Low temperature optical spectroscopy of low-spin ferric hemeproteins

We report the Soret absorption spectra (500-350 nm) of the cyanomet derivatives of human hemoglobin and horse myoglobin, in the temperature range 300-20 K and in two different solvents (65% v/v glycerol-water or 65% v/v ethylene glycol-water). In order to obtain information on stereodynamic properties of active site of the two hemeproteins, we perform an analysis of the band profiles within the framework of electron-vibrations coupling. This approach enables us to single out the various contributions to the spectral bandwidth, such as those arising from non-radiative decay of the excited electronic state (homogeneous broadening) and from the coupling of the electronic transition i) with high frequency modes (that determines the vibronic structure of the band) and ii) with a bath of low frequency modes (that is responsible for the temperature dependence of the experimental spectra). We discuss the relevant parameters and their temperature dependence and compare them with the ones already reported for other derivatives of the same hemeproteins in the same solvents. In particular, non-harmonic contributions to soft modes are found, for cyanomet derivatives, to be larger than those observed for liganded carbonmonoxy but smaller than those observed for unliganded deoxy derivatives. The reported data enable us to obtain information on the dependence of stereodynamic properties of the heme pocket upon iron oxidation state, dimensions of the exogenous ligand and composition of the external matrix. Correspondence to: M. Leone     

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.     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).     

Enantiomeric discrimination of pyrethroic acid esters on polysaccharide derived chiral stationary phases

Enantiomeric separation of pyrethroic acid methyl and ethyl esters was examined on cellulose-based chiral stationary phases (CSPs): chiralcel OD (cellulose tris(3,5-dimethylphenyl carbamate)) and chiralcel OF (cellulose tris(4-chlorophenyl carbamate)). The good resolution of pyrethroic acid esters was achieved on chiralcel OD and OF. Separation factors ranged from 1.19-5.12 for Chiralcel OD and 1.00-1.59 for chiralcel OF. Hexane/2-propanol (100:0.15, v/v %) was used as the eluent. The resolution capability of CSPs was greater chiralcel OD than chiralcel OF in the case of the pyrethroic acid esters. The flow rate was 0.8 ml/min and detection was set at 230 nm. The results of the chromatographic data and molecular mechanics suggest that steric effect was a major factor in the enantioseparation. Furthermore, the hydrogen bond between analytes and CSP played an important role in the chiral recognition.     

Studies on horseradish peroxidase in dimethyl sulphoxide/water mixtures. The activation of hydrogen peroxide and the binding of fluoride.

We studied the variation in spectra and in reactivity towards H2O2 of solutions of horseradish peroxidase in dimethyl sulphoxide/water mixtures, obtained by diluting stock solutions of the enzyme in either water or dimethyl sulphoxide, and assayed the enzyme activity and studied the binding of F- by the peroxidase in 65% (v/v) dimethyl sulphoxide. A broadly similar pattern of changes is observed whether one starts from water or from dimethyl sulphoxide; the changes are essentially reversible, though hysteresis is observed. When the dimethyl sulphoxide content of the solvent mixture is increased, the peroxidase retains its ability to activate H2O2 up to 74% (v/v) dimethyl sulphoxide. The peroxidase in 65% (v/v) dimethyl sulphoxide binds F- together with a proton (or the equivalent loss of HO-), as already established for aqueous solutions. We point out that the occurrence in such solutions of both the ability to activate H2O2 and the inability to bind F- without taking up H+ or losing HO- supports the proposed mechanism for activating H202, whereby the protein binds the substrate in the form of the much more reactive HO2-.     

