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M Ottolenghi

Publications and source records attributed to M Ottolenghi.

11 recordsLinked to original sources
PubMed1992

Properties of trypsin and of acid phosphatase immobilized in sol-gel glass matrices.

Trypsin and acid phosphatase-containing silica sol-gel glasses were obtained by mixing a solution of an enzyme with polyethylene glycol (PEG) 6000 and tetramethoxy orthosilicate at room temperature, followed by gelation and drying. Activity of the immobilized trypsin toward small substrates, such as N-benzoyl-L-arginine-4-nitroanilide at its Km, for the best preparations equaled that of the soluble enzyme. Polylysine (M(r) less than or equal to 13,000) and aprotinin (M(r) = 6,500) inhibited this activity. Larger polylysines as well as soybean trypsin inhibitor (M(r) = 20,100) were ineffective. The sol-gel-entrapped trypsin activity was stable when sol-gel glasses were incubated at ambient temperature (pH 7.5) for several months. In comparison, trypsin, immobilized in sol-gel glass by surface adsorption and incubated under the same conditions overnight, was completely autodigested. The firm interaction between the protein molecules and the silica matrix stabilized the enzymes. Thus, the half-life of sol-gel-entrapped acid phosphatase at 70 degrees C (pH 8.0) was two orders of magnitude larger than that of the enzyme in solution. Transparent, mechanically and chemically stable bioactive sol-gel glasses may be used for the development of robust on-line biochemical photodetection sensors and for the purposes of chemical catalysis.

Acid Phosphatase↗
PubMed1991

Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes.

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes.

Calorimetry, Differential Scanning↗
PubMed1979

Dynamics of pH-induced spectral changes in bacteriorhodopsin.

The kinetics of the spectral shift induced in bacteriorhodopsin by low pH are investigated by using the rapid-mixing, stopped-flow technique. The generation of the acid form of the chromophore (A605) occurs in two distinct steps: a fast process (t1/2I = 21 +/- 4 ms) is followed by a much slower reaction (t1/2II = 6 +/- 2 s). The observations are interpreted in terms of neutralization of an acid group in the neighborhood of the retinyl chromophore, the double-staged kinetics being attributed to cooperative effects between chromophores. The method may serve as a tool for studying the kinetics of proton migration across the purple membrane.

Bacteriorhodopsins↗
PubMed1979

Photoisomerization, energy storage, and charge separation: a model for light energy transduction in visual pigments and bacteriorhodopsin.

A simple model for the early events in visual pigments and bacteriorhodopsin is proposed. The model makes use of the likelihood that a negatively charged amino acid forms a salt bridge with the positively charged nitrogen of the retinylic chromophore. The photochemical event is a cis-trans isomerization in visual pigments and a trans-cis isomerization in bacteriorhodopsin, which in each case cleaves the salt bridge and thus separates charge in the interior of the protein. We propose that this is how the energy of a photon is transduced into chemical free energy of the primary photoproduct. The use of photoisomerization of a flexible chromophore to achieve charge separation provides a general mechanism which may be applicable to other systems. Our model explains many of the fundamental properties of visual pigments and their photoproducts. First, the extraordinarily low rate of thermally populating the ground state of the primary photoproduct, as determined from psychophysical and electrophysiological measurements, is seen as resulting from the large barrier to thermal isomerization about a double bond, perhaps enhanced by electrostatic attraction in the salt bridge. Second, the increase in energy and the spectral red shift that characterize the primary photochemical events are natural consequences of the separation of charge. Proton-dependent processes detected with picosecond techniques are proposed to be ground-state relaxation processes following the primary photochemical event. Finally, the charged groups of the salt bridge, repositioned by photoisomerization, provide a simple mechanism for vectorial proton translocation in bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1977

Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).

Bacteriorhodopsins↗
PubMed1977

On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.

Bacteriorhodopsins↗
PubMed1977

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.

Bacteriorhodopsins↗
PubMed1976

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.

Bacteriorhodopsins↗