Norbert Hampp

Bacteriorhodopsin: a biological material for information processing.

Technology which makes use of biological materials has advanced dramatically in the last few decades. Production of specific biochemicals by selected microbial strains, the use of enzymes for stereospecific biosynthesis of materials and gene technological production of biologically important macromolecules are a few examples of these developments.

The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli

The cDNA clone for strictosidine synthase, the enzyme which catalyzes the stereospecific condensation of tryptamine with secologanin to form the key intermediate in indole alkaloid biosynthesis, strictosidine, has been identified with a synthetic oligodeoxynucleotide hybridization probe in a λgt11 cDNA library of cultured cells of Rauvolfia serpentina. The DNA has been sequenced, revealing an open reading frame of 1032 base pairs encoding 344 amino acids. The sequence of 60 nucleotides in the 5′‐flanking region has been determined by primer extension analysis. The encoded protein has been expressed in E. coli DH5 as detected by immunoblotting of protein extracts with antibodies raised against the native enzyme.

Bacteriorhodopsin wildtype and variant aspartate-96 → aspargine as reversible holographic media

Air dried films of purple membranes (PM) from Halobacterium halobium containing the photochromic protein bacteriorhodopsin (BR) were prepared and the BR-photocycle of this material analyzed. The absorption maxima of the initial state B (lambda(max) = 570 nm) and the photochemical intermediate M (lambda(max) = 412 nm), which is the longest living intermediate in suspension (tau approximately 10 ms), were spectrally well separated. Light-induced population gratings between B and M were used for reversible holographic recording in these dry PM films. The resolution (>5,000 lines/mm) of PM films was comparable to the corresponding values of conventional photochromic recording materials. The longterm stability toward photochemical degradation of PM films is excellent (> 100.000 recording cycles). The spectral bandwidth (400-680 nm) of such films covers nearly the whole visible spectrum. Both the photochemical transition from B --> M with wavelengths in the green-red range and from M --> B with blue light were utilized for holographic recording. The latter possibility (M --> B) seems to be advantageous for several applications because the holographic grating is only formed during reconstruction. Higher reading intensities lead to higher population of the M-state and result in an increase of the fringe contrast instead of decreasing it. New possibilities for the further development of holographic media based on bacteriorhodopsin are raised by the availability of PM variants with modified optical properties. By the use of the variant BR-326, which differs from the wildtype PM by a single amino acid exchange (aspartate-96 --> asparagine), the sensitivity of PM films is increased by approximately 50% from 12 cm(2)/J to 19 cm(2)/J for recording with 568 nm. The sensitivity for recording with 413 nm (33 cm(2)/J) is not influenced by the amino acid exchange. The observed diffraction efficiency eta of PM films with BR-326 is twice that of BR-wildtype (BR-WT) films and is in the range of conventional organic photochromics ( approximately 1%). In dried films of both BR-WT and BR-326 the M-decay was shown to be at least biexponential.

Bacteriorhodopsin films as spatial light modulators for nonlinear-optical filtering

The application of dry purple membrane films for nonlinear-optical filtering is described. The biological photochrome bacteriorhodopsin (BR) is contained in the purple membrane (PM) from Halobacterium halobium. BR has two dominant photoactive states, B and M, which have well-separated absorption bands with maxima at 570 nm (B) and 412 nm (M). Since the local transmission of a PM film depends on the ratio between the forward (B ? M) and the backward (M ? B) photoreactions, PM films can be used as light-controlled absorptive spatial light modulators. A model describing the nonlinear transmission of PM films containing the wild-type form of BR or one of its mutated variants, e.g., BR(D96N), and examples of their application in spatial filtering, e.g., edge enhancement, are presented.

Poly(methylene blue)-modified thick-film gold electrodes for the electrocatalytic oxidation of NADH and their application in glucose biosensors

Electropolymerization of the phenothiazine derivative methylene blue (MB) on screen-printed, thick-film gold electrodes leads to electrocatalytically active and conducting layers of poly(methylene blue) (PMB) in intimate and stable contact with the electrode surface. The catalytic properties of the PMB films allow anodic oxidation of NADH at potentials as low as +200 mV vs. the saturated calomel electrode (SCE) reducing interferences from cooxidizable species as well as minimizing electrode fouling by enabling a simultaneous two-electron transfer mechanism. Dehydrogenase-based biosensors employing PMB-modified thick-film electrodes are obtained either by entrapment of the enzyme into the PMB layer itself or by laminating an enzyme membrane made of an aqueous poly(vinylacetate) dispersion over the PMB-modified electrode. Both methods are used to fabricate glucose biosensors which can be operated at low overpotentials, i.e. +200 mV vs. SCE.

Photochemical conversion of the O-intermediate to 9-cis-retinal-containing products in bacteriorhodopsin films.

