A promiscuous glutathione transferase transformed into a selective thiolester hydrolase

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.     

Photon echo spectroscopy of porphyrins and heme proteins: effects of quasidegenerate electronic structure on the peak shift decay

Three pulse photon echo peak shift spectroscopy and transient grating measurements on Zn-substituted cytochrome c, Zn-tetraphenylporphyrin, and Zn-protoporphyrin IX are reported. The effects of protein conformation, axial ligation, and solvent are investigated. Numerical simulations of the peak shift and transient grating experiments are presented. The simulations employed recently derived optical response functions for square-symmetric molecules with doubly degenerate excited states. Simulations exploring the effects of excited-state energy splitting, symmetric and asymmetric fluctuations, and excited-state lifetime show that the time scales of the peak shift decay in the three-level system largely reflect the same dynamics as in the two-level system. However, the asymptotic peak shift, which is a clear indicator of inhomogeneous broadening in a two-level system, must be interpreted more carefully for three-level systems, as it is also influenced by the magnitude of the excited-state splitting. The calculated signals qualitatively reproduce the data.     

Lab-on-a-chip technology for determination of protein isoform profiles

A novel lab-on-a-chip technique for rapid (<15 min) and quantitative isoform-profile determination is presented. Ion-exchange chromatographic separation of protein-isoforms and a sensitive immunoassay detection are combined in a porous monolith chip. Thin lines of immobilized antibodies are used for specific capturing of target molecules, which can be detected by the reaction with antibodies bound to carbon black nano-strings. The bound carbon black is quantified by the use of an image scanner. As demonstrated with transferrin isoforms, differing only by 0.1 pH unit in their pI, this technology can distinguish minor differences in protein carbohydrate structure and enable specific determination of proteins in a complex environment, requiring only a few picogram of isoform for detection.     

On the Representation of Functions with Gaussian Wave Packets

We introduce Gaussian wave packets in pursuit of representations of functions, in which the representation is invariant under translation, modulation, scale, rotation and anisotropic dilation. Properties of both continuous and discrete representations are discussed. For the discrete (two-dimensional) case, we develop fast algorithms for the application of the analysis and synthesis operators. A main objective for using Gaussian wave packets is to obtain sparse approximations of functions. However, due to the many invariance properties, the representations will have a high degree of redundancy. Therefore, we also introduce sparse methods for highly redundant representations, that employ some of the analytic properties of Gaussian wave packet for gaining computational efficiency.     

Chemical and spectroscopic methods for investigating polymer degradation and stabilization: Oxidative degradation of a hydrogenated nitrile rubber

The oxidation of a hydrogenated butadiene-acrylonitrile (2:1 mole ratio) rubber has been examined by infrared (IR) spectroscopy and the use of γ-initiation to give a controlled initiation step. The resolution and sensitivity of the IR technique was enhanced by the use of chemical derivatization in which carboxylic acids were measured as acid fluorides, hydroperoxides as nitrates and alcohols as nitrites. In all of these compounds, their spectra are sensitive to the precise substituents (primary, secondary or tertiary). Oxidation produced hydroperoxides from both tert. H-C-C=N and sec. H-C-H groups (≈︁1:1.1 mole ratio) as well as chain-end carboxylic acids [-CH₂-C(=O)OH]. From a comparison with measurements of peroxyl radicals produced at low temperature, average kinetic chain lengths for the various oxidation products were estimated and compared with these from other ethylene copolymers.