1500-1800-cm-1 region for solutions in heavy water at pD 1-12. It is shown that absorption of residues of asparagine, glutamine, aspartic and glutamic acids, arginine, and tyrosine must be taken into account in quantitative studies of the infrared spectra of polypeptide and protein solutions in heavy water. Examples of separating out the amide I band for ribonuclease A in heavy water are given.

Notes: Detailed inelastic, incoherent neutron-scattering (INS) spectra are presented for poly-L-alanine in both the α-helical and β-sheet form. Assignments are made from previous normal coordinate calculations. Of particular interest is the methyl torsion at 230 cm-1, which is the strongest peak in the spectrum and is independent of backbone conformation. The height of the potential barrier for methyl group rotation is calculated to be 14 kJ mol-1.

Notes: The conformation of the ribose-phosphate unit for the four principal configurations of the sugar ring has been studied using the extended Hückel theory and the Lennard-Jones empirical potential. Initially, the oxygen atoms of the p3 and p5 groups were not taken into account. With this restriction, the energy of this residue with regard to the structure of RNA has been minimized for the rotational angles ψ and φ′.The equivalence of the sugar puckering C2′endo ≃ C3′exo and C3′endo ≃ C2′exo has been confirmed. We find that the role of the sugar configuration is important and that there is a difference in the rotations around the bond C3′-O3′ (φ′ angle) according to this configuration. The C3′endo sugar form seems to be energetically preferred for this residue of RNA.The oxygen atoms and the electrostatic interactions were then introduced into the empirical potential. The variation of the energy when varying φ′ for different positions of the p5 group was studied for the main C3′endo and C2′endo configurations of the sugar; in this condition the φ′ rotation is considerably restricted but the allowed regions continue to differ according to the configuration of the sugar considered.

Notes: Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)2, cyclo-(Sar-Sar-Gly)2, cyclo-(Sar4), and cyclo-(Sar6) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the Nα-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.

Notes: The interaction between cyclic peptides [cyclo-(Sar4), cyclo-(Pro-Sar-Gly)2, cyclo-(Sar-Sar), and cyclo-(Sar-Gly)] with benzene has been investigated by nmr spectroscopy. The experiment with cyclo-(Sar4) showed that benzene interacted preferentially with the trans peptide bond in a similar manner to the dimethylformamide-benzene interaction. The solvent-induced nmr shift was then applied to the conformational analysis of cyclo-(Pro-Sar-Gly)2 with the aid of the molecular model. The major conformation was proved to possess the C2 symmetry with internally hydrogen-bonded glycine residues, in which all peptide bonds were trans. The interaction of cyclo-(Sar-Sar) and cyclo-(Sar-Gly) with benzene was also studied. The association constant was 0.115-kg solution per mole of cyclo-(Sar-Sar) and 0.089-kg solution per mole of cyclo-(Sar-Gly) in chloroform.

Notes: The high-resolution (300 MHz) proton nmr spectrum of E. coli tRNAfMet has been examined in 0.17M NaCl, with and without Mg2+, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNAfMet, some low field (11-15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A14-S4U8 base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg2+ the tertiary structure base pair is present only to ∼45°C, but in the presence of Mg2+ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A14-S4U8 base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg2+.

Notes: The relaxation kinetics of a staggering zipper model are presented, in which the formation of helical nuclei is regarded to be rate-limiting and in which the sliding of strands along the helices is prohibited. Instead a realignment of staggered chains is only possible via complete uncoiling. While maintaining the third order of the initial reaction as in an all-or-none mechanism, the model predicts a large range of relaxation times, which contribute to the mean relaxation time according to the stability of the individual species and which are weakly coupled in the range of small amounts of helical content. The model can easily be compared to experimental results and agrees well with relaxation data obtained from a triple-helical peptide fragment of collagen. It may be readily expanded to other multistranded helix ⇄ coil transitions with steady-state formation of the individual species and it suggests that the fraying of the helix ends is hidden by the fast-relaxation times due to the equilibration of the shortest helical species.

