Probing the organic-mineral interface at the molecular level in model biominerals

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.     

High-speed droplet generation on demand driven by pulse laser-induced cavitation

We report on a pulse laser-driven droplet generation (PLDG) mechanism that enables on-demand droplet generation at rates up to 10,000 droplets per second in a single-layer PDMS-based microfluidic device. Injected droplet volumes can be continuously tuned between 1 pL and 150 pL with less than 1% volume variation.     

Staged Development of Modified Silicon Dioxide Films

The hydrolytic generation of SiO₂ films from chlorosilanes or alkoxysilanes is interrupted by incorporating labile organic groups which stop SiO₂ formation at a processable prepolymer stage. The monomers for the prepolymer have electron withdrawing substituents in the -position. The organic groups are removed from the prepolymer at low temperature, extruding ethylene. The formation of SiO₂ proceeds by intramolecular condensation of the electronegative substituents which are now in a hydrolytically unstable bond with silicon and hydroxyl groups or ambient moisture. Films of the prepolymer spun onto silicon wafers are converted into uniform SiO₂-rich films at temperatures between 150–400°C.     

The Cycloalkyl Ring Effect on the Spin State of Di(Thiocyanato)Bis(N-Cycloalkyl-Substituted-2-Pyridinaldimine) Iron(II)

A new series of octahedral iron(II) complexes with the composition Fe(II) (N-R-2-pyridinaldimine)₂(NCS)₂, where R=cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, have been synthesized and the spin states of the iron atom have been studied by means of Mössbauer spectroscopy and magnetic measurement.     

Time-resolved spectroscopy of energy transfer and trapping upon selective excitation in membranes of Heliobacillus mobilis at low temperature

Transient absorption difference spectra in the Qy absorption band of bacteriochlorophyll (BChl) g and in the 670 nm absorption band of the primary acceptor A0 in membranes of Heliobacillus mobilis (Hc. mobilis) were measured at 20 K upon selective excitation at 668, 793, 810, and 815 nm with a 5 nm spectral bandwidth. When excited at 793 nm, the spectral equilibration of excitations from shorter to longer wavelength-absorbing pigments occurred within 3 ps and mostly localized at the band centered around 808 nm. When excited at 668 nm, the excitation energy transfer from the 670 nm absorbing pigment to the Qy band of BChl g took less than 0.5 ps, and the energy redistribution occurred and localized at 808 nm as in the case of the 793 nm excitation. All of the excitations were localized at the long wavelength pigment pool centered around 810 or 813 nm when excited at 810 or 815 nm. A slower energy transfer process with a time constant of 15 ps was also observed within the pool of long wavelength-absorbing pigments upon selective excitation at different wavelengths as has been observed by Lin et al. (Biophys. J. 1994, 67, 2479) when excited at 590 nm. Energy transfer from long wavelength antenna molecules to the primary electron donor P798 followed by the formation of P+ took place with a time constant of 55-70 ps for all excitations. Direct excitation of the primary electron acceptor A0, which absorbed at 670 nm, showed the same kinetic behavior as in the case when different forms of antenna pigments were excited in the Qy region. This observation generally supports the trapping-limited case of energy transfer in which the excitations have high escape probability from the reaction center (RC) until the charge separation takes place. Possible mechanisms to account for the apparent "uphill" energy transfer from the long wavelength antenna pigments to P798 are discussed.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

Immobilization of Tyrosinase on Chitosan — An Optimal Approach to Enhance the Productivity of L‐DOPA from L‐Tyrosine

Tyrosinase was immobilized on Chitosan (CTS) beads to produce 3,4‐dihydroxy‐L‐phenylalanine (L ‐DOPA) from L ‐tyrosine. Epichlorohydrin (ECH), ethylene glycol diglycidyl ether (EGDE), and glutaraldehyde (GLU) were used as coupling agents, respectively. Ultraviolet/visible measurements on CTS films showed that the reaction intermediate (L ‐dopaquinone) attacked the amino groups on CTS, so the amine residues on chitosan were capped by acetic acid anhydride (Ac) or formaldehyde (Fm) to avoid the deactivation of the immobilized tyrosinase. The pH and temperature of the maximal rate to produce L‐DOPA were investigated. GLU (coupling agent) and Ac (capping agent) were selected for practical utility. A 7.5% (w/v) concentration of GLU was found to attain maximal activity of the immobilized enzyme. The thermal stability of tyrosinase immobilized on CTS‐GLU‐Ac, and after treatment with sodium borohydride, was enhanced to a great extent. The L ‐DOPA converting efficiency in the environmental conditions of this study decreased from 45.1% to 39.9% (between 1^st and 30^th batch). This immobilized tyrosinase can be used practically in the production of L‐DOPA from L‐tyrosine.     

Kinetics and Mechanisms of Acetylcholinesterase and Butyrylcholinesterase Inhibition by Cardiovascular Drugs and Benzodiazepines

The goal of this work is to determine enzyme kinetics and mechanisms of acetylcholinesterase and butyrylcholinesterase inhibition by five cardiovascular drugs, lovastatin, simvastatin, amlodipine besylate, nifedipine, and hydralazine hydrochloride, and two benzodiazepines, diazepam and chlordiazepoxide hydrochloride. All drugs in this study are reversible mixed‐type inhibitors of acetylcholinesterase and butyrylcholinesterase. The pK_i values for acetylcholinesterase and butyrylcholinesterase inhibition by the cardiovascular drugs are linearly correlated with the molecular weights of the drugs with the slopes of 0.005 and 0.0021, respectively. Therefore, van der Waals' interactions between acetylcholinesterase and the cardiovascular drugs are stronger than those between butyrylcholinesterase and the drugs. This is probably due to a smaller active site gorge and a more significant peripheral anionic substrate binding site of acetylcholinesterase than those of butyrylcholinesterase. The fact that the pK_i values for both butyrylcholinesterase and acetylcholinesterase inhibition by the cardiovascular drugs are linearly correlated with each other suggests that both enzyme inhibition reactions proceed via a common mechanism. Furthermore, amlodipine besylate may be useful in Alzheimer's disease treatment similar to donepezil.     

Potential energy surface of O2-(H2O) and factors controlling water-to-O2- binding motifs

The potential energy surface (PES) of O(2)(-)(H(2)O) is investigated by varying the interoxygen distance of O(2)(-) via ab initio calculations with a large basis set. Although two stationary points, C(s) and C(2v) conformers, are found along the interoxygen-distance coordinate, only the C(s) conformer is identified as a minimum-energy species. We find a critical distance, r(c), separating these two conformers in the PES. The C(s) conformer prevails at interoxygen distances of O(2)(-) that are less than r(c), while the C(2v) conformer dominates at the distances larger than r(c). The structural features of these two conformers are also discussed. Although the water deformation energy is shown to be the stabilization source responsible for the prevalence of the C(s) cluster conformer at the interoxygen distances of O(2)(-) less than r(c), the ionic hydrogen bonding is the major driving force for transformation of the water binding motif from C(s) to C(2v) when the interoxygen distance of O(2)(-) increases.