Structures of intact glycoproteins from vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra for the glycoproteins alpha1-acid glycoprotein (AGP) and bovine submaxillary mucin (BSM), have been measured in D2O solutions and for the films prepared from aqueous (H2O) buffer solutions in the 1800 to 900 cm(-1) region. The solution VCD results revealed that AGP has beta-sheet structure, along with a significant amount of alpha-helix as evidenced from a W pattern in the amide I region. The VCD of BSM solution suggested a polyproline II type structure, characterized by the appearance of strong negative couplet in the amide I region. The film VCD results on AGP and BSM suggested that the secondary structures of polypeptide fold in the film state are similar to those in the solution. The absence of any significant film VCD in the low frequency region (1200-900 cm(-1)), suggested that the dominant linkage for carbohydrate residues is likely to be a beta linkage. VCD spectroscopy gains importance in the secondary structural analysis of polypeptide fold in glycoproteins due to the absence of interfering VCD from the carbohydrate residues in the conformationally sensitive amide I region. Also, film VCD studies permit measurements in the low wavenumber region (1200-900 cm(-1)) that reveal the dominant type of linkage for carbohydrate residues. Such clear structural information is unlike that from ECD, where ECD bands of acylated amino sugar residues interfere with those of polypeptide backbone in the conformationally sensitive far-UV region.     

Vibrational circular dichroism spectra of protein films: thermal denaturation of bovine serum albumin

Vibrational circular dichroism (VCD) spectroscopy has been used for the first time to investigate the thermal denaturation of proteins in H(2)O solutions. Films prepared from heated aqueous solutions were used for these investigations. A well-known alpha-helical protein, bovine serum albumin (BSA), is used for this first study. Both VCD and infrared absorption results obtained for BSA films indicate that the heat treatment of BSA induces significant amounts of beta-sheet structure and that the denaturation process is irreversible. To verify the irreversible nature of thermal denaturation, optical rotation was also measured as a function of temperature in both heating and cooling cycles. These results also indicate that thermal denaturation of BSA in solution is irreversible. This study establishes the usefulness of films for VCD investigations and offers new avenues for VCD studies on biologically important systems.     

Conformational studies of the smallest structural motifs of DNA detectable via vibrational circular dichroism: cytidylyl-(3''-5'')-guanosine and guanylyl-(3''-5'')-cytidine.

The infrared absorption and vibrational circular dichroism (VCD) spectra of buffered aqueous solutions of cytidylyl-(3'-5')-guanosine (5'(CG)3') and guanylyl-(3'-5')-cytidine (5'(GC)3') are reported. Under low ionic strength conditions, these dinucleotides exhibit VCD features that can be predicted qualitatively from structural data of (CG)2 and (GC)2 sequences of poly(dG-dC).poly(dG-dC), using the exciton model for infrared VCD intensities.     

Matrix isolation of free radicals from carbohydrates. II. Reactions of H. with glucose and derivatives in acidic glasses

Photolytically produced H.-atoms in 6 mol dm-3 H2SO4/H2O glasses trapped at 77 K react upon annealing to 130 K with dissolved carbohydrates to form carbon-located free radicals by abstraction of carbon-bound protons. Analysis of electron spin resonance (e.s.r.) spectra at various annealing stages from alpha- and beta-D-glucose together with 6,6-d2-D-glucose, 6-deoxy-D-glucose, 2-deoxy-D-glucose, glucose-1-phosphate, D-xylose, D-allose and D-mannose indicates radical formation at all possible carbon sites with a strong preference for C1 and a somewhat enhanced contribution of C4 over the statistical expectation. The corresponding component spectra are analysed either by spectra isolation or simulation and their parameters are given. Intramolecular radical transformation at temperatures of 140-160 K is explained by acid-catalysed H2O-elimination. The findings are discussed in relation to the radiation-chemistry of aqueous glucose solutions. We thus show that the system of photolyzed Fe2+ in acidic glasses at low temperatures containing 10 mmol dm-3 carbohydrate is suitable for studying H(D.)-reactions by means of e.s.r. spectroscopy. Unlike previously used glasses containing carbohydrates, contributions of oxidation and reduction by direct effects or mixtures of direct and indirect effects and phase-effects due to incomplete glass formation are avoided.     

