bluuebunny/crossref_metadata_2025 huggingface dataset:
Static and dynamic quenching of protein fluorescence. II. Lysozyme
Authors: McGuire Robert , Feldman Isaac
Published: December 2004

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half‐width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin.

The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure‐breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction.

Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride‐induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.
The magnitude of intramolecular DNA optical absorbance with variation in [Na+] and GC base composition: For special application to DNA reassociation studies
Authors: Marx Kenneth A.
Published: December 2004

The magnitude of intramolecular DNA optical absorbance is presented as a function of [Na+] and GC composition of the DNA. The data are presented as a ratio of absorbances A0/A0 for the DNA at the denaturing temperature (Td) and renaturing temperature (Tr) under the given conditions. All ratios were determined for Tr corresponding to the temperature optimum (Tm − Tr = 25°C) in DNA reassociation rate. This fact, coupled with the convenient A0/A0 ratio representation, permits the quick estimation of the magnitude of this optical effect in DNA reassociation reactions over a wide range of experimental conditions.
A theoretical estimate of the energy barriers between stable conformations of the proline dimer
Authors: Venkatachalam C. M. , Price B. J. , Krimm S.
Published: December 2004

Semi‐empirical energy calculations for an internal Pro‐Pro dimer are presented that take into account the nature of the flexibility of the proline ring due to its puckering. Calculations show that three stable conformations are available for the dimer: the cis (ω = 0°, ψ = 160°); the trans (ω = 180°, ψ = 160°, also referred to as trans′); and the cis′ (ω = 180°, ψ = −40°) conformations. The best conformational pathways between these stable conformations are determined. Calculations also show that the barrier for cis′–trans′ conversion is of the same order of magnitude as that for cis–trans conversion.
On the displacement of cooperative transitions of linear biopolymers by an electric field
Authors: Schwarz Gerhard
Published: December 2004

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.
Circular dichroism of flow‐oriented nucleic acids. II. Comparison between observed and calculated spectra
Authors: Chung Su‐Yun , Holzwarth G.
Published: December 2004

The ultraviolet circular dichroism (CD) of oriented DNA and RNA molecules is calculated by an extension of Johnson and Tinoco's theory [(1969) Biopolymers 7, 727–749] for unoriented molecules. The calculations are carried out for molecular models of A‐DNA, B‐DNA, planar B‐DNA, C‐DNA, and RNA‐11‐α. The calculated curves are compared to measured spectra [(1975) J. Mol. Biol. 92, 449–466] Chung and Holzwarth, for oriented solutions of DNA in buffer, DNA in 6 M LiCl or in ethylene glycol, and double‐stranded viral RNA. The calculation, which considers only base–base interactions, predicts that the CD of B‐DNA, measured with light propagating parallel to the helix axis, should be large and semiconservative, whereas the CD for light propagating perpendicular to the helix axis should be nonconservative. These predictions agree qualitatively with the experimental observations for DNA in buffer; agreement improves if one assumes the bases to be exactly perpendicular to the helix axis. For the other geometries, agreement is less satisfactory, but qualitative agreement with experiment is obtained and the signs of the specific CD spectra are in accord with observations.
A vibrational study of poly (L‐proline) I and II in the far infrared
Authors: Rabolt John F. , Wedding William , Johnson Kenneth W.
Published: December 2004

The far infrared spectra of poly(L‐proline) I (190–35 cm−1) and II (400–35 cm−1) were obtained in the solid state at both 300° and 110°K. A significant difference in the region below 100 cm−1 was observed. A very intense band located at 60 cm−1 in the infrared spectrum of form II has no counterpart in form I. This indicates the sensitivity of low‐frequency vibrations to the difference in conformation assumed by both forms in the solid state.

Additional bands observed in this study are correlated with ir and Raman data previously reported and tentative assignments are made using the results of normal mode calculations (in the single‐chain approximation) which have been reported.
Configuration‐dependent properties of randomly coiling polynucleotide chains. II. The role of the phosphodiester linkage
Authors: Olson Wilma K.
Published: December 2004

The dependence of the unperturbed dimensions of randomly coiling polynucleotides on the rotations about the phosphodiester linkages of the chain has been examined in order to understand the conformational discrepancies, set forth in paper I, regarding these angles (ω′ and ω). Large values of the characteristic ratio 〈r2〉0/nl2, which agree with the experimental behavior of the chain, are obtained only if a sizeable proportion of the polymer residues have trans ω′ values. The asymmetric torsional potential that is believed to arise from gauche effects associated with the P‐O bonds has been approximated using a hard core model. The calculated characteristic ratio exhibits a strong dependence upon the magnitude of this torsional barrier (separating trans and gauche conformations) and shows agreement with experimental values for polyribonucleotides only if this energy difference is 1 kcal/mol or less.
Optical rotatory dispersion and circular dichroism of silver(I):Polyribonucleotide complexes
Authors: Arya S. K. , Yang Jen Tsi
Published: December 2004

