Immobilization of thiabendazole-specific monoclonal antibodies to silicon substrates via aqueous silanization

Monoclonal antibodies specific for thiabendazole were immobilized to silicon, silicon dioxide, stoichiometric silicon nitride, and silicon-rich silicon nitride surfaces. This work provides the foundation for the development of a homogeneous sensor system for rapid detection and quantification of thiabendazole residues in produce and animal tissue. Immobilization was performed via aqueous silanization of the substrate followed sequentially by treatment with glutaraldehyde and contact with antibody solution in the presence of detergent. Surfaces were challenged with thiabendazole-horseradish peroxidase conjugate in an ELISA format to estimate immobilized antibody load. A stable and reproducible surface loading of 2 x 10¹¹ antibodies/cm² was obtained only after surfaces received postimmobilization treatments to remove nonspecifically adsorbed antibody. No difference in surface loading was noted when using 30% hydrogen peroxide rather than nitric acid for silanol activation. Little difference was noted among the antibody loadings achieved on the various silicon substrates. Bound antigen-enzyme conjugate was eluted with 0.1N acetic acid and reproducible surface activity was measured for up to four consecutive antigen challenges. Immobilized antibody surfaces were stabilized with 2% sucrose, dehydrated at 37‡C and stored in vacuum or stored at 4‡C in phosphate buffered saline containing 0.01% sodium azide without significant loss of activity.     

Purification and characterization of lamb pregastric lipase

Lamb pregastric lipase was purified from a commercial source using delipidation, solubilization with KSCN, acid-precipitation, pepsin-digestion, affinity chromatography with agarose-Cibacron Blue F3GA, gel filtration, and elution from a native 10% (w/v) polyacrylamide gel. The enzyme had a single subunit of 68,000 Da with maximum esterase activity when measured at pH 6.0 and 30 degrees C. The enzyme preferentially hydrolyzed short- and medium-chain (C4, C6, and C8) synthetic esters and short-chain (C4 and C6) monoacid triglycerides. The NH2-terminal sequence demonstrated high homology with gastric and lingual lipases.     

Thermodynamics of the dissociation of 2-aminopyridinium ion in synthetic seawater and a standard for pH in marine systems

Buffer solutions composed of 2-aminopyridinium chloride and 2-aminopyridine in synthetic seawater are useful as a supplement to buffers of Tris (pH 8.2) and Bis (pH 8.8) in standardizing measurements of hydrogen ion concentration (pm _H or pH(SWS)) in oceanography. The dissociation constant of 2-amino-pyridinium ion over the range of salinities (S) from 30 to 40 has now been determined from the emf of cells without liquid junction at eight temperatures (T) from 278.15 to 313.15 K. The results fit the equation pK=2498.31/T–15.3274+2.4050 lnT+S(0.012928–2.9417×10^–5T) with a standard deviation of 0.0023. Thermodynamic constants for the dissociation process and standard reference values of pm _H and pH(SWS) were derived from the data. The pm _H of the buffer consisting of 2-aminopyridinium chloride and 2-aminopyridine (each 0.04 molal) in synthetic seawater of salinity 35 varies from 7.356 at 278.15 K to 6.601 at 313.15 K.     

Cytochrome-c detection

Following a myocardial infarction (MI) cells die or are damaged and their contents leak into the blood circulation, resulting in elevated serum levels of various enzymes, proteins, and organic molecules. Over the past few decades, it has become standard practice to employ the detection of these elevated substances as markers for the confirmation of MIs and to monitor MI patients’ response to treatment. Although it has previously been shown that cytochrome-c, a small respiratory protein, is among those elevated, the lack of a suitable detection system has prevented its routine use in the diagnosis of MIs. We present a preliminary study in which chemiluminescence was employed to detect elevated levels of cytochrome-c in the serum of MI patients. The technique, which is specific for c-type proteins, is approx 30 times more sensitive than the traditional Coomassie blue stain and can detect as little as 0.03 μg of protein. It also has potential for diagnostic use in other diseases that are characterized by mitochondrial damage.     