Notes: Interaction between the sodium salt of a DNA extracted from salmon sperm (41% GC) with [Pt(NH3)4]Cl2, [Pt(NH2—(CH2)2—NH—(CH2)2—NH2Cl]Cl, cis-Pt(NH2—(CH2)2—NH2)Cl2, cis-Pt(NH3)2Cl2, trans-Pt(NH3)2Cl2, K[Pt(C2H4)Cl3], and K2[PtCl4) indicates at least three types of complexation. A correlation is found between the change of pH and the number of platinum atoms fixed per (AT + GC) unit. The first binding site is located on the G-C pairs (guanine-cytosine), most likely the N-7(G) site, as it was shown in a previous study of the guanosine-platinum salts. The fixation of the second platinum atom by the pair (AT + GC) takes place with liberation of protons. In the case of the complexes cis-Pt(NH2—(CH2)2—NH2)Cl2, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 the second interaction seems to involve simultaneously the N-7(A) and the N-1(G) and N-3(C) sites. This latter intercrosslink between guanine and cytosine obviously liberates protons and the decrease of pH is related in this case to the trans effect of the platinum compounds. The first two platinum atoms in the reaction of K2PtCl4] or the Zeise salt, K[Pt(C2H4)Cl3] with DNA are fixed on the G-C pairs. A maximum of six platinum atoms per (AT + GC) unit were fixed in this case. Preliminary experiments with a DNA extracted from bacteria Micrococcus lysodeikticus (72% GC) give similar results.

Notes: The optical constants of bovine plasma albumin in the form of a dry solid film have been determined in the 2-82 eV region of photon energy. The extinction coefficient k was obtained by transmission measurements on thin films on a calcium fluoride substrate and on ultrathin films of polystyrene supported by mesh screen. The refractive index n was determined by a Kramers-Kronig analysis using the measured values of k. The complex dielectric function ε and the energy-loss function -Im(1/ε) for charged particles were derived from the data of n and k. The result for k exhibit, in addition to the well-known absorptions at 4.4, 5.5, and 6.4 eV, a small peak at 7.6 eV, a shoulder around 9.7 eV, and a strong peak at 14.0 eV. A prominent peak at 22.7 eV in the energy-loss function exhibits a large degree of collective nature.

Notes: Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris-sodium acetate-potassium chloride at 25°C. The pKA of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5-8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 103 M-1 and 0.136 × 103 M-1, respectively, for native DNA and 7.48 × 103 M-1 and 0.938 × 103 M-1, respectively, for denatured DNA. The pKA value of the neutral red-DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.

Notes: The heat capacities of L-Alanine, tri(L-alanine), and poly (L-alanine) (α helicoidal form and β pleated sheet structure) have been measured between 1.5 and 300°K with a standard adiabatic calorimeter.In the solid state, the heat capacity is in general dut to three parts which are additive in first-order approximation. (1) The lattice vibrations or “acoustical modes” which are the largest at low temperatures. The low-temperature lattice specific heat is proportional to T, T2, or T3 for an ideal one-, two- or three-dimensional solid, respectively. (2) The so-called group vibrations or “optical modes” which, due to their high frequencies, usually take effect only at higher temperatures. (3) The defects and unharmonic effects.The α-amino acid and its trimer present a specific heat thermal variation characteristic of molecular solids which is correctly fitted with an empirical law proposed by Kitaigorodskii. This author assumes that, for such solids, the molecular lattice point has six degrees of freedom (three of translation and three of rotation). Thus the lattice contribution of the specific heat satisfies the Debye approximation in agreement with the doubling of the number of degrees of freedom per molecule (compared with atomic crystals).The polypeptide behavior is, however, different. The specific heat for each form exhibits a thermal dependence connected with a strong vibrational anisotropy. The model proposed earlier by Tarasov accounts well for these results. In the case of the β form, we have observed the predicted three- and two-dimensional behavior due to the intermolecular H bondings responsible for the sheet structure. For the α form we observed a one-dimensional pattern at higher temperature, since each peptidic chain vibrates separately.The comparison with other spectroscopic and theoretical investigations shows a large discrepancy. However, we have attemped to account for the “optical contribution” to the specific heat of poly(L-alanine) by using a continuum of mean frequencies as suggested by Wunderlich. Vibrational frequency spectra are proposed to explain our results, but the overlapping of acoustical and optical branches in the case of the α form outlines the limits of macroscopic models. It is quite likely that the acoustical spectrum is greatly affected by the intramolecular H bonding.At low temperature the specific heat is a physical property sensitive to the long-range order of the macromolecule, and therefore further spectroscopic and theoretical investigations are necessary to explain correctly these experimental results.