Optical rotatory dispersion (ORD) and circular dichroism (CD) spectra of single‐ and multistranded polyribonucleotides undergo extensive changes on binding of the silver ion. These changes are consistent with the proposition that Ag(I) binds to the heterocyclic bases and not to the phosphate groups of polynucleotides. ORD and CD of silver complexes of poly(A)·poly(U) and double‐helical rice dwarf viral RNA display negative Cotton effects when there is more than one Ag(I) per two nucleotide residues in solution. These observations suggest a significant distortion of the double‐helical conformation as a result of Ag(I) binding. Silver(I) binding sites of pyrimidine polynucleotides are apparently saturated when there is one Ag(I) per two nucleotide residues and those of purine polynucleotides at one Ag(I) per nucleotide in solution. These data are consistent with the supposition that some Ag(I) binding sites exist on the pyrimidine ring and additional sites on the imidazole ring of polynucleotides. The sedimentation coefficient of poly(A) increases by severalfold when one Ag(I) is present per nucleotide residue. Silver(I) may introduce intra‐ and interstrand cross‐links (through bidentate chelates) in single‐stranded polynucleotides, resulting in structures with high sedimentation coefficients. Among the polynucleotides studied, poly(U) was an exception. Silver(I) did not affect the optical properties (absorbance, ORD, and CD) of poly(U) at neutral pH.
Preparation and characterization of α‐L‐glutamic acid oligomers
Authors: Rinaudo M. , Domard A.
Published: December 2004

The preparation and characterization of α‐L‐glutamic acid oligomers with degree of polymerization (DP) up to 12 are described. The preparation of polymers with low DP corresponding to various A/I ratios (where A and I are monomer and initiator concentrations, respectively) with end groups blocked is given. The conditions of the fractionation, which separates the different oligomers by ion‐exchange chromatography, are discussed. Finally, the isolation from salt solutions of the pure acidic form is given. Each polymer obtained for a given A/I is characterized at the end of the polymerization by its molecular‐weight distribution. The average DP values calculated are compared to the A/I values; agreement is very good. Potentiometric behaviour during neutralization is obtained as a function of the degree of polymerization and the elaboration of the polyelectrolytic phenomenon is discussed.
Investigation of fibrous structures. II. General method for the elucidation of the conformations of the complementary nucleic acids
Authors: Tumanyan V. G. , Esipova Natalia G.
Published: December 2004

The problem of deducing the DNA structure with correct base pairing and specific symmetry is formulated in the form of algebraic equations. In this way the number of independent variables determining double‐helix conformation can be reduced from six to four. The effectiveness of the method is illustrated by the computations for A‐DNA and B‐DNA. The method allows one to predict all possible conformations of the complementary nucleic acids.
Low Values of the Scheraga–Mandelkern β parameter for proteins. An explanation based on porous sphere hydrodynamics
Authors: McCammon J. A. , Deutch J. M. , Bloomfield Victor A.
Published: December 2004

Several globular proteins have values of the Scheraga–Mandelkern β parameter significantly below the theoretical minimum value, β0 = 2.112 × 106, for an impermeable sphere. Using the Felderhof–Deutch generalization of the Debye–Bueche–Brinkman theory of hydrodynamics of porous spheres, we have shown that values of β slightly below this supposed minimum are theoretically expected. A porous sphere of uniform density has a minimum β of 2.084 × 106 at a Debye shielding ratio of 6.5, corresponding, for example, to a sphere radius of 11 Å and an inverse hydrodynamic shielding length of 0.6 Å−1, values not far from those of small proteins. A two‐layer porous sphere model gives similar results. Although this is the first theoretical explanation of values of β below β0, the theory is incomplete since β values as low as 2.03 × 106 are observed.
Determination of the relaxation time of the helix–coil transition of poly(γ‐benzyl‐L‐glutamate) by the nmr technique
Authors: Nagayama Kuniaki , Wada Akiyoshi
Published: December 2004

NMR measurements of poly(γ‐benzyl‐L‐glutamate) are reported in several different strengths of magnetic field to determine the relaxation time of the helix–coil transition. Nmr spectra of various samples had line shapes varying from the double to single, depending on the extent of the polydispersity of the sample. This result indicated that the correct line shape of a polypeptide is obscured in the overlapping of multipeaks, which are due to the heterogeneity of the molecular weight in the sample. Thus, the conventional line‐shape analysis could not be applied to the kinetic study of the helix–coil transition of polypeptides without consideration of this polydispersity effect on the line shape.

