Simultaneous haemo-filtration and haemo dialysis

Raman and vibrational Raman optical activity (ROA) spectra have been calculated for three molecules: H2O2, CHDTF and CHDTOH. The effects of electron correlation on the ROA parameters of these relatively small systems have been investigated by means of the multiconfigurational self-consistent field (SCF) approach. A range of correlation-consistent basis sets has been used in the calculations. The basis set convergence of the calculated ROA parameters is fairly well achieved when sets of at least d-aug-cc-pVTZ quality are used. On the other hand, the aug-cc-pVDZ set seems to be sufficient for a qualitative analysis. Diffuse functions, possibly a double set, are essential to ensure a good quality of the calculated ROA spectrum. In most cases the sign and approximate magnitude of the ROA are correctly predicted at the SCF level, and the corresponding ROA spectra should be considered qualitatively accurate.

Circular dichroism of κ- and λ-type Bence-Jones proteins at various pH values and temperatures

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Vibrational Spectra:Principles and Applications with emphasis on optical activity

Natural resonance electronic Raman optical activity (ROA) is observed for the first time. Coincidently, the first example of vibrational ROA enhanced by low‐lying electronic transition is reported. These new phenomena were measured using the rare‐earth complex Eu(tfc)3 (+)‐tris[3‐trifluoroacetyl‐D‐camphorato]europium(III), where electronic resonance occurs between the 532‐nm laser excitation and the 7F1 → 5D1 transition of the Eu3+ metal center. Electronic Raman spectra involve the Raman transitions terminating on the low‐lying electronic states of Eu(tfc)3. The observed vibrational ROA spectra are enhanced relative to typical ROA spectra by the proximity of vibrational states of Eu(tfc)3 to its low‐lying electronic states with significant magnetic‐dipole character, whereas the parent vibrational Raman spectra do not appear to be resonance‐enhanced since the 532‐nm vibrational Raman spectrum has similar relative intensities to the corresponding Raman spectrum measured with 1064‐nm laser excitation. Copyright © 2010 John Wiley & Sons, Ltd.

Infrared vibrational circular dichroism (VCD) and vibrational Raman optical activity (ROA) have been measured and compared quantitatively over the frequency range from 835 to 1345 cm−1 for trans-pinane, cis-pinane, α-pinene, and β-pinene. For these molecules in this region of spectral overlap between VCD and ROA, the average ratio of VCD or ROA to its parent vibrational intensity favors ROA by a factor of two to three. Several vibrational modes in each molecule yield both large VCD and large ROA, while several other modes show little propensity toward significant VCD or ROA intensity. Beyond this general property of a few strongly chiral and strongly achiral vibrational modes, little additional correlation between VCD and ROA intensity is found. This quantitative compilation of VCD, infrared, ROA, and Raman intensities provides an experimental basis for computational intensity studies of VCD, ROA, and their theoretical comparison.

At neutral pH the far ultraviolet circular dichroism (CD) spectrum of alkaline mesentericopeptidase is dominated by two negative bands: at 208 and 220 nm. The near ultraviolet CD spectrum is characterized by a large negative band at 277nm, a shoulder near 286nm and a small positive band at 297nm. The introduction of phenylmethanesulfonyl (PMS) group on the serine residue at the active site does not alter the intensity and the wavelength position of the bands.In contrast to the other alkaline bacterial proteases, which are remarkably resistant to denaturation by urea, mesentericopeptidase was unfolded in the presence of this reagent at neutral pH. The denaturation reactions in the presence of 9.5 M urea of both native and PMS‐mesentericopeptidase followed first order kinetics. The introduction of PMS‐group on the active‐site serine lowered the conformational stability of the enzyme. In the first 3h PMS‐mesentericopeptidase was denaturated 2.5 times faster than native protease.In the presence of 9.5 M urea at natural pH alkaline mesentericopeptidase lost its catalytic activity. The loss of enzymatic activity correlated with the unfolding of the protein molecule and also followed first‐order kinetics.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle chi(2,1) for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.