Metal clips induce folding of a short unstructured peptide into an alpha-helix via turn conformations in water. Kinetic versus thermodynamic products

Short peptides corresponding to two to four alpha-helical turns of proteins are not thermodynamically stable helices in water. Unstructured octapeptide Ac-His1-Ala2-Ala3-His4-His5-Glu6-Leu7-His8-NH(2) (1) reacts with two [Pd((15)NH(2)(CH(2))(2)(15)NH(2))(NO(3))(2)] in water to form a kinetically stable intermediate, [[Pden](2)[(1,4)(5,8)-peptide]](2), in which two 19-membered metallocyclic rings stabilize two peptide turns. Slow subsequent folding to a thermodynamically more stable two-turn alpha-helix drives the equilibrium to [[Pden](2)[(1,5)(4,8)-peptide]] (3), featuring two 22-membered rings. This transformation from unstructured peptide via turns to an alpha-helix suggests that metal clips might be useful probes for investigating peptide folding.     

Effects of H2O and D2O on polyproline II helical structure

The interaction of solvent with a polypeptide chain is one of the primary factors controlling protein folding and stability. In biologically relevant systems, this solvent is most often water. Experimental estimates of the role of water in peptide folding can be obtained from solvent perturbation experiments. The simplest perturbant for H2O water is its isotopic D2O form. The solvation of peptides known to form PII helices with D2O versus H2O increases their propensity to adopt the PII conformation.     

Heterometallic lead-titanium oxyalkoxide precursors to lead titanate thin films

Two lead titanium oxyalkoxides with composition Pb₂Ti₄O₂(O₂CCH₃)₂(OC₂H₅)₁₄ and Pb₂Ti₂O₂(O₂CCH₃)₂(OCH(CH₃)₂)₈ have been isolated and characterised by elemental analysis, IR, ²⁰⁷Pb NMR and molecular weight measurements. X-ray structural analysis of the 1:2 complex confirmed the presence of two Pb₂Ti₂O units which are linked by a common Pb···Pb edge and are held together by alkoxide and acetate bridges. The acetate groups have migrated from the Pb to the Ti centres during the reaction. Hydrolysis of the 1:1 Pb/Ti complex produced clear gels providing the H₂O/complex ratio was less than 4. Heat treatment of the gel results in loss of residual organics below 400°C. The XRD pattern indicates the presence of the pyrochlore and perovskite phases after heating at 500°C but the sample is poorly crystalline. Complete conversion to the perovskite phase of lead titanate occurs on heating to 600°C for 1 hour. Thin films of lead titanate were deposited by dip-coating a solution of this complex in isopropanol. Analyses of the films, carried out using electron microprobe, Scanning Auger Spectroscopy and Rutherford Backscattering, indicated that they were of excellent quality, crystalline after heating at 600°C and with relatively sharp substrate-coating interface.     

How metallylenes activate small molecules

We have studied the activation of dihydrogen by metallylenes using relativistic density functional theory (DFT). Our detailed activation strain and Kohn–Sham molecular orbital analyses have quantified the physical factors behind the decreased reactivity of the metallylene on going down Group 14, from carbenes to stannylenes. Along this series, the reactivity decreases due to a worsening of the back-donation interaction between the filled lone-pair orbital of the metallylene and the σ*-orbital of H₂, which, therefore, reduces the metallylene–substrate interaction and increases the reaction barrier. As the metallylene ligand is varied from nitrogen to phosphorus to arsenic a significant rate enhancement is observed for the activation of H₂ due to (i) a reduced steric (Pauli) repulsion between the metallylene and the substrate; and (ii) less activation strain, as the metallylene becomes increasingly more predistorted. Using a rationally designed metallylene with an optimal Group 14 atom and ligand combination, we show that a number of small molecules (i.e. HCN, CO₂, H₂, NH₃) may also be readily activated. For the first time, we show the ability of our H₂ activated designer metallylenes to hydrogenate unsaturated hydrocarbons. The results presented herein will serve as a guide for the rational design of metallylenes toward the activation of small molecules and subsequent reactions.

Quantum chemical analyses reveal how model metallylene catalysts activate H₂. This is the first step towards the rational design of metallylenes for the activation of small molecules and subsequent reactions.     

Deactivation of vibrationally excited CD3H using laser-induced fluorescence

Infrared fluorescence observed after exciting to ν₆ (ν=1) of CD₃H with a Q-switched CO₂ laser yields the exponential deactivation rate constant of 0.84 ms^?1 torr^?1. Rate constants for deactivation of CD₃H by rare gases vary from 1.4 (for He) to 0.029 (for Xe) ms^?1 torr^?1.