To overcome this difficulty, we measured linewidths of nmr spectra for fairly monodisperse samples, using various nmr spectrometers, having field strengths from 60 to 220 MHz. The results were analyzed by a quadratic equation, which involves an additional term proportional to the frequency difference of two sites. The equation differs from the conventional quadratic equation, usually utilized in the case of the fast‐exchange limit, only in this additional term. This modification is required to evaluate correctly the unusual broadening of the linewidth resulting from the polydispersity effect and to determine the relaxation time reflected in nmr.

Nmr spectra of three samples (DP‐35, 85, and 250) were measured by 220‐, 100‐, and 60‐MHz spectrometers in trifluoroacetic acid/chloroform at 28°C and linewidths were analyzed. Relaxation times of the helix–coil transition obtained at the transition midpoint are 2.5 × 10−4, 7 × 10−4, and 1.1 × 10−3 sec, for DP‐35, 85, and 250, respectively.
Synthesis of N‐protected peptides by the o‐nitrophenylsulfenyl group using o‐nitrophenylsulfenyl N‐carboxy α‐amino acid anhydrides
Authors: Katakai Ryoichi , Oya Masanao
Published: December 2004

N‐protected peptides, which are important intermediates as a carboxyl component in the fragment condensation method, have been prepared in high yields by the reaction of o‐nitrophenylsulfenyl (Nps) N‐carboxy α‐amino acid anhydrides with unprotected peptides and amino acids in aqueous organic systems. An Nps hexapeptide ester was prepared by the fragment condensation of an Nps tripeptide with a tripeptide ester. It was demonstrated that the synthesis of unprotected peptides by the NCA method, followed by N‐protection by the Nps‐NCA, is a rapid and very useful method for preparing Nps peptides.
Stability of steady states in nucleic acid poly[d(A‐T)] synthesis and the stirred flow reactor
Authors: Schneider Friedemann W.
Published: December 2004

The template directed synthesis of poly[d(A‐T)] from the nucleoside triphosphates in the presence of DNA polymerase I is carried out continuously in a stirred flow reactor for the first time. The initial objective is to test the kinetic stability of the established steady states at various flow rates. Graphical analysis predicts instable steady states for certain high flow rates. As a consequence of instabilities multiple steady states and steady‐state hysteresis may occur. Steady‐state hysteresis has now been found experimentally. For a different enzyme fraction of low exonuclease activity we found the steady‐state absorbance at 260 nm to be almost invariant with flow rate at high enzyme concentrations even if the flow rate was increased by a large factor. We call this phenomenon kinetic buffering. Relaxation of a large flow perturbation approaches the steady state in a sigmoidal fashion. Concentration oscillations at 260 nm occurred in one experiment using an enzyme fraction of low exonuclease activity after perturbing the steady state by monomer (dATP). Advantages of the stirred flow reactor method over serial transfer are discussed.
Raman studies of bovine serum albumin
Authors: Lin V. J. C. , Koenig J. L.
Published: December 2004

The Raman Spectra of bovine serum albumin have been obtained in the solute state, in alkaline and acidic solutions, and in the gel. The reversible denaturations of bovine serum albumin solutions by heat, acid's, and alkali were studied and a new mechanism for heat denaturation has been proposed based on a continuous unfolding of the α‐helices.
Low‐frequency Raman spectra of lysozyme
Authors: Genzel L. , Keilmann F. , Martin T. P. , Wintreling G. , Yacoby Y. , Fröhlich H. , Makinen Marvin W.
Published: December 2004

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm−1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm−1. However, a low‐frequency band at 25 cm−1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.
Molecular forces in ribosomes and polynucleotide complexes
Authors: Bresler S. E. , Saminsky E. M. , Chernajenko V. M.
Published: December 2004

The stability constant of the complex of tRNA with 50S subunits of ribosomes was compared in ordinary and heavy water. A considerable effect (about fourfold) was observed, showing the importance of hydrogen bonds in this interaction. In addition, the isotope effect of complementary polynucleotide interaction was measured for two examples. In the case of the binary complex of heptainosinic acid oligomers with poly(C) in the presence of 10−3 M MgCl2, the transfer from ordinary to heavy water gave an increase of the stability constant of about 5%. But in the case of a ternary complex of hexaadenylic acid with poly(U) under the same conditions, the stability constant in D2O increased threefold. The isotope effect depends strongly on magnesium ion concentration and is possibly due to some specific mechanism of magnesium ion complexing involving water molecules.
Relative stability of the α‐helix of deuterated poly(γ‐benzyl‐L‐glutamate)
Authors: Karasz F. E. , Gajnos G. E.
Published: December 2004