New flavonoid glycosides from Aconitum naviculare (Brühl) Stapf, a medicinal herb from the trans-Himalayan region of Nepal

Three new flavonoid glycosides, 3-O-[beta-D-glucopyranosyl-(1-->3)-(4-O-trans-p-coumaroyl)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranosyl]-7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl]kaempferol, 3-O-[beta-D-glucopyranosyl-(1-->3)-(4-O-trans-p-coumaroyl)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranosyl]-7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl]quercetin and 7-O-[beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl]quercetin were isolated from the aqueous extract of the aerial parts of Aconitum naviculare. Their structures were elucidated by spectral analysis (HRAPI-TOF MS, 1H, 13C NMR, HMQC, HMBC, DFQ-COSY, ROESY and TOCSY).     

Application of infrared spectroscopy (attenuated total reflection) for monitoring enzymatic activity on substrate films

Infrared film analysis, a method based on infrared spectroscopy in the mode of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), is demonstrated as a novel analytical method for monitoring enzymatic activity on surface-attached substrate films in the mid infrared range (400-4000 cm(-1)). The ATR-FTIR technique is sensitive to molecules within a distance of approximately 1 microm from the ATR sampling unit surface (a 7 cm(2) hydrophobic ZnSe crystal). Applying a 0.2-0.3 microm thick film on the ATR unit surface, any chemical changes within this film as well as at the interface can be continuously monitored, even having an aqueous phase on top of the film. Infrared film analysis is considered especially useful for studying detergent enzymes, which act on surface bound films consisting of food component like vegetable oils (triacylglycerols) and carbohydrates (e.g. starch). Experimental data are presented for hydrolysis of a triacylglycerol film (triolein) by use of a triacylglycerol lipase (cutinase), and starch film degradation by use of an alpha-amylase.     

Structural transition during thermal denaturation of collagen in the solution and film states

Temperature dependent vibrational circular dichroism (VCD) spectra of type I collagen, in solution and film states, have been measured. These spectra obtained for solution sample suggest that the thermal denaturation of collagen results in transition from poly-L-proline II (PPII) to unordered structure. The PPII structure of collagen is identified by the presence of negative VCD couplet in the amide I region, while the formation of unordered structure is indicated by the disappearance of VCD in the amide I region. The temperature dependent spectra obtained for the supported collagen film indicated a biphasic transition, which is believed to be the first vibrational spectroscopic report to support a biphasic transition during thermal denaturation of collagen film. The temperature dependent spectra of collagen films suggest that the thermal stability of collagen structure depends on its state and decreases in the order: supported film > free standing film > solution state. These observations are believed to be significant in the VCD spectroscopic analysis of secondary structures of proteins and peptides.     

Surface structure of chitosan and hybrid chitosan-amylose films-restoration of the antibacterial properties of chitosan in the amylose film

The surface structure of films prepared by casting aqueous solutions of mixtures of water soluble chitosan (WSC) and amylose as well as a fully deacetylated chitosan was studied. Zeta potential measurements indicated that the surface of WSC and fully deacetylated chitosan films is positively charged but very weakly, whereas, a film of amylose blended with WSC exhibited an obvious positive charge. X-ray photoelectron spectra of these films suggest that less amino groups are exposed on the surface of WSC and fully deacetylated chitosan films, whereas, more amino groups are exposed on the surface of a WSC film blended with amylose. A sheet structure in which free amino groups are less exposed on the surface of the film of WSC or fully deacetylated chitosan is proposed. This accounts for the loss of antibacterial activity of chitosan on the WSC film surface. When blended with amylose, the morphology of the film may be disrupted, resulting in strong antibacterial properties.     