Vibrational Raman optical activity (ROA) is measured in our laboratory as a small intensity difference in Raman scattered light from chiral molecules in right and left circularly polarized incident light.[l, 2] Recently, a series of instrumental advances [3] has opened up the realm of biopolymers, including proteins, polysaccharides and now nucleic acids, to this novel chiroptical technique. For proteins and polysaccharides it has already been demonstrated that ROA can probe key structural elements more incisively than conventional Raman spectroscopy because it can cut through the complexity of the conventional spectra by probing directly the crucial chiral elements of biomolecular structure. In this contribution we focus on some of our recent results on polynucleotides and as an example present the Raman and ROA spectra of poly(rG)•poly(rC) in H2O between 650 and 1750 cm −1 in Figure 1.

Chirality plays a crucial role in drug discovery and development. As a result, a significant number of commercially available drugs are structurally dissymmetric and enantiomerically pure. The determination of the exact 3D structure of drug candidates is, consequently, of paramount importance for the pharmaceutical industry in different stages of the discovery pipeline. Traditionally the assignment of the absolute configuration of druggable molecules has been carried out by means of X-ray crystallography. Nevertheless, not all molecules are suitable for single-crystal growing. Additionally, valuable information about the conformational dynamics of drug candidates is lost in the solid state. As an alternative, vibrational optical activity (VOA) methods have emerged as powerful tools to assess the stereochemistry of drug molecules directly in solution. These methods include vibrational circular dichroism (VCD) and Raman optical activity (ROA). Despite their potential, VCD and ROA are still unheard of to many organic and medicinal chemists. Therefore, the present review aims at highlighting the recent use of VOA methods for the assignment of the absolute configuration of chiral small-molecule drugs, as well as for the structural analysis of biologics of pharmaceutical interest. A brief introduction on VCD and ROA theory and the best experimental practices for using these methods will be provided along with selected representative examples over the last five years. As VCD and ROA are commonly used in combination with quantum calculations, some guidelines will also be presented for the reliable simulation of chiroptical spectra. Special attention will be paid to the complementarity of VCD and ROA to unambiguously assess the stereochemical properties of pharmaceuticals.

We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

Experimental and ab initio theoretical vibrational Raman optical activity of alanine

Determination of the absolute handedness, known as absolute configuration (AC), of chiral molecules is an important step in any field related to chirality, especially in the pharmaceutical industry. Vibrational optical activity (VOA) has become a powerful tool for the determination of the AC of chiral molecules in the solution state after nearly forty years of evolution. VOA offers a novel alternative, or supplement, to X-ray crystallography, permitting AC determinations on neat liquid, oil, and solution samples without the need to grow single crystals of the pure chiral sample molecules as required for X-ray analysis. By comparing the sign and intensity of the measured VOA spectrum with the corresponding ab initio density functional theory (DFT) calculated VOA spectrum of a chosen configuration, one can unambiguously assign the AC of a chiral molecule. Comparing measured VOA spectra with calculated VOA spectra of all the conformers can also provide solution-state conformational populations. VOA consists of infrared vibrational circular dichroism (VCD) and vibrational Raman optical activity (ROA). Currently, VCD is used routinely by researchers in a variety of backgrounds, including molecular chirality, asymmetric synthesis, chiral catalysis, drug screening, pharmacology, and natural products. Although the application of ROA in AC determination lags behind that of VCD, with the recent implementation of ROA subroutines in commercial quantum chemistry software, ROA will in the future complement VCD for AC determination. In this review, the basic principles of the application of VCD to the determination of absolute configuration in chiral molecules are described. The steps required for VCD spectral measurement and calculation are outlined, followed by brief descriptions of recently published papers reporting the determination of AC in small organic, pharmaceutical, and natural product molecules.

Chapter 16 - Raman optical activity