The coil‐to‐helix transition temperatures of hydrogen bearing and deuterated poly(γ‐benzyl‐L‐glutamate) in 1,3‐dichlorotetrafluoroacetone/H2O and/D2O mixtures, respectively, have been determined. Together with previously obtained data for the conformational transition of this polypeptide in normal and deuterated dichloroacetic acid, these results have been used in an analysis of the effect of deuterium substitution on the intrinsic stability of the α‐helical form of poly(γ‐benzyl‐L‐glutamate). The findings, consistent for both solvent systems, showed that the deuterated polypeptide is some 5% more stable than the normal protonated poly(γ‐benzyl‐L‐glutamate), while the polypeptide‐active solvent interaction enthalpy is also slightly increased by deuterium substitution in the respective molecules. A consideration of available data for poly(β‐benzyl‐L‐aspartate) reveals an anomaly with respect to the present analysis.
Synthesis and characterization of an ionizable polyhexapeptide collagen model
Authors: Sigler Gerald F. , McMillin Carl R. , Walton Alan G.
Published: December 2004

Poly(Lys(HBr)‐Gly‐Pro‐Pro‐Gly‐Pro) has been synthesized and studied by circular dichroism (CD) spectroscopy. It is apparently the first polyhexapeptide collagen model reported with an ionizable side chain. The monomer (ε‐(p‐nitrobenzyloxycarbonyl)‐Lys‐Gly‐Pro‐Pro‐Gly‐Pro‐p‐nitrophenyl‐ester) was prepared by a stepwise strategy employing active esters. Polymerization in N,N‐dimethyl formamide, followed by removal of the Lys side chain protection with HBr/acetic acid, gave a polydisperse product. Fractionation was accomplished by gel filtration chromatography. The polydisperse material had a molecular weight (Mr = 5–17,000). High molecular weight fractions from triple helices under concentrated conditions at 2°C. The triple helical structure gives a CD pattern very similar to that of collagen and its triple helical analogs. However, unlike collagen, the polyhexapeptide undergoes spontaneous dissociation at temperatures substantially below the melting temperature from a triple helical form to single chains. This process is promoted at low concentrations, high temperature, neutral pH, and low molecular weight, and is apparently due, in large part, to unfavorable ionic side‐chain interactions. In addition to this relatively slow “ionic” dissociation the triple helical polypeptide may be thermally dissociated in a manner similar to collagen. The thermal denaturation is a relatively fast process compared with ionic dissociation.

A high molecular weight fraction (3 × Mr = 48,000) was found to melt at 42°C at neutral pH but increased to 54°C at pH 12 where the lysyl side chains are predominantly deprotonated. Furthermore, reconstitution of triple helices appeared to be more readily achieved at high pH. Thus it is concluded that ionic repulsion between side chains causes destabilization of the triple helix and hinders reconstitution.
Étude thermodynamique de la transition hélice–pelote statistique en solvants mixtes de différents esters de l'acide poly(L‐glutamique)
Authors: Daoust Hubert , Choquette Michel , Hoduc Nga
Published: December 2004

Helix–coil transition of poly(γ‐methyl‐L‐glutamate), poly(γ‐ethyl‐L‐glutamate), and poly(γ‐benzyl‐L‐glutamate) has been studied in mixed solvents by calorimetry, polarimetry, and viscometry. The experimental data have allowed the evaluation of solvation enthalpy Δhb, equilibrium constant K for hydrogen bond formation between the active solvent component and CO and NH groups, and the cooperativity parameter σ. The conformational transition of polypeptides in solution in a mixed solvent containing enough active solvent to maintain the coiled conformation has been produced by dilution with the helix‐supporting solvent for the measurements of enthalpy of transition Δhs. The average value for Δhs is 3550 ± 300 J/mol and is practically independent of the nature of the side chain for the dichloroacetic acid‐ethylene dichloride solvent pair at 25°C. A noticeable concentration effect exists in the case of poly(γ‐benzyl‐L‐glutamate). The helical conformation is less stable for poly(γ‐ethyl‐L‐glutamate), and this is explained by a steric effect hindering the access of dichloroacetic acid to side chains. Constant K has been calculated using polarimetric data and also from values of Δhs obtained at different temperatures using the Bixon and Lifson theory on the one hand and that of Sayama and coworkers on the other hand. Values of σ for poly(γ‐ethyl‐L‐glutamate) have been calculated according to both theories mentioned, and the results show that the two sets of values are quite similar. The constant σ depends on the nature of the active solvent, on temperature, and on the binary‐solvent composition. These conclusions are confirmed by viscometric results. Values of Δhb calculated from constant K are 5230 J/mol when Bixon and Lifson theory is used and 5569 J/mol when the theory at Sayama and coworkers is used. In both cases the value for Δhb is much lower than that of an intramolecular hydrogen bond. Experimental results suggest that the solvation mechanism would proceed in a manner so that mechanisms described in both theories are involved.
Specificity in the interaction between poly(L‐glutamate) and iron (III) complex ions in aqueous solution
Authors: Branca M. , Marini M. E. , Pispisa B.
Published: December 2004