Peridinin chlorophyll a protein: relating structure and steady-state spectroscopy

Peridinin chlorophyll a protein (PCP) from Amphidinium carterae has been studied using absorbance (OD), linear dichroism (LD), circular dichroism (CD), fluorescence emission, fluorescence anisotropy, fluorescence line narrowing (FLN), and triplet-minus-singlet spectroscopy (T-S) at different temperatures (4-293 K). Monomeric PCP binds eight peridinins and two Chls a. The trimeric structure of PCP, resolved at 2 A [Hofmann et al. (1996) Science 27, 1788-1791], allows modeling of the Chl a-protein and Chl a-Chl a interactions. The FLN spectrum shows that Chl a is not or is very weakly hydrogen-bonded and that the central magnesium of the emitting Chl a is monoligated. Simulation of the temperature dependence of the absorption spectra indicates that the Huang-Rhys factor, characterizing the electron-phonon coupling strength, has a value of approximately 1. The width of the inhomogeneous distribution function is estimated to be 160 cm(-)(1). LD experiments show that the two Chls a in PCP are essentially isoenergetic at room temperature and that a substantial amount of PCP is in a trimeric form. From a comparison of the measured and simulated CD, it is concluded that the interaction energy between the two Chls a within one monomer is very weak, <10 cm(-)(1). In contrast, the Chls a appear to be strongly coupled to the peridinins. The 65 cm(-)(1) band that is visible in the low-frequency region of the FLN spectrum might indicate a Chl a-peridinin vibrational mode. The efficiency of Chl a to peridinin triplet excitation energy transfer is approximately 100%. On the basis of T-S, CD, LD, and OD spectra, a tentative assignment of the peridinin absorption bands has been made.     

OD1, the first toxin isolated from the venom of the scorpion Odonthobuthus doriae active on voltage-gated Na+ channels

In this study, we isolated and pharmacologically characterized the first alpha-like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage-gated Na+ channels expressed in Xenopus laevis oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80+/-14 nM) while Na(v)1.2/beta1 still was not affected at concentrations up to 5 microM. Na(v)1.5/beta1 was influenced at micromolar concentrations.     

Comparison of the efficacy of 1% flutrimazole cream twice a day with 1% flutrimazole cream once a day for the treatment of superficial dermatophytoses

Different clinical studies have demonstrated flutrimazole's efficacy in the treatment of superficial dermatomycoses when administered either twice daily or once daily for four weeks. The aim of the present study was to compare both dosing schedules for the treatment of superficial dermatomycoses. In this randomized, controlled, double blind study, we included 84 patients suffering superficial dermatophytosis (confirmed by microscopic examination (KOH) and culture) susceptible for topical monotherapy. Forty-one patients received flutrimazole 1% twice daily (TD) and forty-three once daily (OD) for four weeks. The efficacy of treatment was evaluated by clinical and mycological criteria at the end of treatment (D28) and after four weeks without treatment (D58). Clinical and mycological cure rates on D28 were 50% with TD and 65% with OD treatment. Only considering clinical evaluation, clinical cure rates on D28 were 63% (TD) and 70% (OD). Also, clinical and mycological cure rates on D56 were 65% with TD and 72% with OD treatment. Only considering clinical evaluation, clinical cure rates on D56 were of 68% (TD) and 72% (OD). The overall tolerability was similar in both treatment groups. The efficacy assessment at the end of treatment (D28) and four weeks after treatment discontinuation (D56) showed that the OD treatment is not inferior to the TD treatment, with similar clinical and mycological cure rates and clinical cure rates in both cases. The OD administration of flutrimazole cream 1% is the most appropriate dosing schedule as it provides the same efficacy, it improves patient's compliance and the cost per day of treatment.     

