Characterization of the Native Lysine Tyrosylquinone Cofactor in Lysyl Oxidase by Raman Spectroscopy

Lysine tyrosylquinone (LTQ) recently has been identified as the active site cofactor in lysyl oxidase by isolation and characterization of a derivatized active site peptide. Reported in this study is the first characterization of the underivatized cofactor in native lysyl oxidase by resonance Raman (RR) spectrometry. The spectrum is characterized by a unique set of vibrational modes in the 1200 to 1700 cm^(−1) region. We show that the RR spectrum of lysyl oxidase closely matches that of a synthetic LTQ model compound, 4-n-butylamino-5-ethyl-1,2-benzoquinone, in aqueous solutions but differs significantly from those of other topa quinone-containing amine oxidases under similar conditions. Furthermore, we have observed the same ^(18)O shift of the C=O stretch in both the lysyl oxidase enzyme and the LTQ cofactor model compound. The RR spectra of different model compounds and their D shifts give additional evidence for the protonation state of LTQ cofactor in the enzyme. The overall similarity of these spectra and their shifts shows that the lysyl oxidase cofactor and the model LTQ compound have the same structure and properties. These data provide strong and independent support for the new cofactor structure, unambiguously ruling out the possibility that the structure originally reported had been derived from a spurious side reaction during the derivatization of the protein and isolation of the active site peptide

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. © 2014 American Chemical Society.Embargo Period 12 month

Electron Tunneling through Pseudomonas aeruginosa Azurins on SAM Gold Electrodes

Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH_3(CH_2)_nSH:HO(CH_2)_mSH (n=m=4,6,8,11; n=13,15 m=11) self-assembled monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6 to 11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn 47 caused by deprotonation of nearby surface residues

Development of a Copper-electrodeposited Gold Electrode for an Amperometric Creatinine Sensor to Detect Creatinine in Urine without Pretreatment

A copper-electrodeposited gold electrode that can quantitatively detect creatinine without being affected by urine components and can use collected urine as it is produced. In this study, the effect of interfering compounds was eliminated, and the linear range was expanded by increasing the concentration of Nafion covering the electrodes. Furthermore, by extending the electrodeposition time, the linear range was further expanded, and it was possible to measure concentrations up to 12.3 mM (M = mol dm−3), which is equivalent to the creatinine concentration range in the urine of healthy individuals

Design of Phosphonium-Type Zwitterion as an Additive to Improve Saturated Water Content of Phase-Separated Ionic Liquid from Aqueous Phase toward Reversible Extraction of Proteins

We designed phosphonium-type zwitterion (ZI) to control the saturated water content of separated ionic liquid (IL) phase in the hydrophobic IL/water biphasic systems. The saturated water content of separated IL phase, 1-butyl-3-methyimidazolium bis(trifluoromethanesulfonyl)imide, was considerably improved from 0.4 wt% to 62.8 wt% by adding N,N,N-tripentyl-4-sulfonyl-1-butanephosphonium-type ZI (P555C4S). In addition, the maximum water content decreased from 62.8 wt% to 34.1 wt% by increasing KH2PO4/K2HPO4 salt content in upper aqueous phosphate buffer phase. Horse heart cytochrome c (cyt.c) was dissolved selectively in IL phase by improving the water content of IL phase, and spectroscopic analysis revealed that the dissolved cyt.c retained its higher ordered structure. Furthermore, cyt. c dissolved in IL phase was re-extracted again from IL phase to aqueous phase by increasing the concentration of inorganic salts of the buffer solution

Immobilization of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenase with a Polyion Complex and Redox Polymer for a Bioanode

A bioanode for ethanol oxidation was prepared by immobilizing the recombinant pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase from Pseudomonas putida KT 2440 (PpADH) with polyion complex (PIC) and redox polymer. The PIC based on poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) was suitable for immobilizing PpADH on the electrode. PpADH was immobilized using only one redox polymer, aminoferrocene, which was attached to the PGA backbone (PGA-AmFc) on the electrode. The anodic current density at 0.6 V (vs. Ag/AgCl) was 22.6 μA·cm−2. However, when the number of the cycles was increased, the catalytic current drastically decreased. PpADH was immobilized using PGA-AmFc and PIC on the electrode. The anodic current density at 0.5 V (vs. Ag/AgCl) was 47.3 μA·cm−2, and the performance maintained 74% of the initial value after five cycles. This result indicated that the combination of PIC and PGA-AmFc was suitable for the immobilization of PpADH on the electrode. In addition, the long-term stability and catalytic current density were improved by using the large surface area afforded by the gold nanoparticles