Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Synthesis, characterization, and DNA binding and cleavage properties of copper(II)-tryptophanphenyl-alanine-1,10-phenanthroline/2,2'-bipyridine complexes

The mononuclear dipeptide‐based Cu^II complexes [Cu^II(trp‐phe)(phen)(H₂O)] ⋅ ClO₄ ( 1 ) and [Cu^II(trp‐phe)(bpy)(H₂O)] ⋅ ClO₄ ( 2 ) (trp‐phe=tryptophanphenylalanine, phen=1,10‐phenanthroline, bpy=2,2′‐bipyridine) were isolated, and their interaction with DNA was studied. They exhibit intercalative mode of interaction with DNA. The intercalative interaction was quantified by Stern Volmer quenching constant (K_sq=0.14 for 1 and 0.08 for 2 ). The Cu^II complexes convert supercoiled plasmid DNA into its nicked circular form hydrolytically at physiological conditions at a concentration as low as 5 μM (for 1 ) and 10 μM (for 2 ). The DNA hydrolysis rates at a complex concentration of 50 μM were determined as 1.74 h⁻¹ (R=0.985) for 1 and 0.65 h⁻¹ (R=0.965) for 2 . The rate enhancement in the range of 2.40–4.10×10⁷‐fold compared to non‐catalyzed double‐stranded DNA is significant. This was attributed to the presence of a H₂O molecule in the axial position of the Cu complexes.     

Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA

Preparations of circular plasmid DNA in either supercoiled or nicked circular form often are contaminated with undesired linear DNA fragments arising from shearing/degradation of chromosomal DNA or linearization of plasmid DNA itself. We report a simple enzymatic method, using a combination of λ exonuclease and RecJ_f, for the selective removal of linear DNA from such mixtures. λ exonuclease digests one strand of linear duplex DNA in the 5′ to 3′ direction, whereas RecJ_f, a single-strand-specific exonuclease, digests the remaining complementary single strand into mononucleotides. This combination of exonucleases can remove linear DNA from a mixture of linear and supercoiled DNA, leaving the supercoiled form intact. Furthermore, the inability of λ exonuclease to initiate digestion at nicks or gaps enables the removal of undesired linear DNA when nicked circular DNA has been enzymatically prepared from supercoiled DNA. This method can be useful in the preparation of homogeneous circular plasmid DNA required for therapeutic applications and biophysical studies.     

Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard: Microalgal pcna as the Model Gene

Quantitative real-time PCR (qPCR) has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear) used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form) and linear DNA standards (linearized plasmid DNA or PCR amplicons), using proliferating cell nuclear gene (pcna), the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.     

Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli

A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error‐prone PCR or molecular shuffling, and a linear vector backbone was prepared by high‐fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap‐extension PCR with a pair of 5'‐phosphorylated primers. Third, full‐length linear plasmids with phosphorylated 5'‐ends were self‐ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high‐efficiency transformation. Self‐made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10⁵ cfu/µg of the self‐ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6‐phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5‐fold improved catalytic efficiency (k_cat/K_m) on NAD⁺ as compared to the wild‐type. This protocol is DNA‐sequence independent, and does not require restriction enzymes, special E. coli host, or labor‐intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.     

Internal Dynamics of Supercoiled DNA Molecules

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

Synthesis and enantiopreferential DNA-binding profile of late 3d transition metal R- and S-enantiomeric complexes derived from N,N-bis-(1-benzyl-2-ethoxyethane): Validation of R-enantiomer of copper(II) complex as a human topoisomerase II inhibitor