Fourier transform vibrational circular dichroism as a decisive tool for conformational studies of peptides containing tyrosyl residues

Previous UV-circular dichroism (UV-CD) and NMR studies showed that Ac-AAAAAAAEAAKA-NH(2) has an alpha-helical structure in 50% (v/v) aqueous trifluoroethanol. Replacement of Ala(1) to Ala(6) with Tyr results in spectra that show an apparent loss of helicity in the same solvent. This apparent loss of helicity could be attributed to the coupling of the tyrosyl side chain chromophore with the backbone amide. However, such electronic coupling does not affect the vibrational CD (VCD) spectra. The VCD spectra of the peptides with tyrosyl residues were identical to that of the peptide containing no Tyr, which shows the same alpha-helical structure. Because it is now clear that Tyr replacement does not change the backbone conformation of peptides, UV-CD measurements should be complemented by VCD to determine the secondary structure when electronic effects can disturb the UV-CD spectrum of the inherent structure.     

Vibrational coupling as diagnostic for intramolecular hydrogen bonds of carbohydrates in aqueous solution

The coupling of nuC-O and deltaO-D vibrations in the 1200-900 cm(-1) IR range leads to band shifting in opposite directions, which provides information on intramolecular hydrogen bonding of carbohydrates in aqueous solution. The aqueous solution IR spectra of 2-acetamide-1,6-anhydro-2-deoxy-D-glucopyranose and cis-1,2-cyclopentanediol and tetrahydrofuran-ethanol mixtures are reported. Frequency upshifting upon deuteration is observed for the nuC-O bands of both a hydrogen bond acceptor and donor in ether-hydroxyl and hydroxyl-hydroxyl contacts. The 1200-900 cm(-1) range can be used to reveal the presence of intramolecular hydrogen bonds in aqueous carbohydrates, unlike the OH stretching region, which cannot be used in this sense due to carbohydrate band masking by the strong nuOH IR absorption of water.     

Chirality of camphor derivatives by density functional theory

Infrared (IR) and vibrational circular dichroism (VCD) spectra of chiral camphor, camphorquinone and camphor-10-sulfonic acid (CSA), known as standard compounds for electronic circular dichroism (ECD) spectroscopy, are measured and their vibrational frequencies, infrared intensities, and rotational strengths are calculated using density functional theory (DFT). The observed IR and VCD spectra of chiral camphor and camphorquinone in carbon tetrachloride solution are reproduced by the DFT calculations, but those of CSA are not. DFT calculations of hydration models, where an anionic CSA specifically binds a few water molecules, are carried out. The average of the simulated VCD spectra in the hydration models is more consistent with the observed spectra. In addition, the wavelengths and dipole and rotational strengths for chiral camphor, camphorquinone, anionic CSA, and the hydration models were calculated by time-dependent DFT. In the region of 280-300 nm, the calculated wavelengths of the ECD bands for chiral camphor and camphorquinone coincide with the observed wavelengths that have been reported, and the calculated wavelengths for the hydration models are closer to the observed wavelengths reported than are those calculated for chiral anionic CSA. Consequently, the analysis combined with VCD and ECD spectroscopy using DFT calculations can elucidate the chirality of optically active molecules, even in an aqueous solution.     

Infrared CD of deoxy oligonucleotides. Conformational studies of 5''d(GCGC)3'', 5''d(CGCG)3'', 5''d(CCGG)3'', and 5''d(GGCC)3'' in low and high salt aqueous solution.

Infrared (vibrational) circular dichroism (VCD) spectra have been obtained for the self-complementary tetranucleotides, 5'd(CGCG)3', 5'd(GCGC)3', 5'd(CCGG)3', and 5'd(GGCC)3'. In buffered aqueous solution at low salt concentration, these tetramers exhibit spectra associated with right-handed polymers, although the spectra differ significantly for the four species. In high salt solution, a B-->Z transition occurs in 5'd(CGCG)3', while the other tetranucleotides appear only slightly altered. Temperature dependent studies of these oligonucleotides reveal a greater amount of thermal stability for the tetramers in low salt than for the high salt solutions. VCD intensities computed via the exciton formalism are compared with observed results.     

