Low-frequency Raman spectra of lysozyme.

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm^?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm^?1. However, a low-frequency band at 25 cm^?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm^?1 which displaces the tryptophan peak at 1010 cm^?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1-25 was compared with that of a truncated homodimer (dSP-B_8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm^− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B_1-25 dimer. In contrast, the truncated dimer dSP-B_8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm^− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

A laser Raman study of lysozyme denaturation

The Raman spectrum of chemically denatured lysozyme was studied. The denaturants studied included dimethyl sulfoxide, LiBr, guanidine · HCl, sodium dodecyl sulfate, and urea. Previous studies have shown that the amide I and amide III regions of the Raman spectrum are sensitive to the nature of the hydrogen bond involving the amide group. The intensity of the amide III band at 1260 cm^?1 (assigned to strongly hydrogen-bonded α-helix structure) relative to the intensity of the amide III band near 1240 cm^?1 (assigned to less strongly hydrogen-bonded groups) is used as a parameter for comparison with other physical parameters used to assess denaturation. The correlation between this Raman parameter and denaturation as evidenced by enzyme activity and viscosity measurements is good, leading to the conclusion that the amide III Raman spectrum is useful for assessing the degree of denaturation. The Raman spectrum clearly depends on the type of denaturant employed, suggesting that there is not one unique denatured state for lysozyme. The data, as interpreted, place constraints on the possible models for lysozyme denaturation. One of these is that the simple two-state model does not seem consistent with the observed Raman spectral changes.     

Structure in solution of biscyclo(dipeptide) restricted by CSSC gauche effect

Biscyclo(Cys-Sar) [I] and biscyclo(Cys-Pro) [II] were prepared by the extension of amino acid moieties from cystine. Compound I equilibrates between two rotamers around the CSSC bond of the cystine residue in methanol, showing dual negative CD transitions (CS-SC) at 270 and 255 nm, and dual S-S vibrations at 507 and 539 cm^?1 in Raman spectra. In contrast, a conformation of P-helix type is suggested in CH₃CN, which shows a distinct negative CD at 270 nm and only one Raman band at 507 cm^?1 for the S-S bond. Compound II rotates freely in dimethylsulfoxide (Me₂SO) but takes a relatively stable conformer of P-helix type in methanol, chloroform/Me₂SO (9:1), and chloroform/trifluoroacetic acid (9:1). The conformation in chloroform is retained even on addition of trifluoroacetic acid. A more complete conformation of compound II in water was determined from the negative CD of S-S transition and ¹H-nmr spectra of the Cys-β-CH₂ protons.     

The conformation of polycytidylic acid, polyguanylic acid, polyinosinic acid, and their helical complexes in aqueous solution from laser Raman scattering

The Raman spectra of the double helical complexes of poly C–poly G and poly I–poly C at neutral p^H are presented and compared with the spectra of the constituent homopolymers. When a completely double-helical structure is formed in solution a strong sharp band at 810–814 cm^?1 appears which has previously been shown to be due to the A-type conformation of the sugar–phosphate backbone chain. By taking the ratio of the intensity of the 810–814 cm^?1 band to the intensity of the 1090–1100 cm^?1 phosphate vibration, one can obtain an estimate of the fraction of the backbone chain in the A-type conformation for both double-stranded helices and self-stacked single chains. This type of information can apparently only be obtained by Raman spectroscopy. In addition, other significant changes in Raman intensities and frequencies have been observed and tabulated: (1) the Raman intensity of certain of the ring vibrations of guanine and hypoxanthine bases decrease as these bases become increasingly stacked (Raman hypochromism), (2) the Raman band at 1464 cm^?1 in poly I is asigned to the amide II band of the cis-amide group of the hypoxanthine base. It shifts in frequency upon base pairing to 1484 cm^?1, thus permitting the determination of the fraction of I–C pairs formed.     

A novel xylose isomerase from Neurospora crassa

Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg^-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg²⁺ whereas 2 mM Co²⁺ afforded protection against inactivation by heat. The K _m for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose.     