To evaluate the biological preference of chiral drug candidates for molecular target DNA, new potential metal‐based chemotherapeutic agents 1 , 1a , 1b , 2 , 2a , 2b , 3 , 3a , 3b of late 3d transition metals Ni(II), Cu(II), and Zn(II), respectively, derived from (R)‐ and (S)‐2‐amino‐2‐phenylethanol with  CH₂ CH₂ linker were synthesized and thoroughly characterized. Interaction studies of 1 , 1a , 1b , 2 , 2a , 2b , 3 , 3a , 3b with calf thymus DNA in Tris buffer were studied by electronic absorption titrations, luminescence titrations, cyclic voltammetry, and circular dichroism. The results reveal that the extent of DNA binding of R‐enantiomer of copper 1a was highest in comparison to rest of the complexes via electrostatic interaction mode. The nuclease activity of 1a , 1b with supercoiled pBR322 DNA was further examined by gel electrophoresis, which reveals that complex 1a exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322DNA, and the cleavage activity of both enantiomers of complex 1 was significantly enhanced in the presence of activators. The activating efficiency follows the order Asc > H₂O₂ > MPA for 1a , and reverse order was observed for 1b , because of the differences in enantioselectivity and conformation. Further, it was observed that cleavage reaction involves singlet oxygen species and superoxide radicals via oxidative cleavage mechanism. In addition, complex 1a exhibits significant inhibitory effects on the topoisomerase II (topo II) activity at a very low concentration ∼24 μM, which suggest that complex 1a is indeed catalytic inhibitor or (poison) of human topo II. Chirality 2011 © 2011 Wiley‐Liss, Inc.     

Statistics of branching and hairpin helices for the dAT copolymer

At low temperatures a single strand of dAT copolymer tends to form a double helix with a loop at one end (hairpin). At slightly higher temperatures a branched structure (consisting of many hairpins connected by coiled regions) appears. The equilibrium properties of this branched structure are studied here by a propagator method. In particular the radius of gyration R_G is calculated; under typical conditions, R_G is expected to show a minimum at a temperature slightly below melting, in agreement with viscosity measurements. The present calculation is restricted to rather sharp transitions, for which the size of each helical region is large, even at the melting point. Then, in a rather broad range of molecular weights, M, R_G is proportional to M^1/4(as in the Zimm-Stockmayer theory of simple branched polymers).     

The recF-dependent endonuclease from Escherichia coli K12. Formation and resolution of pBR322 DNA multimers

Summary The instability of supercoiled pBR322 DNA obtained from different cells has been investigated. Partially purified plasmid DNA species from rec ⁺, recA and recBC sbcB cells are converted in vitro first to relaxed and then to linear molecules. The recA and recBC sbcB cells produce the best conditions for the monomerization of the pBR322 DNA and the stable maintenance of plasmids. The supercoiled pBR322 DNA from the recBC sbcB recF144 cells has been isolated preferentially in multimeric from (circular oligomers). These DNA forms are not converted to plasmid monomers and are converted to linear molecules three-fold slower than the monomer linearization in the case of the recBC sbcB cells.On the other hand, incubation of the pure pBR322 DNA with the recF-dependent protein Z (Krivonogov and Novitskaja 1982) results in the ATP-independent conversion of supercoiled plasmid DNA to relaxed and linear molecules. These results demonstrate an endonuclease activity of the recF-controlled protein Z, which may be involved in general recA-dependent recombination and formation of the pBR322 monomers in the cell.The results also show that the recF144 mutation in recBC sbcB recF and recF cells leads to the absence of detectable amounts of a 49,000 molecular weight protein.     

Monte carlo computation of the supercoiling energy,the sedimentation constant,and the radius of gyration of unknotted and knotted circular DNA

Closed random Gaussian polygonal chains of N (6 < N < 150) bonds of equal length b and thickness d have been generated on a computer. The knot type, the writhing number w, the radius of gyration, and the average of the inverse of the distance between two apices have been determined for each chain. For all the studied knot types—0, 3₁, 4₁, 5₁, and 5₂—the probability density of finding a given w is Gaussian. The Gaussian is centered about 0 for the amphichiral knots. Therefore, for long circular DNAs, the contribution to the supercoiling energy, which depends on w only, may be considered as purely entropic and may be expressed as ARTw²/N, in agreement with previous semiempirical considerations. The parameter A increases with chain thickness, it decreases as N gets larger but rapidly reaches a plateau. Comparison with experimental data from the literature would suggest that the ratio of the writhing to the constraint increases with ionic strength. The ratio of sedimentation constant of the supercoiled DNA to the sedimentation constant of the nicked DNA varies as N^1/4 (w/N)², and therefore depends on the writhing density and on the length of the DNA.     

