Raman spectroscopic investigations of sarcoplasmic reticulum membranes

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000–1130 cm^?1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the same technique. The fluidity of the membrane also manifests itself in the amide I portion of the membrane spectrum with a strong 1658 cm^?1 band characteristic of CC stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H₂O and in ²H₂O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm^?1 attributable to membrane-associated carotenoids.     

Infrared absorption and raman scattering of sulfate groups of heparin and related glycosaminoglycans in aqueous solution

The i.r. spectra for aqueous solutions of sulfated glycosaminoglycans and model compounds in the transmittance “window” region of the solvent (1400-950 cm^?1) are dominated by the strong and complex absorption centered at ～1230 cm^?1 and associated with the antisymmetric stretching vibrations of the SO groups. Primary and secondary O-sulfate groups absorb at somewhat higher frequencies (1260-1200 cm^?1) than N-sulfates (～1185 cm^?1). Each sulfate band lends itself to quantitative applications, especially within a given class of sulfated polysaccharide. Laser-Raman spectra of heparin and model compounds have been obtained in aqueous solution and in the solid state. The most-prominent Raman peak (at ～1060 cm^?1) is attributable to the symmetrical vibration of the SO groups, with N-sulfates emitting at somewhat lower frequencies (～1040 cm^?1) than O-sulfates. The Raman pattern in the 950-800 cm^?1 region (currently used in the i.r. for distinguishing between types of sulfate groups) also involves vibrations that are not localized only in the COS bonds.     

Infrared and raman studies of the methyl cis undecenoates and of the methyl undecynoates

The infrared spectra (of CCl₄ solutions) and the Raman spectra (of the neat liquids) of the eight isomeric methyl cis-undecenoates, of methyl undec-10-enoate, and of the nine isomeric methyl undecynoates have been obtained. Conjugation of the double bond with the carbonyl group lowers the wavenumber value of ν(CC) by 8 cm^?1 from the mean value of 1657 cm^?1. Conjugation of the triple bond with the carbonyl group gives rise to a single Raman band due to ν(CC) at 2241 cm^?1 whereas the non-conjugated, non-terminal triple bond gives a Fermi resonance doublet at 2234 and 2293 cm^?1.     

Raman spectroscopic analysis of the sodium salt of kappa-carrageenan and related compounds in solution

Laser-Raman spectra of Na⁺ kappa-carrageenan, Na⁺ neocarrabiose 4-sulphate, and neocarrabiose in the region 700–1500 cm⁻¹ are reported for solutions in H₂O and D₂O. The C-1-H-1α vibration, coupled with COH related modes, is assigned to a band at 840 cm⁻¹, close to the maximum of the symmetrical COS stretching (∼850 cm⁻¹). The symmetrical SO stretch is proposed to occur near 1040 cm⁻¹ and is probably coupled with COH vibrations which give rise to strong bands in the region 1000–1100 cm⁻¹. The intense band in the region 730–740 cm⁻¹ is ascribed to a complex ring vibration.     

Pre-resonance Raman spectra and conformations of nystatin in powder,solution and phospholipid-cholesterol multilayers

A Raman scattering study of the channel-forming polyene antibiotic nystatin, is reported in the solid state, in organic and aqueous solutions as well as in phospholipid and phospholipid-cholesterol multilayers. Measurements of the solid and solution spectra as a function of excitation wavelengths in the 459.7–514.5 nm range, and the phospholipid spectra as a function of temperature in the 10–60°C range, have also been made. The spectral features indicate a pre-resonance-enhanced Raman spectrum in which the CC and CC stretching modes of the polyene segment of nystatin are dominant. However, in contrast to previously published results on the nearly isostructural polyene antibiotic amphotericin B, a line at 1610 cm^?1 assignable to the CO stretching mode is also observed to be strongly resonance enhanced. This is explained by a postulated ground-state conformation model in which a twisting of the molecule is facilitated by the break in the polyene chain. This allows the CO group at one end of the molecule to be aligned along the polyene unit at the other end, and the CC stretching vibration, which is strongly modulated by the polyene π → π1 excited state, to mix with the CO stretching vibration. The peak frequencies and intensities of the CC and CC stretching modes in nystatin, however, remain essentially unchanged compared with amphotericin B, indicating that the polyene units in nystatin remain planar and trans both in the ground and excited states.The intensity of the CO mode with respect to the CC stretching mode was observed to vary appreciably with nystatin environment, indicating a     

Evidence for a structural change in the substrate preceding hydrolysis of a chymotrypsin acyl enzyme: application of the resonance Raman labelling technique to a dynamic biochemical system.