Notes: Poly(L-tyrosine) [(L-Tyr)n] has been characterized in aqueous solution using circular dichroism (CD) and infrared (ir) spectroscopy, and ultracentrifugal analysis. Most of the experiments were carried out at 0.01% polymer or less to avoid the complications caused by precipitation previously encountered by others. This permitted us to study solutions of (L-Tyr)n at lower pH values than had been attained previously. Our results show that a transition to an antiparallel-β conformation occurs at pH 11.32 upon titration from higher pH. The β structure is intramolecular when first formed and aggregates with time or upon titration below pH 11. Ultracentrifugal analysis of the intramolecular β conformation shows that it is quite compact, with a frictional coefficient ratio, f/fmin, of 1.09. In addition to the β structure, a nonordered form of the polymer has been obtained below pH 11 by rapid titration of the ionized polyelectrolyte. This form is nonaggregated and was found to have an f/fmin of 1.01, and is therefore almost spherical. The aggregated β form was found to be thermodynamically more stable than the nonordered form at pH 10.7.

Notes: Semi-empirical molecular orbital calculations have been carried out for the free base and cationic forms of imidazole so as to obtain data which are required for the calculation of the chiroptical properties of molecules that contain this chromophoric group. The polarization, energy, and monopolar charge distribution are reported for the lowest energy electronic transitions. The absorption spectra for imidazole have been determined to 180 nm and circular dichroism spectra for L-histidinol and L-2-amino-1-butanol have been measured.

Notes: The cross-sectional radius of gyration of the deoxyribonucleoprotein (DNP) threads was measured by small-angle X-ray scattering in a wide range of ionic strengths (from 0.0005 to 2 M NaCl). For DNP in a solution of low ionic strength, this value is 30 Å. The increase of ionic strength results in partial deproteinization of DNP, while the cross-sectional radius of gyration varies from 25 Å for DNP in 0.7 M NaCl to 10 Å for DNP in 2 M NaCl. It is suggested that gradual deproteinization by the increase of NaCl concentration causes conformational changes, which are associated with the alteration of the DNP superstructure. The data are interpreted on the basis of the superhelical model of DNA packing in DNP; however, the coexistence of superhelical and unfolded regions in the DNP structure is also a possibility.

Notes: The buoyant density and potentiometric hydrogen ion titration curves of human immuno-gamma globulin in 3M cesium chloride have been recorded. In addition, the amino acid analysis of the IgG employed has been completed. The hydration of the protein and the variation of the hydration with pH have been calculated from the buoyant density data. The potentiomtric hydrogen ion titration curve has been employed to estimate the intrinsic pK′s of the acidic and histidyl residues of the molecule, and to confirm the hypothesis that it does in fact conform to the oil drop model of protein conformation. Correlations have been drawn between the three sets of data in the following manner. The results of the potentiometric hydrogen ion titration have been checked against the amino acid analysis to determine whether the numbers of groups observed to titrate and the numbers of groups observed in the amino acid analysis do correspond. Second, previous hypotheses as to the direct correlation between potentiometric hydrogen ion titration behaviour and buoyant density titration behaviour have been investigated and substantially confirmed.

Notes: A basic theory for equilibrium properties of cooperative transitions (particularly helix-coil transformations) of sufficiently long linear biopolymers under the influence of an electric field is developed. General relations for the calculation of the distribution of uninterrupted sequences of elementary subunit states (e.g., helical fragments) as well as the overall degree of transition θ are given. Strong cooperativity is found to permit simplifications. It is shown that only in this case can a practically significant field effect be expected. Numerical results are presented for a special example of experimental interest.

Notes: The electron-mediated spin-spin coupling constant J between the amide NH and the α-CH protons in the dipeptide fragment Cα—CO(NH—CαH)R—C′ONH—Cα is dependent on the dihedral angle of rotation (Φ) around the N—C bond. Measurement of J in a series of zwitterionic dipeptides H3N+—CHR1—CONH—CHR2—CO2- (which is conformationally similar to the dipeptide fragment) in TFA solution shows that J is independent of R1, but dependent on the steric bulk of R2. The data are interpreted in terms of a model that assumes that what we measure is an average value of J—a thermal average over all the possible rotamers. The groups R1 and R2 are, in most cases, sterically kept apart by the trans and planar amide bonds, and hence the independence of J of R1. This model is consistent with the theoretical calculations done on the dipeptide fragment. The effect of the structural characteristics of the side chains (e.g., the effect of lengthening and branching the side chains) on the J values in dipeptides is discussed in the light of the existing results of theoretical calculations.Study of 〈J〉 values in tripeptides (C6H5CH2OCONH—CHR1—CONH—CHR2—CO2CH3, essentially three linked peptide units) shows that electrostatic interaction between the two amide bonds modifies the potential energy surface and the 〈J〉 value of a dipeptide subunit in the tripeptides. Also in some cases, direct steric interaction between the two side chains in the two adjacent dipeptide subunits in the tripeptide affects the potential energy surfaces of the individual dipeptide subunits and hence the 〈J〉 values. The influence of the structural characteristics of the side chains of individual amino acids on structure formation at or beyond the dipeptide level is discussed at various points.The J(NH—αCH) values of CH3CONH—CHR—CONH2 and CH3CONH—CHR—CO2CH3 with the same R are quite different for R = valine, leucine, phenylalanine, methionine, but equal for R = glycine. This, coupled with the fact that one of the carboxamide NH resonances has a chemical shift different from its counterpart in simple amides like CH3CONH2 and the other carboxamide NH has the same chemical shift as its counterpart in CH3CONH2, suggest the presence of a hydrogen bond in dipeptide CH3CONH—CHR—CONH2 with carboxamide NH as the donor. Theoretical evidence for two seven-membered hydrogen-bonded rings with the carboxamide NH as donor and the acetyl oxygen as acceptor is summarized. Our data cannot suggest the number of such hydrogen-bonded rings, nor can they conclude the relative proportion of these rings in a particular dipeptide. A discussion of the difficulty of interpretation is presented and the data are discussed under certain simplifying assumptions.