The molecular structure and solution conformation of an acidic heteropolysaccharide from Auricularia auricula-judae

A water soluble acidic heteropolysaccharide named WAF was isolated from Auricularia auricula‐judae by extracting with 0.9% NaCl solution. By using gas chromatography, gas chromatography‐mass spectrometry, and NMR, its chemical structure was determined to be composed of a backbone of α‐(1→3)‐linked D ‐mannopyranose residues with pendant side groups of β‐D ‐xylose, β‐D ‐glucose, or β‐D ‐glucuronic acid at position O6 or O2. Six fractions prepared from WAF with a weight‐average molecular mass (M_w) between 5.9 × 10⁴ and 64.7 × 10⁴ g/mol were characterized with laser light scattering and viscometry in 0.1M NaCl at 25°C. The dependence of intrinsic viscosity ([η]) and radius of gyration (R_g) on M_w for this polysaccharide were found to be [η] = 1.79 × 10^?3M_w^0.96 cm³ g^?1 and R_g = 6.99 × 10^?2 M_w^0.54 nm. The molar mass per unit contour length (M_L) and the persistence length (L_p) were estimated to be 1124 nm^?1 and 11 nm, respectively. The WAF exhibited a semirigid character typical of linear polysaccharides. Molecular modeling was then used to predict the ordered and disordered states of WAF; the simulated M_L and L_p were however much smaller than the experimental values. Taken altogether, the results suggested that WAF formed a duplex in solution. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 217–227, 2011.     

Introduction of axial chirality in a planar aromatic ligand results in chiral recognition with DNA

Dimerization of a hydroxycarbazole produces an axially chiral biaryl, BICOL ( 2 ). One enantiomer (R)‐ 2 , is capable of enantioselective binding to different polymorphs of DNA. The biaryl (R)‐ 2 was shown by fluorescence and circular dichroism to induce a shift of Z‐DNA to B‐DNA. The opposite enantiomer (S)‐ 2 shows no specific binding. The significant difference in behaviour between the two enantiomers (S)‐ 2 and (R)‐ 2 is in line with molecular modelling studies which show two very different binding geometries between the enantiomers with each polymorph of DNA. Copyright © 2010 John Wiley & Sons, Ltd.     

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular‐Docking Method

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular‐docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three‐dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular‐docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH₃ in R₁ had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8‐anilino‐1‐naphthalenesulfonic acid, was changed with norgestrel addition.     

Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels.

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.     

MITOCHONDRIAL GENOME CONFORMATION AMONG CW‐GROUP CHLOROPHYCEAN ALGAE1

Most green algal taxa have circular‐mapping mitochondrial genomes, whereas some have linear genome‐ or subgenomic‐sized mitochondrial DNAs (mtDNA). It is not clear, however, if the circular‐mapping genomes represent genome‐sized circular molecules, if such circular molecules and the linear forms are the predominant in vivo mtDNA structures, or if the linear forms arose only once or multiple times among extant green algal lineages. We therefore examined the DNA components detected with homologous mtDNA probes after pulsed‐field gel electrophoresis of total cellular DNA from the chlorophycean basal bodies displaced clockwise(CW)‐group taxa Chlamydomonas reinhardtii and Chlamydomonas moewusii. For C. reinhardtii, the 15.8‐kb linear mtDNA was the only DNA component detected, and there was no evidence of circular or large linear precursors of this DNA. In the case of C. moewusii, which is known to have a circular‐mapping 22.9‐kb mitochondrial genome, three DNA components were detected; these appeared to be circular (relaxed and supercoiled) and genome‐sized linear DNA molecules, the latter of which likely resulted from random double‐strand breaks in the circular forms during DNA isolation. In further studies, DNA from additional CW‐group taxa was examined using conventional gel electrophoresis and DNA‐filter blot analysis with C. reinhardtii and C. moewusii mtDNA probes. We conclude that all taxa from the “Volvox clade” (sensu Nakayama et al. 1996 of the CW‐group have genome‐ or subgenomic‐sized linear mtDNAs as their predominant mtDNA form and that these arose from a genome‐sized circular form in an ancestor that existed near the base of this clade.     