By using a flow system, resonance Raman spectra have been obtained of the acyl enzyme 4-amino-3-nitro-cinnamoyl-α-chymotrypsin over the pH range where it shifts from a stable to an active state. The band at 1625 cm^?1 was assigned to the CC stretch in the acryloyl residue

and bands in the 1200 to 1450 cm^?1 region were shown to be associated with the aromatic residue. Studies with analogues of the substrate established that the CC and the aromatic bands could independently monitor events in the

and aromatic moieties, respectively. Thus, events in the acyl enzyme leading to spectral changes involving the catalytic site could be distinguished from those involving the aromatic site. The data strongly suggested that the structure of the acylating group in the stable acyl enzyme at pH 3.0 resembled that of the substrate in aqueous solution, the conformation about the ethylenic bonds being essentially planar, trans and probably s-trans about the CCCO single bond. However, on shifting the acyl enzyme to pH values of 5.7 to 7.0 a large change occurred in the 1625 cm^?1 band without detectable changes in the aromatic bands. Thus, the enzyme produces a change in a submolecular grouping which contains the bond being hydrolysed. The change occurred within the mixing and observation time of about ten seconds. Since it was identical at essentially inactive (pH 5.7) and active (pH 7.0) pH values it must precede and be independent of the rate-controlling step in deacylation. The change in the 1625 cm^?1 band thus probably reflects an ionization in the acyl enzyme between pH 3.0 and 5.7. Consequently, above pH 5.7 deacylation proceeds from a structure differing from that at the stable pH of 3.0. The resonance Raman spectra of this intermediate are consistent with either deformation to reduce planarity within the acryloyl residue, or of the stabilization of resonance structures of the form

Either of these mechanisms would produce an intermediate, the carbonyl group of which would be probably intrinsically more reactive relative to the substrate in solution or to the stable acyl enzyme. The resonance Raman provides some support for Henderson's (1970) prediction that distortion occurs in the acryloyl residue prior to deacylation.     

Preparation and characterization of oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) complexes of 3,3′-(1,2-phenylenediimino)diacrolein

The oxovanadium(IV), acetatomanganese(III), chloroiron(III), nickel(II), copper(II), zinc(II) and palladium(II) of 3,3′-(1,2-phenylenediimino)diacrolein were prepared and investigated by means of mass, electronic, vibrational, NMR and ESR spectroscopy as well as magnetic susceptibility measurements. The acetatomanganese(III) and chloroiron(III) complexes were confirmed to be of high spin type. The absorption bands appearing in the energy range greater than 23 000 cm⁻¹ were attributed to π→π^* transitions within a ligand molecule and charge- transfer transitions from metal to ligand. The metal complexes assume the square-planar configuration type since the ligand-field bands were detected in the 12 700–18 500 cm⁻¹ region. Strong bands appearing at 1601 and 1627 cm⁻¹ were assigned to the CC and CO stretching vibrational modes, respectively, and were shifted to lower frequency upon metal-coordination. A VO stretching band was observed at 982 cm⁻¹ for the oxovanadium(IV) complex and a CO stretching band was observed at 1547 cm⁻¹ for the acetatomanganese(III) complex. Upon complex formation the amine proton signal is found to vanish and the aldehydic methine proton signal in the lowest field is shifted upfield for the nickel(II), zinc(II) and palladium(II) complexes. ¹³C NMR spectra support the coordination structure of the complexes which is revealed by ¹H NMR spectra. As judged by the spin Hamiltonian parameters, the oxovanadium(IV) complex is of a square- planar type with an unpaired electron in the d_xy orbital and the copper(II) complex assumes a distorted square-planar coordination due to the presence of five- and six-membered chelate rings with an unpaired electron in the d_x2−y2 orbital.     

