¹H NMR Evaluation of Polar and Nondeuterated Ionic Liquids for Selective Extraction of Cellulose and Xylan from Wheat Bran

Cellulose and xylan, extracted from wheat bran with polar ionic liquids (ILs), were quantified using ¹H NMR spectroscopy. Both No-D NMR and solvent suppression techniques were applied to realize direct analysis of extracts in nondeuterated ILs. As models of extracts, mixtures of cellulose and xylan dissolved in ILs were measured with ¹H NMR spectroscopy. There was a linear relation between mixing ratio and specific peak area of each polysaccharide. Extracts from bran in ILs were analyzed with the obtained calibration curve. This NMR analysis was confirmed to be applicable to three representative ILs used for extraction of polysaccharides. A relation between extracted amount and extraction conditions was obtained

Synthesis and Characterization of Alkanethiolate-Coordinated Iron Porphyrins and Their Dioxygen Adducts as Models for the Active Center of Cytochrome P450: Direct Evidence for Hydrogen Bonding to Bound Dioxygen

Two kinds of novel cytochrome P450 models, which have alkanethiolate axial ligands and hydroxyl groups inside molecular cavities, were designed and synthesized as functional O2 binding systems. A superstructured porphyrin, designated as “twin-coronet” porphyrin, was used as the common framework of the model complexes. This porphyrin bears four binaphthalene bridges on the both sides and forms two pockets surrounded by the bulky aromatic rings. Thiobenzyloxy and thioglycolate moieties, which contain an alkanethiolate group exhibiting various electron-donating abilities and degrees of bulkiness, were covalently linked to twin-coronet porphyrin to yield thiolate-coordinated hemes, TCP-TB and TCP-TG (twin-coronet porphyrin with thiobenzyloxy and thioglycolate groups), respectively. Both ferric complexes exhibited high stability during usual experimental manipulation under air and were characterized by MS, UV/vis, ESR spectroscopies, and CV. The ESR spectra exhibited low-spin signals (TCP-TB:  g = 2.334, 2.210, 1.959; TCP-TG:  g = 2.313, 2.209, 1.966). The cyclic voltammogram of TCP-TB in CH3CN gave a quasi-reversible wave which corresponds to the FeIII/FeII redox couple:  Ep/2 = −1.35 V (vs Fc/Fc+). On the other hand, TCP-TG showed a fine reversible wave:  E1/2 (FeIII/FeII) = −1.12 V. The stable dioxygen adducts were formed in the reaction of the ferric complexes with KO2 under an oxygen atmosphere and characterized by UV/vis and resonance Raman (RR) spectroscopies. In the RR spectra, the ν(O−O) bands of the dioxygen adducts were observed at 1138 cm-1 (TCP-TB) and 1137 cm-1 (TCP-TG). The hypothesis that hydrogen bonding between the bound oxygen and the hydroxyl groups of the binaphthyl moieties could increase their stability was verified by RR spectroscopy. When all hydroxyl groups were deuterated, only the frequencies of the ν(O−O) bands were upshifted by 2 cm-1 without any perturbation in the porphyrin skeleton. This work shows the first direct evidence for a hydrogen bond to dioxygen in an oxy form of a thiolate-coordinated heme model system. These results are discussed in context of the process of dioxygen binding and activation in cytochrome P450

Real-Time Dynamic Adsorption Processes of Cytochrome <i>c</i> on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

<div><p>An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome <i>c</i> (cyt <i>c</i>) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt <i>c</i> adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt <i>c</i> molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt <i>c</i> using EC-HS-AFM. The root mean square roughness (<i>R</i>_RMS) from the AFM images and the number of the electrochemically active cyt <i>c</i> molecules adsorbed onto the electrode (<i>Γ</i>) simultaneously increased in positive cooperativity. Cyt <i>c</i> molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.</p></div

Real-time cyt <i>c</i> desorption processes from the MUA-modified gold electrode.

<p>(A) Continuous AFM images of desorbing cyt <i>c</i> molecules. Frame rate, 2 frames/s; image area, 150 × 150 nm². (B) Time evolution of <i>R</i>_RMS values at the higher ionic strengths. (C) Schematic of desorbing cyt <i>c</i> molecules from the MUA-modified electrode. </p

Time-course analysis of <i>R</i>_RMS and <i>Γ</i> values.

<p>(A) Time evolution of <i>R</i>_RMS values from the HS-AFM images (open circle) and the level of electrochemically active cyt <i>c</i> (<i>Γ</i>) from the cyclic voltammograms (open square). (B) Schematic of the adsorbing cyt <i>c</i> molecules on the MUA-modified electrode at each time point.</p

AFM images show (A) the MUA-modified gold surface and (B) cyt <i>c</i> adsorbed on the MUA SAM at 450 sec.

<p>Continuous AFM images of adsorbing cyt <i>c</i> molecules with real-time labels. Frame rate, 2 frames/s; image area, 150 × 150 nm². The cross sections of the images in (A) and (B), along the short white line, are shown in the lower right. </p

CVs of cyt <i>c</i> adsorbed on the electrode.

<p>(A) CVs of cyt <i>c</i> molecules adsorbed on the MUA electrode in a 10 mM phosphate buffer solution (pH 7.0) from 333 to 522 s (from −0.35 V to 0.1 V, each segment is 4.5 s). (B) Background-subtracted CVs from the voltammogram from 313 to 324 s and 333 to 522 s (from inside to outside, each segment is 4.5 s). The voltammograms were collected at a scan rate of 100 mVs⁻¹. Ag wire was used as a reference electrode. </p

Titration of the apo-form of DH_PDH with PQQ.

The purified apo-form of DHPDH (50 nM) was pre-incubated with various concentrations of PQQ in 50 mM MES buffer, pH 6.5, containing 1 mM CaCl2. After 1 min, the enzyme activity was determined according to the procedure described in the Experimental Procedures.</p

UV-visible absorption spectra of the apo- and holo-forms of DH_PDH.

<p>Dotted line, apo-form of DH_PDH; black solid line, holo-form of DH_PDH; blue solid line, reduced form by addition of 1 mM l-fucose. All spectra were recorded in 50 mM HEPES buffer, pH 7.0 at room temperature.</p

Cyclic voltammogram of <i>Cc</i>PDH immobilized on a plastic-formed carbon electrode modified with 27 nm carbon nanoparticles.

The voltammogram was obtained in 100 mM HEPES buffer, pH 7.0, at a scan rate of 20 mV/s.</p