Triterpenoid saponins from Meryta lanceolata

Four new oleanane-type saponins and a known one were isolated from the leaves and stems of Meryta lanceolata. The new saponins were characterised by spectroscopic means and chemical hydrolysis as 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]oleanolic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[beta-D- glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]oleanolic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-6-O-acetyl glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl]oleanolic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester and 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl]echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester. The NMR assignments were made by means of HOHAHA, 1H-1H COSY, HMQC, HMBC and NOE difference studies.     

Triterpenoid saponins from Ardisia mamillata

Two saponins were isolated from the roots of Ardisia mamillata HANCE. Their structures were established on the basis of MALDI-TOFMS, 1H, 13C NMR and 2D NMR (COSY, HOHAHA, HETCOR, HMBC and ROESY) spectra, and on chemical evidence, to be ardisimamilloside A, 3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 4)-[beta-D-glucopyranosyl-(1 --> 2)]-alpha-L-arabinopyranosyl]-3beta, 16alpha,28alpha-trihydroxy-13beta,28-epoxy-oleanan+ ++-30-al; and ardisimamilloside B, 3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 4)-[beta-D-glucopyranosyl-( 1 --> 2)]-alpha-L-arabinopyranosyl]-beta3-hydroxy-13beta,28- epoxy-oleanan-16-oxo-30-al.     

Comparison of mid-infrared Fourier transform vibrational circular dichroism measurements with single and dual polarization modulations

Fourier transform infrared vibrational circular dichroism (FTIR-VCD) measurements have gone through major advances in the last decade. A major thrust in these advances was to find ways that can minimize the VCD spectral artifacts and obtain the VCD signals at enhanced signal quality. However, all these advances are not incorporated in FTIR-VCD instruments manufactured by some commercial manufacturers. In this article, we compare the measurements obtained with single and dual polarization modulations to seek the attention of the users of single polarization modulation method in minimizing the artifacts in such measurements. We also report that the VCD spectrum of a home-made collagen film can serve as a simple and convenient sample for routine verification of the correct functioning of the VCD spectrometers.     

Observation and characterization of a specific vibrational circular dichroism band in phenyl glycosides

Application of vibrational circular dichroism (VCD) spectroscopy to structural analysis of carbohydrates has recently progressed. However, few studies on glycoconjugates VCD have thus far been reported, despite the fact that naturally occurring carbohydrates exist as various glycoconjugates. To further explore the application of the VCD technique, we have measured a series of aromatic glycosides and found that axial aromatic glycosides exhibited a negative band at around 1230 cm(-1) while equatorial ones showed flat features in this region. This is the first structure-spectra relationship on glycoconjugate VCD that distinguishes the stereochemistry of the sugar anomers. Several model compounds were prepared and their vibrational properties calculated by using the density functional theory (DFT) method, which assigned the vibrational mode of this band based on the stretching motion of the glycosidic oxygen and aromatic carbon. This concept that aglycan parts can reflect stereochemical information of sugar moieties may encourage further VCD studies on glycoconjugates to realize practical structural analysis of carbohydrates.     

Saponins and acylated saponins from Dizygotheca kerchoveana

Four new triterpenoid saponins were isolated from the leaves and stem of branches of Dizygotheca kerchoveana along with seven known ones. The new saponins were respectively characterized as 3-O-[beta-D-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid, 3-O-[beta-D-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[beta-D-3-O-trans-p-coumaroyl-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester and 3-O-[beta-d-3-O-cis-p-coumaroyl-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester. Their structures were elucidated by 1D and 2D NMR experiments, FAB-MS as well as chemical means.     

Noncovalent interactions of peptides with porphyrins in aqueous solution: conformational study using vibrational CD spectroscopy.