Conformational geometry and vibrational frequencies of nucleic acid chains.

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

Raman studies of conformational changes in model membrane systems

Laser Raman spectra of concentrated samples of phosphatidyl choline and phosphatidyl ethanolamine were taken at approximately 10° intervals over a temperature range of 90°–19°C. The spectral region from 30 to 3300 cm^?1 was investigated. Several new spectral features were discovered which are correlated to phospholipid liquid crystalline structure. It is shown that 1) frequency shifts occur in the $\text{PO}{}_2{}^{\mathrm{?}}$ symmetric stretch band which suggest a change in exposure of the PO₂ group to the solvent upon melting, 2) the frequency of the translational hydrocarbon mode around 150 cm^?1 appears to indicate the degree to which the hydrocarbon chain is extended, 3) the methyl and methylene stretch bands at 2890 and 2850 cm^?1 very clearly demonstrate hydrocarbon chain melting, and 4) the 720 cm^?1 band, previously assigned to the symmetric OPO diester stretch, appears to be due instead to the symmetric CN stretch of choline.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

Resonance Raman on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm^?1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm^?1 in the RR spectrum with D-alanine was shifted to 1675 cm^?1 upon [¹⁵N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm^?1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.     

Electrostatically induced change of the conformational order of charged lipid membranes

Raman spectroscopy and X-ray diffraction are used to investigate the influence of surface charges on the structure of ionizable lipid membranes of dimyristoylmethylphosphatidic acid. The membrane surface charge density is regulated by varying the pH of the aqueous phase. Changes of the conformational order of the lipid chains are determined from the intensity of the CC stretch chain vibrations around 1100 cm^?1 in a lipid Raman spectrum. In going from an electrical neutral to a negatively charged membrane, the conformational order is reduced by 5% in the ordered and by 9% in the fluid membrane phase, corresponding to 0.6 and 0.8 CC bonds, respectively, which change from a trans to a gauche conformation. The electrostatically induced conformational change is mainly concentrated at the lipid chain ends as indicated by the spectral variations of the 890 cm^?1 CH₃ rocking band of the chain termini. The X-ray diffraction experiments show that increasing the surface charge density in the ordered membrane phase leads to a lateral expansion of the packing of the lipid polar groups, whereas the packing of the lipid chains in a plane perpendicular to the chain axes remains constant, indicating an increase of the tilt of the lipid chains from δ = 10° (pH 3) to δ = 27° (pH 9).     

Vibrational Raman optical activity of alanyl peptide oligomers: A new perspective on aqueous solution conformation

The vibrational Raman optical activity (ROA) spectra of di- and tri-L -alanine in the range 650–1750 cm^?1 have been measured in H₂O and D₂O solution at high, neutral, and low pH and pD. Corresponding ROA spectra for tetra- and penta-L -alanine have also been obtained, but over a more restricted set of pH and pD conditions. There are similarities with the ROA spectrum of L -alanine below ～ 1200 cm^?1, but the spectra are very different above this wavenumber due to the influence of the vibrational coordinates of the peptide group. The similar overall appearance of the di-, tri-, and tetrapeptide ROA under selected conditions of pH and pD, and of all four peptide ROA spectra in DCl and HCl solutions, in the backbone skeletal stretch region ～ 1050–1200 cm^?1 and the extended amide III region ～ 1250–1350 cm^?1, suggests that the backbone conformation is approximately the same in all four structures. One difference, however, is a shift of a large positive ROA band in H₂O at ～ 1341 cm^?1 in the dipeptide, assigned to C_α–H and in-plane N–H deformations, down to ～ 1331 cm ^?1 in the tripeptide and to ～ 1315 cm^?1 in the tetrapeptide and pentapeptide (the last in HCl due to insufficient solubility in H₂O), which indicates increasing delocalization of the corresponding normal mode with increasing chain length. Our results do not support the suggestion that stabilizing interactions of the zwitterionic end groups in tri-L -alanine at neutral pH leads to a different solution structure to that at high pH. © 1994 John Wiley & Sons, Inc.     