Notes: An electric birefringence apparatus with an He-Ne laser is described, which permits a high sensitivity of measurement for field strengths up to 20 kV/cm and pulse durations between 10-5 to 1 sec. It is applied to poly(γ-benzyl-L-glutamate) and poly(β-benzyl-L-aspartate) in a nonaqueous solvent mixture, especially under the conditions of the helix-coil equilibrium. A theoretical treatment of the problem shows that the usual neglect of end effects is only justified for comparatively long chains. On the basis of a computational procedure taking into account the finite chain lengths, the experimental data for 15 kV/cm reveal an obvious enhancement of the coil-to-helix transition caused by the electric field. This effect is quantitatively confirmed by a pertinent theory. The cooperative parameter σ may be best determined from the initial linear increase of the bire-fringence versus the square of the field strength where the conformational equilibrium is not affected.

Notes: The first uv absorption band hypochromism of poly(dA) · poly(dT), poly(dG) · poly(dC), poly(dA), poly(dT), poly(dG), and poly(dC) is calculated with the help of perturbation theory on the basis of monomer characteristics computed by the Pariser-Parr-Pople method taking into account all singly excited configurations. The theoretical results obtained are in good agreement with experimental values of hypochromism. The origin of the hypochromic effect in the double-stranded polynucleotides is investigated. It is shown that intrastrand interactions between the bases make the main contribution to hypochromism (60-76%), while the contribution of the Watson-Crick-pair formation is small (2-12%). The essential part of hypochromism (22-28%) is due to the interstrand interactions between the bases that are not coupled by hydrogen bonds. The discussion of the experimental data shows that the present theoretical investigation could serve as a basis for the correct treatment of experimental data.

Notes: An X-ray study of the synthetic polypeptide poly(L-homoarginine hydrochloride) has been made to investigate whether, like the chemically related polypeptides poly(L-lysine hydrochloride), poly(L-arginine hydrochloride), and poly(L-ornithine hydrobromide), it can undergo conformational transitions merely from variations in its degree of hydration. X-ray photographs of powder and oriented specimens containing one to 15 molecules of water per L-homoarginine hydrochloride residue showed that this polymer forms only a β-pleated-sheet structure. The pleated sheets, formed by antiparallel polypeptide chains hydrogen-bonded to each other, are piled up along the b axis in an alternating sequence (“sandwich structure”). This structure did not appreciably change with variations of the degree of hydration, and the observed reflections at 56% relative humidity (1.8 molecules of water per residue) could be indexed satisfactorily in terms of a monoclinic unit cell, of space group P21, with a = 9.34 Å, b = 40.07 Å, c = 6.94 Å, and γ = 106°. These dimensions are shown by models to be compatible with the proposed structure, and the calculated density of 1.27 g/cm3 agrees well with the experimental value of 1.29 g/cm3. Removal of the last molecule of water results in a very diffuse pattern, while specimens containing 20 molecules of water per residue show only reflections due to water.

Notes: The helix-coil conformational transition undergone by poly(γ-benzyl-L-glutamate) in solutions of trifluoroacetic acid and deuterated chloroform was studied by proton and carbon-13 nmr. The results indicate that in the case of the solvent-induced helix-coil transition, the side chain assumes a helical conformation before the backbone. In the thermally induced helix-coil transition, the results indicate the existence of an intermediate state, which is between the α-helix and random coil and is free from intramolecular hydrogen bonding.