Effect of preoptic lesions on behavioral thermoregulation of green sunfish,Lepomis cyanellus,and of goldfish,Carassius auratus

Summary Sunfish (Lepomis cyanellus) and goldfish (Carassius auratus) acclimated to 5°, 15° and 25 °C were placed individually in a horizontal temperature gradient from 2° to 30 °C where the fish could swim freely. They spent large proportions of time (70%) at temperatures near their acclimation temperature and avoided the extremes. By behavioral selection, internal body temperature was maintained relatively constant. After bilateral medial and lateral preoptic lesions, fish spent random amounts of time at all temperatures available in the gradient and did not maintain a stable body temperature.Support from grants NSF PCM 76-15861 and HEW PHS GM 7143 is acknowledged.     

Phytase production by the yeast, Pichia anomala

Pichia anomala, isolated from dried flower buds of Woodfordia fruticosa, produced a high activity of an intracellular phytase, at 68 U per g dry biomass, when grown at 20 °C for 24 h in a medium containing glucose (40 g l^–1) and beef extract (10 g l^–1) supplemented with Fe²⁺ (0.15 mM). Partially purified phytase was optimally active at 60 °C and pH 4 with a half life of 7 days at 60 °C. It retained 85% of its activity at 80 °C for 15 min. The enzyme is suitable for supplementing animal feeds to improve the availability of phosphate from phytate.     

A quantitative bioapatite/collagen calibration method using Raman spectroscopy of bone

Numerous calibration models were developed and tested for the quantitative analysis of collagen and bioapatite in bone using Raman spectroscopy. The ν₁ phosphate vibration at 960 cm^–1 was used as indicator of the content of bioapatite while for collagen three markers were used: the C–H₂ band at 2940 cm^–1, the amide I band at 1667 cm^–1 and the vibrations of proline and hydroxyproline at 855 and 878 cm^–1, respectively. Also a calibration model based on the PLS algorithm was developed, too. Validation of the derived calibration models indicated that the model that makes use of the height ratio of the peaks 960/(855+878) exhibits the best accuracy. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A resonance Raman band assignable to the O–O stretching mode in the resting oxidized state of bovine heart cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

In the resting oxidized state (the fully oxidized “as-isolated” state) of cytochrome c oxidase (CcO) preparation, a resonance Raman band is observed at 755 cm^-1 upon 647.1 nm excitation in resonance with an absorption band at 655 nm. Addition of cyanide eliminates the Raman band concomitant with loss of the absorption band at 655 nm. These results strongly suggest that the Raman band at 755 cm^-1 originates from the O−O stretching mode of the bridging peroxide (Fe−O^-−O^-−Cu) in the O₂ reduction site of the fully oxidized “as-isolated” CcO. Although the peroxide bridged structure has been proposed on the basis of X-ray crystallography and reductive titration experiments, the present vibrational spectroscopic analyses reveal conclusively the chemical nature of the bridging ligand at the O₂ reduction site of the fully oxidized “as-isolated” bovine heart CcO.     

On the origin of the 1602 cm–1 Raman band of yeasts; contribution of ergosterol

The 1602 cm^–1 Raman signature, which we call the “Raman spectroscopic signature of life” in yeasts, is a marker Raman band for cell metabolic activity. Despite the established fact that its intensity sensitively reflects the metabolic status of the cell, its molecular origin remained unclear. In this work, we propose ergosterol as the major contributor of the 1602 cm^–1 Raman signature. The theoretical isotope shift calculation for ergosterol agreed with previous observations. Furthermore, experiments showed that the Raman spectrum of ergosterol corresponds very well with the depleting spectral component in yeast that behaves together with the 1602 cm^–1 signature when the cells are under stress. This work implies that the 1602 cm^–1 Raman signature could serve as an intrinsic ergosterol marker in yeasts for the study of sterol metabolism in vivo and in a label‐free manner, which could not be done by any other techniques at the current stage. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5–300 mg ml^?1). No changes in the amide‐I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm^?1 and the ratios of the intensities of the Tyr bands at 856 and 837 cm^?1, the Trp bands at 870 and 877 cm^?1, and the bands at 2940 (CH stretching) and 3420 cm^?1 (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high‐concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 237–246, 2015.     