A vibrational spectroscopic study of the self-association of 5′-GMP in aqueous solution

The Raman spectra of highly concentrated solutions of 5′-GMP at neutral and acid pH were recorded in order to better characterize the structure of the self-aggregates formed in these solutions and their melting behavior. Vibrational coupling of the C?O stretching vibrations in tetrameric units at neutral pH is shown to yield a characteristic pattern of two Raman bands at ca. 1730 and 1680 cm^?1 (1708 and 1664 cm^?1 in D₂O), and an iractive mode at 1678 cm^?1 in D₂O. From the intensity of the 1730-cm^?1 band, proportional to tetramer concentration, and that at 1485 cm^?1, which reflects the stacking of the bases, the thermal stability of the self-associates formed at neutral pH is shown to be higher for stacked tetramers. At acid pH, the melting of the helical aggregates responsible for the formation of a gel is preceded by the freeing of the hydrogen-bonded phosphate groups, accompanied by a change of conformation from C3′-endo to C2′-endo in some of the associated ribose units. Previous spectroscopic results suggesting the formation of tetramers as an intermediate step in the melting of the gel were not reproduced in this study.     

Fatty acids,part X. Raman spectra of all dimethylene interrupted methyl cis,cis-octadecadienoates and octadecadiynoates

The Raman spectra of all the dimethylene interrupted methyl cis, cis-octadecadienoates and octadecadiynoates have been studied. The Raman band positions and their relative intensities for the ν(CC), ν(CC), ν(CO), ν(CH) and δ(CH₂) modes are recorded. The height intensity of the bands arising from ν(CC) relative to ν(CO) provides a means of determining the number of cis-ethylenic bonds in a mono-ester. In the acetylenic series, the intensity of the bands arising from ν(CC) relative to ν(CO) failed to indicate with certainty the number of acetylenic bonds in the mono-esters studied, due to the weak intensity of the band due to ν(CO). However a better correlation between the relative intensities of the ν(CC) and δ(CH₂) bands is established instead. An attempt to correlate the areas under the bands due to ν(CC), (CC), (CO) and δ(CH₂) failed to produce any significant results. The Raman spectra of the methyl octadec-cis-10-en-5-ynoate and methyl octadeca-5, 10-diynoate are also recorded.     

Resonance Raman evidence for Fe(IV) in compound II of horseradish peroxidase

Resonance Raman spectra have been obtained for Compound II of horseradish peroxidase. Its prophyrin vibrational frequencies are consistent with a planar low-spin heme containing Fe(IV). The oxidation-state marker band is found at the unprecedentedly high value of 1382 cm^?1. This band was also observed in solutions of myoglobin and cytochrome c peroxidase to which H₂O₂ had been added. No evidence was found for an actual FeO double bond in Compound II.     

Laser-Raman study of valinomycin-doped and omega-CD3-labeled phosphatidylcholine liposomes

The relative intensities of the CH stretching vibrations are used to study the interaction of lecithin liposomes with valinomycin, a mobile carrier for alkali ions. In the case of dipalmitoyl lecithin liposomes, the lipid phase transition is not significantly affected by valinomycin. However, in dimyristoylphosphatidylcholine liposomes, the phase transition is broadened by the addition of 1 mol% valinomycin even at low K⁺ concentrations. This indicates that the carrier interacts with the hydrophobic core of the bilayer. In addition, these experiments showed that the lipid phase transitions which are reflected by the methylene groups and the terminal methyl groups are nearly equivalent. Therefore a reevaluation of the assignment of the CH stretching bands seemed necessary. Our Raman spectroscopic investigation of ω-deuterated dipalmitoyl lecithin liposomes improves the assignment of CH stretch vibrations to methylene and methyl groups. The deuteration displaces the methyl group vibrations to the 2050–2250 cm^?1 region and produces gross intensity changes of the bands at 2883 and 2936 cm^?1. These changes lead to the conclusion that both bands arise from vibrations which can be attributed simultaneously to the methylene and methyl groups of the fatty acid chains. The displacement of the CH₃ group vibrations from their original positions enhances the intensity ratios (per centimeter), $\text{2883}\text{2847}$ and $\text{2936}\text{2847}$, for the CH₂- groups which are used to monitor the lipid phase transition, and implies that the contributions of the CH₃ groups to the phase transition curves are unimportant. Our finding that the -CD₃ groups reflect no phase transition supports this statement.     