Comparative studies between covalently immobilized and coated chiral stationary phases based on polysaccharide derivatives for enantiomer separation of N‐protected α‐amino acids and their ester derivatives

Liquid chromatographic enantiomer separation of several N‐benzyloxycarbonyl (CBZ) and N‐tert‐butoxycarbonyl (BOC) α‐amino acids and their corresponding ethyl esters was performed on covalently immobilized chiral stationary phases (CSPs) (Chiralpak IA and Chiralpak IB) and coated‐type CSPs (Chiralpak AD and Chiralcel OD) based on polysaccharide derivatives. The solvent versatility of the covalently immobilized CSPs in enantiomer separation of N‐CBZ and BOC‐α‐amino acids and their ester derivatives was shown and the chromatographic parameters of their enantioselectivities and resolution factors were greatly influenced by the nature of the mobile phase. In general, standard mobile phases using 2‐propanol and hexane on Chiralpak IA showed fairly good enantioselectivities for resolution of N‐CBZ and BOC‐α‐amino acids and their esters. However, 50% MTBE/hexane (v/v) for resolution of N‐CBZ‐α‐amino acids ethyl esters and 20% THF/hexane (v/v) for resolution of N‐BOC‐α‐amino acids ethyl esters afforded the greatest enantioselectivities on Chiralpak IA. Also, liquid chromatographic comparisons of the enantiomer resolution of these analytes were made on amylose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IA and Chiralpak AD) and cellulose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IB and Chiralcel OD). Chiralpak AD and/or Chiralcel OD showed the highest enantioselectivities for resolution of N‐CBZ‐α‐amino acids and esters, while Chiralpak AD or Chiralpak IA showed the highest resolution of N‐BOC‐α‐amino acids and esters. Chirality 2009. © 2008 Wiley‐Liss, Inc.     

Sedimentation of DNA in ethanol-water solutions within the interval of B to A transition.

Sedimentation of DNA ethanol-water solutions has been studied over the range of ethanol concentrations corresponding to the B to A transition (65-80% ethanol, v/v). High ethanol concentrations (more than 75%) have been found to promote aggregate formation in solution. The molecular weight of DNA under fixed ionic conditions in solution (5x10(-4)M NaCl) has been shown to influence the value of ethanol concentration at which aggregates appear. On the other hand, the fact that DNA molecular weight has not been found to exert any influence on B to A transition curves obtained from CD measurements suggests that the changes observed in DNA CD spectra on adding ethanol to the solution are independent of aggregate formation. The date obtained show that, first, aggregation is not a necessary condition for the DNA transition from the B to the A-conformation and, second, changes in CD spectra of DNA under the influence of ethanol are not related to the process of aggregation.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more alpha-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivity indicated that the active sites of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-Trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more α-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivation indicated that the active site of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Intramolecular hydrogen bonds and conformation of the lincomycin molecule in organic solvents

IR spectra (1600-1800 and 3000-3650 cm-1) of lincomycin base solutions in inert (CCl4 and C2Cl4), proton acceptor (dioxane, dimethylsulfoxide and triethyl amine) and proton donor (CHCl3, CD3OD and D2O) solvents were studied. Analysis of the concentration and temperature changes in the spectra revealed that association in lincomycin in the inert solvents was due to intramolecular hydrogen linkage involving amide and hydroxyl groups. Disintegration of the associates after the solution dilution and temperature rise was accompanied by formation of intramolecular bonds stabilizing the stable conformation structure of the lincomycin molecule. The following hydrogen linkage in the conformation was realized: NH...N (band v NH...N at 3340 cm-1), OH...O involving the hydroxyl at C-7 and O atoms in the D-galactose ring (band v OH...O at 3548 cm-1), a chain of the hydrogen bonds OH...OH...OH in the lincomycin carbohydrate moiety (band v OH...O at 3593 cm-1 and v OH of the end hydroxyl group at 3625 cm-1). Bonds NH and C-O of the amide group were located in transconformation. Group C-O did not participate in the intramolecular hydrogen linkage.     

Normal phase chiral HPLC of methylphenidate: comparison of different polysaccharide-based chiral stationary phases.