Evidence for a folded conformation of the cell wall protein fromAcetabularia (Polyphysa) cliftonii

Summary The cell wall protein fromAcetabularia has a non-random structure in aqueous solution at pH 5.3, as determined on the basis of intrinsic viscosity, sedimentation velocity and small angle X-ray scattering experiments. This non-random structure is stable in a pH range of 4.5–6.8, as observed on the basis of circular dichroism and viscosity measurements, supporting that the cell wall protein has a specific folded structure. All hydrodynamic measurements, including small angle X-ray scattering in solution, in this pH range are consistent with a prolate ellipsoid model for the shape of this protein, with overall dimensions ofc=86.0 Å,b=7.0 Å, anda=7.5 Å, and with a radius of gyration ofR=39.5 Å. The possibility of a coiled shape was investigated using a worm-like chain model, but it was inconsistent with the experimental data. Instead, a filled particle with uniform density which is equivalent in the scattering behavior is proposed. By a comparison of the observed radius of gyration, R_g=39.5 Å, and the radius of gyration of the cross section,R _c =7.5 Å, we were able to describe the cell wall protein in terms of a prolate ellipsoid of revolution. Comparisons of the experimental scattering curve, plotted as logl (h) versus logh, with the corresponding plots of normalized intensities, calculated for particles of particular shape and various axial ratios indicate a very asymmetric shape for the cell wall protein fromAcetabularia.This research was supported by a grant of the Deutsche Forschungsgemeinschaft.     

Small-angle x-ray scattering of bovine nasal cartilage proteoglycan in solution

The proteoglycan subunit (PGS) from bovine nasal cartilage was examined in water and in 0.15 N LiCl by small-angle x-ray scattering (SAXS). The molecular weight of 2.5 × 10⁶ and the radius of gyration, R_g = 493 Å, in 0.15 N LiCl, obtained by SAXS, are in good agreement with values reported by others for similar preparations. Values of the radius of gyration of the cross section, mass per unit length, and persistence length of the PGS are also reported. The low value of intrinsic viscosity ([η]) found in 0.15 N LiCl, and a comparison of the experimental distance distribution function to that of the theoretical distance distribution function for sphere, suggest that the PGS in salt solution approaches spherical symmetry. The much higher value of [η] in water suggests a prolate ellipsoid of low axial ratio.     

Absolute configuration and biological profile of pyrazoline enantiomers as MAO inhibitory activity

A new racemic pyrazoline derivative was synthesized and resolved to its enantiomers using analytic and semipreparative high‐pressure liquid chromatography. The absolute configuration of both fractions was established using vibrational circular dichroism. The in vitro monoamine oxidase (MAO) inhibitory profiles were evaluated for the racemate and both enantiomers separately for the two isoforms of the enzyme. The racemic compound and both enantiomers were found to inhibit hMAO‐A selectively and competitively. In particular, the R enantiomer was detected as an exceptionally potent and a selective MAO‐A inhibitor (K_i = 0.85 × 10^?3 ± 0.05 × 10^?3 μM and SI: 2.35 × 10^?5), whereas S was determined as poorer compound than R in terms of K_i and SI (0.184 ± 0.007 and 0.001). The selectivity of the enantiomers was explained by molecular modeling docking studies based on the PDB enzymatic models of MAO isoforms.