Notes: Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L-cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5-7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2-0.6 at pH 6-10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.

Notes: Statistical copolymers (Lysx,Alay)n were synthesized by copolymerization of N-carboxyanhydrides of L-amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lysx,Alay)n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.

Notes: Both Raman spectra and X-ray diffraction patterns have been obtained from oriented fibers of sodium deoxyribonucleic acid (Na-DNA) as a function of salt content and relative humidity. We have confirmed the previously reported X-ray results that, for oriented fibers, the A-form always exists between 75 and 92% relative humidity and that the conformation will change to the B-form at 92% relative humidity only if an excess (3-5%) of added salt is present. Oriented fibers containing low amounts of added salt remain in the A-type conformation at 92% relative humidity and higher. An exact correlation has been found between the familiar A- and B-type X-ray diffraction patterns of DNA fibers and the Raman spectra previously reported without X-ray verification from this laboratory for the A- and B-forms. In particular, a band at 807 cm-1 was always present when a fiber showed the A-type diffraction pattern, and this band shifts to 790 cm-1 in the B-form. Using the Raman spectrum to determine the specific conformation of DNA in samples less amenable to X-ray analysis, we have studied the A ⇄ Btransformation in unoriented fibrous masses of DNA and in concentrated, oriented gels. We find that in unoriented fibrous masses, the A ⇄ B transition always occurs at 92% relative humidity even at very low salt concentration (0-4%). However, in oriented DNA gels at low salt, the A-form can persist as a metastable state to concentration as low as 20% DNA. The origin of the bands at 807 and 790 cm-1 and the possible biological implications of these findings are discussed.

Notes: Analysis of experimental equilibrium constants for the oxygenation of hemoglobin leads to a plausible mechanism for the effect of pH and of chloride ions on cooperativity in hemoglobin. According to this mechanism, the structural changes responsible for cooperativity in chloride- and 2,3-diphosphoglycerate-free hemoglobin are affected only slightly by changes in pH, and the effect of chloride can be accounted for by sequential binding and release of chloride ions during oxygenation.

Notes: Normal coordinate analysis of diethyl phosphate has been made, which predicts all observed Raman frequencies in the range 170-1300 cm-1. The force constants from this calculation have been transferred to a vibrational calculation for a simplified model of the backbone of nucleic acids, which also involves the —O—PO2-—O phosphate group and the —C5′—C4′—C3′—linkage of the ribose. The coordinates of these atoms are those recently given by Arnott and Hukins, which place the ribose ring of B-DNA in a C3′-exo conformation. This simple polymer model appears to be able to describe adequately the frequency-dependent changes observed in the Raman spectra arising from the backbone vibrations of nucleic acid in going from the B- to A-form. The symmetric —O—P—O— diester stretch increases in frequency from about 787 cm-1 in the B-form to 807 cm-1 in the A-form. The increased frequency characteristic of the A-form is due to the combining of the diester stretch with vibrations involving the C5′, C4′, and C3′ nuclei. The frequency of the symmetric —O—P—O— diester stretch is shown to be very dependent on the conformation of the ribose ring, indicating that in polynucleotides the ribose ring takes on one of two rigid conformations: C3′-endo for A-form or C3′-exo for B-form and “disordered” polynucleotides. The calculation lends confirmation to the atomic coordinates of Arnott and Hukins since the use of other geometries with the same force constants failed to give results in agreement with experimental evidence. The calculations also demonstrate the lowering effect of hydration on the anionic PO stretching frequencies. Experimental results show that the 814-cm-1 band observed in the spectra of 5′GMP gel arises from a different vibrational mode than that of the 814-cm-1 band of A-DNA.

Notes: The molecular weights and radii of gyration of Streptococcus salivarius levan fractions were obtained from light-scattering measurements in water. Sedimentation coefficients and partial specific volumes of the fractions were also obtained. Double logarithmic plots of [η] versus M̄w and S0 versus M̄w yielded slopes having values of 0.17 and 0.62, respectively. The data and various calculated parameters show that levan from Streptococcus salivarius is highly branched and behaves hydrodynamically as a compact particle of spherical symmetry.

Notes: The molar magnetic susceptibility of ferrihemoglobin solutions, as a function of pH and temperature using the nmr technique of Evans, in water and 5% t-butanol has been determined. Results suggest that within a small pH region, the conventional analysis of magnetic susceptibility in terms of high-spin and low-spin contributions breaks down. The implication of this is discussed in terms of possible conformational change involving two subspecies. The effect of t-butanol is also discussed in terms of the t-butanol effect on water.