Noncovalent interactions of poly(L-lysine) (PL), oligopeptides L-lysyl-L-alanyl-L-alanine and (L-lysyl-L-alanyl-L-alanine)(2) with meso-tetrakis(4-sulfonatophenyl)porphine (TPPS), and poly(L-glutamic acid) (PLGA) with meso-tetrakis(1-methyl-4-pyridyl)porphine tetra-p-tosylate (TMPyP) in aqueous solutions have been studied using combination of spectroscopic methods: Vibrational circular dichroism (VCD) spectroscopy in the mid-infrared region provides a direct information on conformational changes of the polypeptides and oligopeptides caused by interactions with porphyrins; ultraviolet-visible absorption, fluorescence, and electronic circular dichroism (ECD) reveal the aggregation characterization of the porphyrin part of the complexes. Interactions of TPPS with tripeptide, hexapeptide, and PL containing about ten amino acid residues in the molecular chain are accompanied with the changes of VCD patterns in the amide I' region. In these cases, the conformation of the oligopeptide part of complexes is obviously influenced by interactions with TPPS and partial changes of random coil structure are observed in VCD. When PL was composed of the hundreds of lysine residues, just a weak intensity decrease was detected and the shape of VCD spectrum typical for the random coil structure was preserved. As follows from the uv-vis absorption and fluorescence spectra, porphyrin molecules are attached to peptides by electrostatic interaction as a monomer or dimer and interaction between porphyrin and peptide depends on the polypeptide chain length. For the PLGA-TMPyP system with PLGA containing from tens to hundreds of glutamic acid residues in the chain, the VCD spectra were unchanged when TMPyP was presented in the aqueous solution of PLGA and random coil conformation of PLGA-TMPyP aggregates was preserved.     

FTIR detection of water reactions during the flash-induced S-state cycle of the photosynthetic water-oxidizing complex

Photosynthetic water oxidation is performed via the light-driven S-state cycle in the water-oxidizing complex (WOC) of photosystem II (PS II). To understand its molecular mechanism, monitoring the reaction of substrate water in each S-state transition is essential. We have for the first time detected the reactions of water molecules in WOC throughout the S-state cycle by observing the OH vibrations of water using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. Moderately hydrated (or deuterated) PS II core films from Synechococcus elongatus were used to obtain the FTIR difference spectra upon the first, second, third, and fourth flash illumination, representing the structural changes in the S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transitions, respectively. In the weakly H-bonded OH region, bands appeared at 3617/3588 cm(-1) as a differential signal in the first-flash spectrum and at 3634, 3621, and 3612 cm(-1) with negative intensities in the second-, third-, and fourth-flash spectra, respectively. These bands shifted down by approximately 940 cm(-1) upon deuteration and by approximately 10 cm(-1) upon H(18)O substitution, indicating that they arise from the OH stretches of water including the substrate and its intermediates. Strongly D-bonded OD bands of water were also identified as broad features in the range of 2600-2200 cm(-1) by taking the double difference between the spectra of D(2)(16)O- and D(2)(18)O-deuterated films. In addition, broad continuum features that probably arise from the large proton polarizability of H-bonds were observed around 3000, 2700, 2550, and 2600 cm(-1) in the first-, second-, third-, and fourth-flash spectra, respectively, of the hydrated PS II film, revealing changes in the H-bond network of the protein. The negative OH intensities upon the second to fourth flashes might be related to proton release from substrate water. The results presented here showed that FTIR detection of water OH(D) bands can be a powerful method for investigating the mechanism of photosynthetic water oxidation.     

Oleanolic glycosides from Pometia ridleyi

Six triterpenoid saponins were isolated from the stem bark of Pometia ridleyi along with two known saponins, acutoside A and calenduloside C. Their structures were established using one- and two-dimensional NMR and mass spectrometry as 3-O-beta-D-apiofuranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-alpha-L-arabinopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-alpha-L-arabinopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-, 3-O-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-oleanolic acid. The EtOH and EtOAc extracts of the stem bark showed no cytotoxic activity. At a concentration of 23 microg/ml, the saponin mixture showed haemolytic activity and caused 50% haemolysis of a 10% suspension of sheep erythrocytes.