Conformational dependence of the Raman scattering intensities from polynucleotides. 3. Order-disorder changes in helical structures

Raman spectra are presented on ordered and presumably helical structures of DNA and RNA as well as the poly A·poly U helical complex, polydAT, and the helical aggregates of 5′-GMP and 3′-GMP. The changes in the frequency and the intensity of the Raman bands as these structures undergo order-disorder transitions have been measured. In general the changes we have found can be placed into three categories: (1) A reduction in the intensities of certain ring vibrations of the polynucleotide bases is observed when stacking or ordering occurs (Raman hypochromism). Since the ring vibrational frequencies are different for each type of base, we have been able to obtain some estimate of average amount of order of each type of base in partially ordered helical systems. (2) A very large increase in the intensity of a sharp, strongly polarized band at about 815 cm^?1 is observed when polyriboA and polyriboU are formed into a helical complex. Although this band is not present in the separated chains at high temperature, a broad diffuse band at about 800 cm^?1 is present. The 815 cm^?1 band undoubtedly arises from the vibrations of the phosphate-sugar portions of the molecule and provides a sensitive handle to the back-bone conformation of the polymer. This band also appears upon ordering of RNA, formation of the helical aggregate of 5′-riboGMP, and to some extent in the selfstacking of the polyribonucleotides polyA, polyU in the presence of Mg⁺⁺, PolyC, and polyG. No such intense, polarized band is found, however, in ordered DNA, polydAT, or the 3′-riboGMP aggregate, although there is a conformationally independent band at about 795 cm^?1 in DNA and polydAT. (3) Numerous frequency changes occur during Conformational changes. In particular the 1600–1700 cm^?1 region in D₂O shows significant conformationally dependent changes in the C?O stretching region analogous to the changes in this region which have been observed in these substances in the infrared. Thus, Raman scattering appears to provide a technique for simultaneously observing the effects of base stacking, backbone conformation and carbonyl hydrogen bonding in nucleic acids in moderately dilute (10–25 mg/ml) aqueous solutions.     

Protocatechuate 3,4-dioxygenase. Resonance Raman studies of the oxygenated intermediate.

Resonance Raman spectra of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have been investigated during the reaction of the enzyme with substrate and oxygen. It is found that the spectrum of the turned-over enzyme is indistinguishable from that of the resting enzyme in the absence of substrate, and is characterized by resonance-enhanced tyrosinate ring vibrational modes at 1263 and 1174 cm^?1. In the ternary ESO₂ complex, however, the tyrosinate vibrational modes are shifted to 1252 and 1165 cm^?1, respectively. There is no evidence for any dioxygen vibrations in the spectra of ESO₂ complexes prepared with ¹⁶O₂, ¹⁸O₂, and ¹⁶O¹⁸O in the region between 1300 and 200 cm^?1. The results of this resonance Raman study are interpreted to indicate that molecular oxygen is attached only to the substrate (but not iron) in the stable intermediate, and that the concomitant rearrangement at C4 of the substrate induces a substantial change in geometry of the tyrosine residues associated with the iron complex. Furthermore, the optical spectrum of the ESO₂ complex (λ_max = 520 nm) is dominated by tyrosinate → Fe(III) charge transfer and contains little or no peroxide → Fe(III) charge transfer. These results invalidate the previously advanced analogy in spectral properties between this enzyme and the respiratory protein, oxyhemerythrin.