Notes: We have measured the ultraviolet CD spectra for covalently closed and linear forms of phage PM2 DNA in solution. We find that increased concentrations of salt or ethanol (up to 50% ethanol by weight) depress the long-wavelength positive CD bands in the spectra of both forms of DNA, although the spectrum of the native covalently closed DNA always has a slightly larger magnitude of these bands than does the spectrum of the linear DNA. In addition, both DNAs are equally capable of undergoing a transition to the A conformation in 70-80% ethanol at low Na+ concentrations. Thus, the constraint imposed by the covalent closure of a DNA molecule does not seem to hinder its conformational response to these changing solution conditions. Lang [(1973) J. Mol. Biol. 78, 247-254] has found by electron microscopy that T7 DNA has an inherent ability to condense into compact particles, suggested to be supercoils of multiple order. Both covalently closed and linear forms of PM2 DNA also become condensed when the DNA, in 0.2M ammonium acetate and 1 mM EDTA, is exposed to ethanol and subsequent drying on specimen grids [Lang, D., Taylor, T. N., Dobyan, D. C. & Gray, D. M. (1976) J. Mol. Biol. 106, 97-107]. Under similar conditions, in solutions of 0.2M ammonium acetate and 1 mM EDTA to which ethanol is added, we have measured the CD spectra of both covalently closed and linear forms of DNA. Below ethanol concentrations at which the DNA obviously precipitates, the CD spectra of both forms have reduced long-wavelength positive CD bands.

Notes: The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.

Notes: The low-temperature dynamic mechanical relaxation of ox Ligamentum nuchae elastin, swollen with water and ethylene glycol at various swelling ratios, has been investigated by means of the Rheovibron DDV II viscoelastometer, at frequencies from 3.5 to 110 Hz. The peak location on the temperature scale, its intensity and activation energy have been studied as a function of solvent content and it has been found that the two solvents influence the dynamic mechanical behavior in a quite different way. A similar interaction of the solvent molecules with the backbone peptide groups is assumed for both solvents, and the differences in the mechanical spectra, which are more marked the higher the solvation degree, are explained in terms of the different size of the protein-solvent relaxing units.

Notes: The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.

Notes: The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m2′)] and poly[A(e2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ〈280 nm (τα) are different from those found at λ 〉280 nm (τβ), indicating the presence of more than two conformational states. The time constants τα increase in the series poly(dA), poly[A(m2′)], constants τβ/τα is approximately 2.5, except in poly(dA) where τβ/τα ≈ 9. Relaxation measurements with r(A)n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D2O reveal a solvent isotope effect for τα of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τβ. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar-phosphate chain. The absence of a seprate relaxation process (corresponding to τβ) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar-phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.

Notes: An algorithm has been developed that permits one to find all possible conformations of the sugar-phosphate backbone for any given disposition of DNA base pairs. For each of the conformations thus obtained, the energy of the helix was calculated by the method of atom-atom potentials. Several isolated regions in the space of the bases′ parameters (Arnott's parameters) have been found for energetically favorable helical structures. Two parameters, the distance of a base pair from the helix axis, D, and the windling angle, τ, allow one to subdivide possible conformations into the families of closely related forms. Two regions (ravines) on the (D, τ) map correspond to the know A and B families. In the B family a continuous transition has been obtained in which the double helix undergoes increasing winding, while the base pairs are moving toward the major (nonglycosidic) groove.Interrelationships between the variables, characterizing the spatial structure of the double helix, D, τ, TL and χ, when going along the bottom of the B ravine, were also obtained.Besides the Known A and B families, several new ones were found to be energetically possible. Among these the strongly underwound helices with the negative D values, as well as the forms with the C4-C5 angle in a trans position, should be mentioned.Biological roles of the different double-stranded conformations, in particular, in protein-nuclei acid interaction are discussed.

Notes: The theoretical basis for the statistical mechanical deconvolution of a thermally induced macromolecular melting profile is presented. It is demonstrated that all the thermodynamic quantities characterizing a multistate macromolecular transition can be obtained from the average excess enthalpy function, 〈ΔH〉, of the system, without any assumption of the particular model or mechanism of the reaction.Experimentally, 〈ΔH〉 is obtained from scanning calorimetric data by direct integration of the excess apparent molar heat capacity function, ΦCp. Once 〈ΔH〉 is known as a continuous function of the temperature, the partition function, Q, of the system can be calculated by means of the equation \documentclass{article}\pagestyle{empty}\begin{document}$$Q_{\left( T \right)} = \exp \smallint _{T_0}^T {\left( {\frac{{\left\langle {\Delta H} \right\rangle }} {{RT^2}}} \right)} dT $$\end{document} From the partition function all the thermodynamic quantities of the system can be obtained. It is shown that the number of discrete macroscopic energy states, the enthalpy and entropy changes between them, and the relative population of each state as a function of temperature can be calculated in a recursive form.

