Michael Ottolenghi

Biochemically active sol-gel glasses: The trapping of enzymes ☆

Abstract We describe the preparation and properties of a biochemically active sol-gel glass, obtained by trapping the enzyme alkaline phosphatase (ALP) in a polymerizing tetramethoxysilane. The immobilized purified ALP from bovine intestinal mucosa had a 30% activity yield and an improved stability to thermal deactivation compared to a solution. The composite bioactive glass was preserved in water at room temperature for two months without loosing activity. A non-Michaelis-Menten kinetics was observed. The concept of preparing bioactive materials by the sol-gel method seems to be general. Thus, other enzymes (chitinase, aspartase, β-glucosidase) were successfully trapped.

Doped sol-gel glasses as chemical sensors☆

Abstract It is reported that by trapping suitable analytical reagents, porous sol-gel glasses can be used for the preparation of a wide variety of chemical-sensing materials. The key to the successful preparation of these materials is based on the observation that a substantial fraction of the trapped molecules is exposed to the liquid or gaseous intra-pore volume. The glass matrix thus serves as a solid support for analytical reagents which interact with analyzates present in the adjacent phase. The phenomenon is quite general, and has been demonstrated for a variety of color tests for metal cations, proton (pH indication), anions, and organic molecules.

CIS-TRANS ISOMERIZATION IN THE PHOTOCHEMISTRY OF VISION

Various models proposed for the primary photoevent in vision are critically discussed. It is concluded that the classical picture of a single cis-trans isomerization step is the only one which satisfactorily accounts for all the available experimental data. Experiments are performed showing that this process is temperature independent over a range of 200°C. Photoisomerization yields for the free protonated Schiff base of 11-cis (and all-trans) retinal are measured as a function of the excitation wavelength. In contrast to the efficient and wavelength independent photobleaching of rhodopsin, the yields of the 11-cis→al-trans isomerization of the free chromophore are small, exhibiting a marked dependence on the excitation wavelength. Potential energy curves for both ground and excited states of rhodopsin are derived from the analysis of the accumulated experimental data. In variance with the behavior of model compounds, photoisomerization in the pigment proceeds via the quantitative population of a common, barrierless, thermally relaxed excited state along the 11-12 torsional coordinate separating the 11-cis (rhodopsin) and all-trans (bathorhodopsin) configurations. In the ground state, interactions with the protein destabilize the all-trans isomerization product, leading to storage of a significant fraction of the photons energy in the primary step.

Two emitting states of excited p-(9-anthryl)-n,n-dimethylaniline derivatives in polar solvents

Abstract Steady-state and time-resolved fluorescence spectroscopy, as well as the S n ← S 1 absorption spectra of three anthryl derivatives of dimethylaniline, I–III, reveal the existence of two emitting states in all these compounds. The primary locally excited (LE) state of the anthracene moiety relaxes in polar solvents to a highly polar excited state. The relaxation involves most probably an internal rotation between the dialkylaniline donor and the anthracene acceptor structural sub-units, to reach a perpendicular conformation of the twisted internal charge-transfer (TICT) excited state. The intersystem crossing occurs preferentally in the TICT state, being followed by a back electron transfer relaxation to a lower lying LE triplet.

Biocatalysis by sol-gel entrapped enzymes

Abstract Attachment of enzymes to insoluble matrix is an essential step in the development of biocatalysts. Transparent xerogels containing various enzymes were obtained by mixing a solution of an enzyme with tetra-methoxy orthosilicate (TMOS) at room temperature followed by gelation and drying. Effective immobilization was usually obtained at initial pH values > 7, where there is a change in the gelation mechanism from predominant hydrolysis/condensation to predominant direct polymerization of silicate precursors. The properties of sol-gel matrix, namely, transparency, large hydrophilic surface and good chemical and thermal stability, make it an ideal material for both biocatalysts and optical sensor devices. An example of a simple optical glucose sensor is demonstrated.

Doped sol-gel glasses as pH sensors

Abstract A series of pH indicators were trapped in sol-gel porous glasses by polymerization of tetramethoxysilane in the presence of a surface active agent. The properties of these novel sensing materials including spectral shifts, shifts in the pH-sensing range, cycle repeatability, leachability, rates of response and isosbestic points are described. A prototype of a pH meter based on a pH-sensing glass was constructed.

On the primary quantum yields in the bacteriorhodopsin photocycle

Pulsed Nd laser experiments in suspensions of the purple membrane of Halobacterium halobium are carried out at room temperature. At sufficiently high laser intensities, a photostationary mixture of bacteriorhodopsin (BR) and its red-shifted (batho) photoproduct (K) is obtained. The spectra of the first three intermediates in the photocycle are reported. The data yield a value of phi1/phi2=0.40 +/- 0.05 for the ratio of the quantum yields of the forward (phi1) and reverse (phi2) processes, setting an upper limit of approximately 0.4 for the quantum efficiency of the cycle at room temperature. This method is generally available for the determination of phi2 in the case of a photoequilibrium: A in equilibrium B, where B is a short-lived transient and phi1 is known from low intensity measurements. Its potential application is of importance for the study of the photophysics of visual pigments at physiological temperatures.

Miniaturization of organically doped sol-gel materials: a microns-size fluorescent pH sensor

Abstract Miniaturization of a reactive doped sol-gel material is described. A reversible fluorescent pH sensor was constructed by placing a pyranine doped SiO 2 sol-gel matrix at a 10 μm tip of a metal coated micropipette. Subsecond response time was achieved by further downscaling to ≈ 1 μm. Using optical near-field microscopy configuration, these observations demonstrate the potential of constructing a variety of miniaturized sensors and chemical manipulators, by doping the sol-gel matrix with a suitable reagent.

On the photoionization of aromatic molecules in polar liquids

Abstract The available flash and pulsed-laser photochemical data, concerning the photoionization of N, N, N′, N′, tetramethyl-para phenylenediamine (TMPD), pyrene and perylene in polar liquids, are analyzed. A model for the photo-ionization of aromatic molecules in polar liquids is presented, involving an intermediate, semi-ionized, state. This state is populated from the Franck-Condon excited state of the system, in competition with relaxation to the fluorescent level of the molecule.

The quantum efficiency of the bacteriorhodopsin photocycle.

The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.