The binding process between sodium poly(L‐glutamate) and trans‐2,2′,2″,2‴‐tetrapyridyl‐Fe(III) complex ions in aqueous solution at pH around 7 has been studied by means of equilibrium dialysis and optical measurements. The binding isotherm indicates the occurrence of a cooperative process, whereby bound molecules facilitate the association of additional molecules. According to circular dichroism (CD) data, this effect is coupled with that which sees a conformational change in the charged polypeptide upon progessive binding of complex counterions. All these features are discussed in the light of the structural characteristics of the interacting species. A stereochemical model of the association “complex” is proposed.
Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions
Authors: Altekar Wijaya
Published: December 2004

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([KQ]eff) and the fraction of fluorescence available ([fa]eff) to the quencher are also calculated.

In contrast to its effect on tryptophan, CH3COO− quenches tyrosyl fluorescence and ClO4− does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan‐containing proteins. (1) NO3−, NO2−, Br−, and I− have high [KQ]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO4− and SCN− also quench fluorescence of tryptophan in proteins and have lower ([KQ]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(fa)eff] = 1. (3) Cl−, CH3COO−, and SO4 do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO4=, CH3COO− ≃ Cl− < ClO4− < SCN− < Br− < I− < NO3− < NO2−.
A general method for evaluation of diffusion constants, dilute‐solution viscoelasticity, and the dielectric property of a rigid macromolecule with an arbitrary configuration. I
Authors: Nakajima Haruhiko , Wada Yasaku
Published: December 2004

As the first part of the study on relaxational behavior of rigid macromolecules in solution, a general method for evaluating diffusion constants of a rigid macromolecule with an arbitrary configuration in a viscous solvent is presented. The evaluation is made by modeling the molecule with a rigid assembly of Stokes spheres. The diffusion constants are expressed by a 6 × 6 matrix which consists of translational, rotational, and cross constants, each being a 3 × 3 submatrix. Procedures of numerical calculation are presented for a given configuration of the molecule, together with some exemplifying results for the rigid rod, rigid sphere, and myosin molecule. The results for the rod and the sphere are compared with analytical results.
Binding of azide ion to methemoglobin at elevated temperatures and the reality of the “compensation” temperature
Authors: Anusiem A. C. I. , Ogunmola G. B. , Beetlestone J. G.
Published: December 2004

We have studied the binding of azide ion to ferrihemoglobin at elevated temperatures. Up to a temperature of 45°C there is no difference in the ligand binding behavior of hemoglobin when compared with the results obtained at lower temperatures. The compensation temperature Tc of 290.6 ± 5.3°K, obtained in this study within the temperature range 303–318°K, confirms that the compensation pattern obtained by Lumry and Rajender is not dependent on the temperature range of the experiment but an intrinsic property of the protein conformation.
Theory of DNA melting curves
Authors: Fixman Marshall , Freire Juan J.
Published: December 2004

Exact algorithms for the calculation of melting curves of heterogeneous DNA with N base pairs apparently require computer time proportional to N2. However, it is shown that a decomposition of the loop entropy factor into a sum of I exponential functions (1) gives an extremely accurate approximation to the loop entropy factor for small values of I and (2) makes the computer time for the exact algorithms proportional to I·N. In effect, exact results for melting curves and lengths of helix or coil stretches are obtained with computer time comparable to that required for the Frank‐Kamenetskii approximation. The remarkable accuracy of the latter for the fraction of helical content (errors of 0.01–0.05) is confirmed, but appreciably larger errors are found for the lengths of helix or coil stretches (typical errors of 30–100%).
Characterization of group C meningococcal polysaccharide by light‐scattering spectroscopy. III. Determination of molecular weight, radius of gyration, and translational diffusional coefficient
Authors: Tsunashima Y. , Moro K. , Chu B. , Liu T. Y.
Published: December 2004

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〉 = 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.
Collagen fibrils and elastic fibers in rat‐tail tendon: An electron microscopic investigation
Authors: Parry D. A. D. , Craig A. S.
Published: December 2004