Notes: The high-resolution 1H-nmr study of the ferrichrome cyclohexapeptides, in d6-DMSO solutions, has been extended to the amide NH spectral region. A total of ten diamagnetic analogues of ferrichrome that differ in the coordinated metal ion (Al3+, Ga3+ or Co3+), the primary structure, the nature of the bidentate hydroxamate moiety, or the isotope compositions (14N, 15N) have been investigated. The 3JαNH values reflect regiorous conformational isomorphism throughout the complete suite of analogues, quite independent of the residue occupancy of each site. Totally resolved amide multiplets have been obtained in most cases and the four-line (doublet of doublets) appearances of glycyl NH resonances has been observed for the first times; these data enabled stereospecific assignment and accurate analysis of the NH-CαH proton spin systems. The high resolution was made possible by the use of a suitable spectral deconvolution shceme at 360 MHz. The fine structure, extraordinarily well displayed in the 15N-peptide spectra, provides a series of parameter values whose consistency has been checked by computer simulation. Since the crystallographic structure for two of the ferric peptides is known to 0.002-Å resolution, a 3J vs θ correspondence could be confidently established. A Karplus curve was derived from the combined x-ray and nmr data: \documentclass{article}\pagestyle{empty}\begin{document}$$ ^3 J_{\alpha {\rm NH}} = 5.4\cos ^2 \theta - 1.3\cos \theta + 2.2{\rm Hz} $$\end{document} It is suggested that seriously nonplanar amides can exhibit 3JαNH values higher than predicted by the ferrichrome curve.

Notes: The general formulas describing the low-angular dependence of polarized (Vv) and depolarized (Hv) light-scattering intensity by a chain macromolecule of arbitrary form with anisotropic polarizability are derived. It is shown that the value dHv/dtHvo (where t = [〈R2〉S/L]2, S being the scattering vector) is an indication of the chain-flexibility mechanism. This permits one to distinguish between the models of a wormlike chain, regular zigzag, or statistical zigzag. The results of numerical calculations useful for the interpretation of experimental data are presented.

Notes: The kinetics of the fluorescence decay of the energy donor in a homologous series of oligopeptides each containing at its ends a donor and an acceptor of electronic excitation energy was investigated in solvent mixtures of different viscosities. The repeating unit in the peptides was N5-(2-hydroxyethyl)-L-glutamine and the chromophores used as donor and acceptor were naphthalene and dansyl, respectively. The number of units in the peptides studied varied from four to nine. The solvents used were mixtures of glycerol and trifluoroethanol in various proportions. The decay rate of the donor fluorescence increases when the solvent viscosity decreases. This behavior is due to the disturbance of the equilibrium end-to-end distribution of distance of the excited molecules by the energy transfer process, which is more favorable foe short than for long distances. The subsequent rearrangement towards the equilibrium distribution by diffusion of the molecular ends relative to one another enhances the efficiency of the energy transfer. Assuming a modified Fick equation to describe this diffusion motion, the fluorescence decay data were analyzed in terms of a diffusion coefficient describing the Brownian motion of the molecular ends. The diffusion coefficients thus evaluated increase systematically upon decreasing the solvent viscosity. For example, for the oligopeptides studied it changes from unmeasurably small values in glycerol solution to values varying between 10-8 to 10-7 cm2/sec at room temperature in a glycerol trifluoroethanol solvent mixture of viscosity of 8 centipoise. The values obtained for the diffusion coefficient are smaller by about an order of magnitude than the values expected for the diffusion coefficients of the free chromophores in solvents of comparable viscosity. It is thus concluded that the backbone of the polymeric chains possesses appreciable internal friction which exerts resistance to the Brownian motion of the polymer chains. The diffusion coefficient of the end-to-end motion is systematically smaller for the shorter than for the longer chains. For example, at room temperature in a solvent mixture of 8 centipoise it is 3×10-8, 5×10-8, 7.6×10-8, and 8.5×10-8 cm2/sec for oligomers containing four, five, eight, and nine N5-(2-hydroxylethyl)-L-glutamine repeating units, respectively. The internal friction thus impedes the motion of the molecular ends more effectively in the shorter chains than in the longer ones.Analysis of the energy-transfer experiments in solvents spanning a wide range of viscosities shows that the orientational factor appearing in Förster's equation for energy transfer does not interfere perceptibly with the evaluation of the end-to-end distances from the fluorescence decay data when naphthalene and dansyl serve as donor and acceptor, respectively. This is due to the fact that both the donor emission and acceptor absorption in the range of spectral overlap are characterized by more than one transition dipole moment, as is evident from the corresponding polarization data.