A comparison of the enantiomeric resolution of (+/-)-threo-methylphenidate (MPH) (Ritalin) was achieved on different polysaccharide based chiral stationary phases. The mobile phase used was hexane-ethanol-methanol-trifluoroacetic acid (480:9.75:9.75:0.5, v/v/v/v). Benzoic acid and phenol were used as the mobile phase additives for the enantiomeric resolution of MPH on Chiralcel OB column only. The alpha values for the resolved enantiomers were 1.34, 1.29, 1.30, and 1.24 on Chiralpak AD, Chiralcel OD, Chiralcel OB (containing 0.2 mM benzoic acid in mobile phase), and Chiralcel OB (containing 0.2 mM phenol in mobile phase) columns, respectively. The R(s) values were 1.82, 1.53, 1.19, and 1.10 on Chiralpak AD, Chiralcel OD, Chiralcel OB (containing 0.2 mM benzoic acid in mobile phase), and Chiralcel OB (containing 0.2 mM phenol in mobile phase), respectively. The role of benzoic acid and phenol as mobile phase additives is discussed.     

Proteomic analysis of a thermostable superoxide dismutase from Bacillus stearothermophilus TLS33

Thermophilic bacterium Bacillus stearothermophilus TLS33 isolated from a hot spring in Chiang Mai, Thailand produces an extracellular superoxide dismutase (SOD). SOD is a free radical metabolizing enzyme that protects the cell membrane from damage by the highly reactive superoxide free radicals. To identify the secreted SOD, we used the systematically proteomic approaches of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis and database searching. The bacterium was grown in a medium containing 0.1% w/v yeast extract and 0.1% w/v tryptone in 100% v/v base mixture at 65 degrees C for 72 h, by assessing their growth by protein and SOD activity. The bacterium produced the highest SOD activity at 65 degrees C for 48 h and the extracellular SOD was run on 2-D PAGE using broad range pH 3-10 immobilized pH gradients (IPGs) and narrow range pH 4-7 IPGs. The isoelectric point and molecular mass of the extracellular SOD were approximately 5.8 and 28 kDa, respectively. In addition, the NH(2)-terminal amino acid sequence was found to be P-F-E-L-P-A-L-P-Y-P-Y-D-A-L-E-P-P-I-I-D, which had a homology of approximately 85% to the Mn-SOD family and 65% to the Fe-SOD family.     

Simultaneous stereoselective high-performance liquid chromatographic determination of 10-hydroxycarbazepine and its metabolite carbamazepine-10,11-trans-dihydrodiol in human urine

An enantioselective HPLC method for the simultaneous determination of the concentration of the enantiomers of the oxcarbazepine metabolites 10-hydroxycarbazepine (MHD) and carbamazepine-10,11-trans-dihydrodiol (DHD) in human urine is described. The method is based on extraction with tert.-butylmethyl ether–dichloromethane (2:1, v/v) under alkaline conditions, separation and evaporation of the organic phase and dissolution of the residue in the mobile phase. Enantiomers are resolved on a Diacel Chiralcel OD column (250 mm×4.6 mm I.D.) under isocratic conditions using as mobile phase n-hexane–ethanol–2-propanol (18:2:1, v/v/v) with addition of glacial acetic acid (0.1%). The enantiomers are detected by UV at 215 nm. The method allows reliable determination of the MHD and DHD enantiomers in human urine with limits of quantification of 0.2 mg/l and 0.4 mg/l, respectively.     

Avidity of IgG antibodies distinguishes primary from non-primary cytomegalovirus infection in pregnant women