Earlier studies by the authors showed that the collagen fibrils in rat‐tail tendon have a bi‐modal distribution of fibril diameters from a time shortly after birth through to the onset of maturity at about 3–4 months. Present work has extended those observations for rats up to the age of 2 years. Histograms of the fibril diameter distributions for mature tail tendon and direct electron microscope observations show that the fibrils break down as the tendon ages. Further work on the constant diameter subfibrils of diameter 140 Å described previously, has confirmed that these are part of the elastic fibers present in tendon at all ages. It has been shown that there is relatively little variation in the collagen fibril diameter distribution as a function of the position of the specimen in the tail, and as the measured percentage of the area taken by the collagen fibrils present at any particular point. Estimation of the fibrillar collagen content of rat‐tail tendon as a function of age indicates that it increases steadily from birth and reaches a maximum at the onset of maturity, beyond which the fibrillar collagen content appears to remain constant.
Conformational studies on the pyridoxal Schiff bases of polypeptides
Authors: Dentini M. , Perretti G. , Savino M. , De Santis P. , Verdini A. S.
Published: December 2004

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.
Conformation of amylose in water. I. Light‐scattering and sedimentation‐equilibrium measurements
Authors: Kodama M. , Noda H. , Kamata T.
Published: December 2004

The conformation of amylose in aqueous solution has been found to be dependent on its molecular weight. When the molecular weight of amylose is outside of the so‐called “dissolving gap” described by Burchard (6500<Mr<160,000) it behaves as a random coil, whereas when its molecular weight is within the “dissolving gap,” it easily aggregates forming a rigid coil which is the B‐type (retrograded) amylose. The conformation of this rigid coil is suggested to be a double helix.
Infrared spectra and structure of synthetic polytripeptides
Authors: Lazarev Yu. A. , Lazareva A. V. , Shibnev V. A. , Esipova N. G.
Published: December 2004

A number of polytripeptides related to collagen, namely, (Gly‐Pro‐Pro)n, (Gly‐Pro‐Hyp)n, (Gly‐Hyp‐Hyp)n, (Gly‐Pro‐Ala)n, (Gly‐Pro‐Leu)n, (Gly‐Pro‐Gly)n,(Gly‐Ala‐Pro)n, (Gly‐Ala‐Hyp)n, (Ala‐Pro‐Pro)n, and (Ala‐Hyp‐Hyp)n were investigated by the method of ir spectroscopy and hydrogen‐deuterium kinetics. Strength and order of interpeptide hydrogen bonds of the polytripeptides in a triple‐helical conformation were found to depend on the amino acid composition and residue sequence in the triplets. Correlation of X‐ray diffraction and spectroscopic data for (Gly‐Pro‐Hyp)n showed that the increase of the helix parameter in the process of dehydration is accompanied with the weakening of interpeptide hydrogen bonds. Influences of bound water on the length and order of interchain hydrogen bonding was also examined. It was shown that the incorporation of water molecules into the triple helix depends on the amino acid composition and residue sequence. Synthetic models and native collagens were compared.
Optical properties and viscosity of hyaluronic acid in mixed solvents: Evidence of conformational transition
Authors: Park J. W. , Chakrabarti B.
Published: December 2004

Circular dichroism, optical rotatory dispersion, and viscosity of hyaluronic acid at various solvents compositions, concentrations, and pH values have been studied. The data show a large change in the molecular properties in organic/water solvents such as ethanol, p‐dioxane, or acetonitrile/water at pH ⩽ pKa. At this pH range of aqueous solution, hyaluronic acid shows a CD minimum near 210 nm whereas in the presence of organic solvent it exhibits a strong negative dichroism (below 200 nm) and a positive band near 226 nm. It undergoes a sharp, cooperative transition with respect to pH and solvent. The observed CD features are assigned to the π‐π* and n‐π* transitions of the amide and carboxyl chromophores. The ORD results show a gradual blue shift of trough at 220 nm with increasing magnitude of rotation when the organic solvents and hydrogen ion concentrations are increased. A one‐term Drude's equation was used to analyze the ORD data, and the result show a variation of dispersion parameters with different solvents in accordance with the observed CD changes. The intrinsic viscosity of hyaluronic acid in mixed solvent at pH 2.6 is lower than that of aqueous solution. All the observed property changes of hyaluronic acid are reversed on addition of foramide in mixed solvents indicating that the hydrogen bonds are involved in this transition. The observed spectroscopic and hydrodynamic features are attributed to a conformational change of hyaluronic acid in a mixed solvent involving intramolecular hydrogen bonding between the acetamido and carboxyl groups. The possible conformational state of hyaluronic acid in solution under various conditions is discussed in terms of the reported helical structure of hyaluronic acid from x‐ray diffraction studies.
Shape of the conformational energy surface near the global minimum and low‐frequency vibrations in the native conformation of globular proteins
Authors: Gō Nobuhiro
Published: December 2004