Raman spectroscopic investigation on the interaction of malignanthepatocytes with doxorubicin

Raman spectroscopy has proven to be a very powerful technique and is currently experiencing a renaissance. In this paper, it is used to explore the interaction between doxorubicin and malignant hepatocytes in vitro. For the addition of doxorubicin, the band intensity at 1609 cm^− 1, mainly assigned to CC in-plane bending mode of phenylalanine and/or tyrosine residues, increases significantly, and the intensities of the bands at 1585 and 1313 cm^− 1, mainly due to the guanine bases, decrease greatly. In addition, Raman spectra are investigated at different doxorubicin concentrations, and the mean areas ratios of the band at 1450 to that at 1003 cm^− 1, A₁₄₅₀/A₁₀₀₃, fluctuate according to the doxorubicin concentration increasing, which suggests that doxorubicin affects the relative content of lipid in cells.     

Purification and characterisation of NAD+-dependent xylitol dehydrogenase from Fusarium oxysporum

An NAD⁺-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M _r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K _m values for d-xylitol and NAD⁺ were 94 mM and 0.14 mM, respectively. Mn²⁺ at 10 mM increased XDH activity 2-fold and Cu²⁺ at 10 mM inhibited activity completely.     

Carboxypeptidase Activity of the Zinc Metalloprotease in the Cement Precursor Secretion of the Barnacle, Chthamalus fragilis Darwin

The liquid cement precursor secretion (CPS) of the adult barnacle, Chthamalus fragilis, exhibits Zn-metalloprotease activity. To assess the bond specificity of the Zn-metalloprotease, samples of liquid CPS were collected by glass micropipette from the exposed bases of adult barnacles and incubated in 50 mM Tris buffer, pH 8.0, containing 10 mM Ca⁺⁺, at 27°C or 20°C for 6 or 24 hr. in the presence of fluorescent dye-labeled synthetic C-1 and A-1 PepTag peptides. These peptides have a net positive charge and migrate toward the anode during agarose gel electrophoresis. When incubated with the C-1 PepTag peptide, CPS samples generated F1 hydrolysis fragments that failed to migrate during electrophoresis in 0.8% agarose gels, indicating the presence of proteolytic activity in the CPS. Proteolysis of the C-1 peptide was inhibited by 2.0 mM orthophenanthroline in the presence of 10 mM Ca⁺⁺ ions. No hydrolysis products were generated when samples of CPS were incubated in the presence of the A-1 PepTag peptide. This suggested that the CPS contained a Zn-metalloprotease that exhibited a preference for the carboxy-terminal lysine of the C-1 PepTag peptide. There were no indications of aminopeptidase or endopeptidase activities in the barnacle CPS. When incubated with hippuryl-lysine and hippuryl-arginine substrates, samples of CPS gave activities ranging from 111 to 319 μmol hippuric acid formed/min/mg of CPS protein. These observations indicate that the CPS of C. fragilis contains a Zn-metallo-exoprotease with a preference for carboxy-terminal basic amino acids.     

Intensity Changes of Base and Backbone Raman Modes in the B to Z Transition of poly(dG-dm5C)

Abstract

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm-⁵C). The transition midpoint was about 37°C for a solvent containing 20 mM Mg²⁺. A 10-fold change in Mg²⁺ concentration altered the transition midpoint by at least 60°C. Raman spectra of the B and Z forms of poly(dG-dm⁵C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm^?1 guanine mode and 835 cm^?1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm^?1, 805 and 819 cm¹. Several bands unique to poly(dG-dm⁵C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green Microalga Botryococcus braunii

Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C₃₀) is metabolized by methylation to give intermediates of C₃₁, C₃₂, C₃₃, and C₃₄, with C₃₄ being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm⁻¹ showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm⁻¹ were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm⁻¹ was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm⁻¹ are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm⁻¹ botryococcene Raman shift.