Background: Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Fetal damage is mostly linked to maternal primary infection. It is therefore important to differentiate primary from non-primary infection in pregnant females. IgM tests often used for this purpose are not reliable enough.Objective: To evaluate an HCMV–IgG urea-elution assay for its ability to distinguish primary from non-primary infection. In this assay, soaking the antigen–antibody complex with an urea containing solution frees antibodies with low avidity but has no influence on those with high avidity. An avidity index (AI) was calculated: AI=(OD with urea/OD without urea)×100.Study design: HCMV–IgG avidity was measured on a single serum of 79 patients with past infection (pregnant women, graft recipients and blood donors) and of 63 patients (78 sera) with documented seroconversion (pregnant women and graft recipients). Sixty-one pregnant women positive or equivocal for HCMV–IgM but without a documented seroconversion were included in this study.Results: Most (72/79) of the patients with past infection had an AI>65% and all but one had an AI>50%. In pregnant women, in the case of a primary infection within the past 3 months, AI are usually (51/53)<50% and never>65%. Among the IgM positive pregnant women who lack a seroconversion history, 38 had AI>65% suggestive of an infection that had occured at least 3 months earlier, 11 had an AI in a grey area between 50 and 65% and 12 had an AI<50%, suggestive of a recent primary infection.Conclusions: In pregnant women, measurement of the IgG avidity may help to date a HCMV infection, an AI >65% highly suggests a past infection while an AI <50% corresponds to a recent primary infection.     

Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase

A systematic investigation on the effects of trifluoroethanol and acetonitrile at various concentrations on cellulase (EC 3.2.1.4) was studied by enzyme assay, intrinsic fluorescence, ANS binding, circular dichroism and ATR-Fourier transform infra red spectroscopy. The results show the presence of molten globule states at 3% (v/v) TFE and 80% (v/v) ACN. Cellulase aggregates at 25% (v/v) TFE and 90% (v/v) ACN, as detected by decrease in intrinsic and ANS fluorescence, loss in tertiary structure and enzyme activity, increase non-native β-sheet structure as evident from far-UV CD and FTIR spectra, increase in Thioflavin T fluorescence and shift in Congo red assay.     

Partially folded intermediate state of concanavalin A retains its carbohydrate specificity

A systematic investigation of the effect of polyethylene glycol (PEG) 200 and 400 on the solution conformation of concanavalin A (con A) was made using circular dichroism (CD), tryptophan fluorescence, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and size-exclusion chromatography. Far-UV CD spectra of con A at 30%(v/v) PEGs show the retention of ordered secondary structure as compared to 70%(v/v) PEGs. Near-UV CD spectra showed the retention of native-like spectral features in the presence of 30%(v/v) PEGs. Intrinsic tryptophan fluorescence studies indicate a change in the environment of tryptophan residues on the addition of PEG. ANS binding was maximum at 30%(v/v) PEGs suggesting the compact "molten-globule"-like state with enhanced exposure of hydrophobic surface area. Size-exclusion chromatography indicates an intermediate hydrodynamic size at 30%(v/v) PEGs. GdnHCl denaturation of these states was a single-step, two-state transition. To study the possible minimum structural requirement in the specific binding, the effect of PEGs on the interaction of con A with ligand was investigated by turbidity measurements. The C50 value was less in PEG 400 suggesting the more inhibitory ability of PEG 400. The C50 value of PEGs was highest for dextran followed by glycogen, ovalbumin, and ovomucoid. From percentage inhibition of con A-ligands at 30%(v/v) PEG, maximum inhibition was in ovalbumin followed by ovomucoid, glycogen, and dextran. To summarize: con A at 30%(v/v) PEGs exists as compact intermediate with molten-globule-like characteristics, viz., enhanced hydrophobic surface area, retention of compact secondary as well as tertiary structure, and a considerable degree of carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and quaternary association in the legume lectin in particular, where precise topology is required for their biological activities.     

Effect of volatile anaesthetics on the electrical activity and the coupling coefficient of weakly electrically coupled neurones

1. The application of the volatile anaesthetics, halothane and isoflurane (1% v/v and 2% v/v), to the CNS of Lymnaea reduced the firing frequency of the small weakly coupled pedal A cluster (PeA) neurones, which eventually become quiescent. There was no change in their resting membrane potential. 2. Met-enkephalin significantly increased the coupling coefficient between PeA neurones. 3. The volatile anaesthetics decreased the coupling coefficient even in the presence of met-enkephalin. 4. These effects were dose dependent and the effects of halothane were more rapid than those of isoflurane, reflecting their different anaesthetic potencies.