Based on the assumption that the conformational energy surface of a protein molecule can be approximated near the global minimum point by a multidimensional parabola, conformational fluctuations in the native state are discussed. In this approximation the conformational fluctuations can be viewed as excitations of coupled harmonic oscillations of dihedral angles. For the purpose of estimating the range of frequencies vibrations, globular proteins are assumed to made of homogeneous continuous elastic material. The number of vibrational modes in such an elastic body, with the wavelength no less than the characteristic length of an amino acid residue, are estimated roughly to be three times the number of amino acid residues in a protein, which is slightly less than the number of variable dihedral angles in a protein. Their frequencies, when converted to the wavenumber of corresponding light, are found to range from 1.8 × 10 cm−1 to 2.1 × 102cm−1 for a protein with the diameter d = 40 Å, when Young's E = 1011 dyne/cm2 is assumed. A significant fraction of the coupled vibrations of dihedral angles in real globular proteins are collective ones, i.e., those involving the whole protein molecules. Based on these results, it concluded that the depth of the global minimum s at least 150 Kcal/mol.
Hydrodynamic properties of macromolecular complexes. IV. Intrinsic viscosity theory, with applications to once‐broken rods and multisubunit proteins
Authors: De La Torre José Garcia , Bloomfield Victor A.
Published: December 2004

We have extended our previous theories of the translational and rotational frictional properties of multisubunit complexes to calculate the intrinsic viscosity of such structures. Our theory is similar to those recently construced by McCammon and Deutch, and by Nakajima and Wada, in that it uses a modified hydrodynamic interaction tensor and solves the system of simultaneous interaction equations by digital computation rather than by successive approximations. However, there are some differences in the formulation and averaging of these equations. Extensive numerical comparison is made between this theory and others that are available—associated with the names of Hearst and Tagami, Abdel‐Khalik and Bird, and Tsuda—using as a basis exact results for prolate ellipsoids of revolution. For large axial ratios, only our theory asymptotically approaches the correct limit; but for small axial ratios, only the Tsuda “shell‐model” theory is adequate, because the other theories neglect the preponderant influence of the sphere located at the center of rotation. Intrinsic viscosities, translational frictional coefficients, and Scheraga‐Mandelkern β parameters, are tabulated for a large number of polygonal and polyhedral subunit structures, with up to eight elements, using both our theory and Tsuda's. Particular application is made to hemerythrin and aspartate transcarbamylase. Finally, the viscosities and friction coefficients o once‐broken rods are calculated and compared with an approximate theory by Wilenski.
Oxidation of dihydronicotinamide adenine dinucleotide by a flavin derivative of polyethylenimine
Authors: Spetnagel Wayne J. , Klotz Irving M.
Published: December 2004

A modified Polyethylenimine has been prepared that has riboflavin attached to it, as well as hydrophobic groups. The catalytic efficiency toward oxidation of6 dihydronicotinamide adenine dinucleotide (NADH) by this flavopolymer is more than 100‐fold greater than observed with small‐molecular riboflavin. The products of the reaction in aerobic solution have been established to be the oxidized nicotinamide NAD+ and hydrogen peroxide. The kinetics fit a Michaelis‐Menten mechanism. Thus, the versatility of modified polyethylenimines as catalysts has been extended from hydrolyses and decarboxylations to oxidation‐reduction reactions also.
Poly(L‐histidyl‐L‐alanyl‐α‐L‐glutamic acid). II. Catalysis of p‐nitrophenyl acetate hydrolysis
Authors: Goren H. Joseph , Fletcher Thea , Fridkin Mati , Katchalsi‐Katzir Ephraim
Published: December 2004

The hydrolysis of p‐nitrophenyl acetate is catalyzed by imidazole, free in solution or as the side chain in poly(His‐Ala‐Glu). This is based on the observations that the reaction is first order in ester and first order in nonprotonated imidazole. Catalysis of p‐nitrophenyl acetate hydrolysis is dependent on solvent conditions. The effect of low concentrations of ethanol, dioxane, and trifluoroethanol were investigated. As the concentration of organic solvent is increased, the second‐order rate constant for imidazole catalysis decreases. The decrease, however, is greater for imidazole than for poly(His‐Ala‐Glu). In 2% trifluoroethanol/water solution, free imidazole has twice the catalytic activity of polymeric imidazole, while in 40% trifluoroethanol/water they have equal activity. Since under the latter solvent conditions poly(His‐Ala‐Glu) is partially α‐helical, the relative improvement in polymeric–imidazole catalysis may be attributed to imidazole hydrogen‐bonded to a carboxylate ion. With this assumption the carboxylate–imidazole hydrogen‐bonded system has been calculated to have three times the base catalytic activity of imidazole.
Linear oligopeptides. XLIII. Study of the relationship between conformation and nature of side chain: Homologous series derived from γ‐branched amino acid residues
Authors: Toniolo Claudio , Bonora Gian Maria , Palumbo Manlio , Peggion Evaristo , Stevens Eugene S.
Published: December 2004