Notes: Raman spectroscopic studies have been carried out on polymers of L-valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40-1750 cm-1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L-alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L-valine.

Notes: The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λmax) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λmax, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λmax of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.

Notes: Group-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4KM KCl and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times at different concentrations using incident wavelengths of 632.8 nm from a He-Ne laser and of 488 nm from an argon-ion laser yield information on aggregation properties, molecular weight (Mr), radius of gyration 〈r0g〉1/2z, translational diffusion coefficient 〈D〉0z, and second virial coefficients A2 and B2 of C-1 polysaccharide.At relatively high ionic strength (0.04M KCl + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution Mr = 5.15 × 105, 〈r2g〉1/2z = 345 Å, A2 = 1.25 × 10-4 ml/g, 〈D〉0z = 1.16 × 10-7 cm2/sec with a corresponding Stokes radius of 240 Å and B2 = 4.4 ml/g. A2 and B2 are the second virial coefficients from intensity- and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils. Viscosity and sedimentation studies further confirm our conclusions that the fractioned C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum at ∼0.2M KCl. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors.

Notes: We extend fluorescence correlation spectroscopy to systems that undergo translation or laminar flow in a sample cell. We include theoretical and experimental results; we consider uniform and nonuniform velocity profiles. Concentration correlation analysis extracts microscopic rate parameters from measurements of the spontaneous concentration fluctutations, which occur even at equilibrium. Fluorescence is one of the most sensitive means of monitoring these fluctuations. Analysis of flowing or translating systems (1) offers a method of measuring number concentrations of selected species, for example, of aggregates or polymers, (2) provides a nonperturbing velocity probe, (3) sometimes allows one to circumvent photolytic degradation, (4) has proved extremely helpful in testing and aliging apparatus for fluorescence correaltion measurement and in verifying theoretical analyses, and (5) may be required for interpretation of results obtained on systems in motion, even though that motion is undesired or initially unsuspected. We include both theoretical and experimental results for combined Poiseuille flow and diffusion in the geometry which is of most practical interest. Theoretical expressions for the much simpler cases of nondiffusive Poiseuille flow as well as uniform flow or translation with or without diffusion constitute limiting cases which are displayed explicitly.

Notes: In the preceding article1 we demonstrated that the partition function of a system is experimentally accessible from scanning calorimetric data. In this article the general results of the deconvolution theory are applied to the general case of cooperative transitions in macromolecules. It is demonstrated that, in the limit of very large systems, all the relevant molecular averages and molecular distribution functions can be directly obtained from the experiment. In doing this, the method of the grand partition function is used. It is shown that in the case of homopolymers, only one parameter, the stability constant, needs to be explicitly specified for a complete description of the system. Since the partition function is directly evaluated from the experiment, no special assumptions or artificial constraints directed to obtain a mathematically solvable model are required. This result offers the unique opportunity of having direct experimental access to statistical averages of systems in which the partition function cannot analytically be solved. Consequently, the theory can be extended to cooperative transitions occurring in two and three dimensions by introducing cluster distribution functions.

Notes: The pyridoxal Schiff bases of the polypeptides poly(L-lysine), poly(L-ornithine), and poly(L-α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L-α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.

Notes: The protein turkey-heart cytochrome c is used as a model protein to study charge effects in sedimentation equilibrium experiments in three-component solutions. Data are given for the dependence of the apparent M (1-υρ) on ρ in solutions of KCl, RbCl, CsCl, and triethylamine hydrochloride. The results show the Donnan effect to have a significant influence on the apparent molecular weight, found by extrapolation of the data to a solution density of one. The apparent molecular weights are for protein at infinite dilution. A theoretical treatment is presented where the magnitude of this effect can be predicted accurately from the formal net charge of the protein as computed from the amino acid composition. The results are shown to be important in computing the preferential hydration of the protein in concentrated salt solutions. For such systems the Donnan effect should be subtracted from the total interaction coefficient for multicomponent system in order to obtain the preferential hydration.