Conformational studies of the three homologous series, from dimer through heptamer, of monodisperse, N‐and C‐protected oligopeptides derived from the γ‐branched α‐amino acid residues L‐leucine, β‐cyclohexyl‐L‐alanine, and L‐phenylalanine are reported. By means of ir absorption and CD in the vacuum‐uv (150nm), the occurrence of intermolecular β‐conformations in the higher oligomers in the solid state was established. In solvents of low polarity at high dilution, the extent of intramolecularly hydrogen‐bonded folded‐structure formation was assessed as a function of chain length and the nature of the side chain. Unordered and intermolecular β‐conformations were found in alcohols and aqueous alcoholic mixtures. The results obtained indicate that—the position of branching being equal–steric requirements, electronic properties, and hydrophobic character of the amino acid side chains are all important in determining the nature and stability of oligopeptide conformations.
Resolution of electronic absorption bands and nucleotide binding processes in actinomycin D
Authors: Auer Henary E. , Pawlowski‐Konopnicki Barbara E. , Chiao Yu‐Chih Chen , Krugh Thomas R.
Published: December 2004

Complexes of actinomycin D with model dexoxynucleoides have been studied by means of absorption spectroscopy and CD spectroscopy and CD spectroscopy. The CD spectra of the complexes of actinomycin D with 5′‐dGMP, pdG‐dT, pdT‐dG, pdG‐dA, and pdA‐dG, respectively, all resemble one another. It is presumed that in solution the interactions between the guanine residues and actinomycin D in these complexes are the same as found in the crystalling 1:2 actinomycin D:dG complex [Jain, S. C. & Sobell, H. M. (1972) J. Mol. Biol. 68, 1–20]. The CD spectrum of the Complexes with pdG‐dC differs from of the complexes just mentioned, and resembles those of the complexes formed by actinomycin D with calf‐thymus DNA and with poly(dG‐dC)‐poly(dG‐dC). These resulls are consisent with, the nontion that pdG‐dC froms a double‐staranded intercalated complex with actinomycin D, and that the dG‐dC sequence is an important binding site for actinomycin D in polydeoxynucleotides.

Titrations of actinomycin D with monodeoxynucleotides were monitored at 380, 425, 440,465, and 480, nm in both absorption and CD modes. Comparisons fo saturation profiles at these wavelengths reveal that the curves obtained at various wavelenths do not superimpose with each other, so that they must reflect different titation processes. In complex formation wiht any given nucleotide, the apparent binding affinity monitored at these wavelengths decreases in the order given above. Based on these resulted and on features noted in the CD spectra of certain complexes, it is concluded that a minimum of theree electronic transitions underlie the visible absorption band of actinomycin D, extending earlier findigs. Comparing the titration proffiles obtained with dAMP and dIMP with the result for these systems in mmr titratins [Krugh, T. R. & Chen, Y. C. (1975) Biochemistry 14, 4912–4922], it is concluded that one transition, centered at 370 nm, monitors preponderantly effects occurring at the 6 (benzenoid) nucleotide binding site and a second transition, located at 490 nm, is sensitive preferentially to processes occurring at the 4 ( quinoid) binding site. The latter is probably closely asscoiated with 2‐amino and /or 3‐carbonyl substituents. The third transition, identified with the absorption maxium at 420–440 nm, appears to reflect contributions arising in the entire phenoxazone chromophore. Using these band assignments, it is concluded that the benzenoid site binds nucleotides 1.5–3 times more avidly than does the quinoid site. CD titrations resolve these processes more effectively than do abscrption titrations. Aspects of the structures of these complexes formed in solution are discussed.
Solid‐phase synthesis of somatostatin and glucagon‐selective analogs in gram quantities
Authors: Rivier J. , Kaiser R. , Galyean R.
Published: December 2004

Somatostatin (SS) and two glucagon selective analogs [D‐Cysl4]‐SS and [D‐Trp8, D‐Cys14]‐SS have been synthesized in gram quantities by the solid‐phase procedure. A general modification of Monahan and Gilon's procedure for esterification of the first protected amino acid onto the chloromethylated resin as well as a general protocol for solid‐phase peptide synthesis on Beckman 990 automatic synthesizer are described. A new general procedure for disulfide formation, which involves the adaptation of the “high‐dilution” principle to the ferricyanide oxidation and the optimization of the sequence of purification steps as applied to somatostatin and its analogs, yields highly purified peptides (≥ 199% pure) as checked by reverse‐phase high‐pressure liquid chromatography—which is shown to be a highly sensitive, resolutive, and quantitative analytical tool for evaluation of the homogeneity of peptides.