The photochemical activity of the O-state was investigated in bacteriorhodopsin (BR) films containing wildtype BR at pH 6.5 in the presence of glycerol. The formation of a photoproduct of O with an absorption maximum at 490 nm and 9-cis-retinal configuration was found. This 490-nm product was named P and shows a slow thermal reaction into a compound with a maximal absorption at 380 nm which was named Q and contains free 9-cis-retinal in the proteins binding site. The photoproducts of O, i.e., P and Q, are very similar, or even identical, to those previously observed in blue membranes. Common to the O-state and blue membrane forms of bacteriorhodopsin is a protonated aspartic acid 85, and we suggest that it is the reduced negative charge around the Schiff base which is responsible for the 9-cis photoisomerization. The release of a proton from aspartic acid 85 is linked to the conversion of the O-state back to the initial state of BR. Therefore the conditions of low proton mobility in BR films containing glycerol favor the accumulation of the O-state. For optical and holographic applications such BR films are very attractive. It is possible to create photoproducts with red light which are thermally stable at room temperature and that can be photochemically erased. Dependent on the light composition both properties can be realized in the same sample material. This feature may bridge the gap between information processing and short-term and long-term storage of information with BR.

Biological photochrome bacteriorhodopsin and its genetic variant Asp96 ? Asn as media for optical pattern recognition.

The biological photochrome bacteriorhodopsin (BR) is contained within the purple membrane (PM) of Halobacterium halobium. Artificial derivatives with improved optical properties can be generated by genetic methods and isolated from mutated halobacterial strains. The use of PM films that contain wild-type BR and BR variants as real-time recording media for various holographic applications has been reported previously, and the advantages of BR variants have been demonstrated. The high reversibility (>> 10(5) record/erase cycles), the fast time scale of its photoconversions (femtoseconds to milliseconds), and the large photochromic shift ( approximately 160 nm) occurring during its photocycle make it a promising material for real-time applications. A dual-axis joint-Fourier-transform (DA-JFT) correlator is used to demonstrate the applicability of PM films in holographic pattern recognition. One major advantage of PM films in this application is their high spatial resolution of more than 5000 lines/mm. Severe restrictions on the overall performance of the DA-JFT correlator system are caused by scattered light and result in a low signal-to-noise ratio. Since PM patches typically have a diameter in the range of the visible wavelengthsthat are used for hologram recording, light scattering is an intrinsic problem of PM films. The polarization recording properties of PM films are employed to overcome this problem. More than 20-fold improvement of the signal-to-noise ratio in the DA-JFT correlator output is obtained.

Homogeneous strictosidine synthase from cell suspension cultures of Rauvolfia serpentina

Abstract Strictosidine synthase, the enzyme which catalyses the stereospecific condensation of secologanin and tryptamine to H-3α( S )-strictosidine, the key intermediate in monoterpenoid indole alkaloid biosynthesis, was purified to homogeneity from suspension cells of Rauvolfia serpentina (920-fold purification, 35% yield). The specific activity is 184 nkat/mg. The isolated enzyme is a single polypeptide, M r 30 000, possessing a 5.3% carbohydrate content. The enzyme has a pH-optimum at 6.5, a temperature optimum at 45°, isoelectric point at pH 4.5, and apparent K M values both for tryptamine and secologanin of 4 mM. The enzyme was immobilized and has, in this form, a half-life of 100 days at 37°.

Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles that Determine Their Interaction with Cells

A homologous nanoparticle library was synthesized in which gold nanoparticles were coated with polyethylene glycol, whereby the diameter of the gold cores, as well as the thickness of the shell of polyethylene glycol, was varied. Basic physicochemical parameters of this two-dimensional nanoparticle library, such as size, ζ-potential, hydrophilicity, elasticity, and catalytic activity ,were determined. Cell uptake of selected nanoparticles with equal size yet varying thickness of the polymer shell and their effect on basic structural and functional cell parameters was determined. Data indicates that thinner, more hydrophilic coatings, combined with the partial functionalization with quaternary ammonium cations, result in a more efficient uptake, which relates to significant effects on structural and functional cell parameters.

A triple-step potential waveform at enzyme multisensors with thick-film gold electrodes for detection of glucose and sucrose

Abstract Significant loss of activity of thick-film gold electrodes is observed when they are used for continuous amperometric detection of H2O2 in 0.1 M sodium phosphate buffer and in serum samples. Triple-step potential waveforms are utilized for pulsed amperometric detection to clean the surface of conventional electrodes from poisoning substances and reactivate them, thus maintaining uniform electrode activity. This technique is applied to a microprocessor-controlled multichannel sensor system with poly(vinylacetate)-based enzyme membranes, which are attached to the thick-film gold electrodes by adhesion. The parameters of a triple-step potential sequence, which are dependent on the electrochemical behaviour of the membrane-coated thick-film gold electrodes, have been determined so that efficient cleaning of the electrode surfaces is obtained and at the same time the characteristics of the enzyme sensors are not influenced. The potential sequence consists of an oxidation potential of 800 mV, a reduction potential of 100 mV and the detection potential of 600 mV (versus Ag/AgCl) for hydrogen peroxide. The triple-step potential waveform is applied to the multichannel system for simultaneous determdination of glucose and sucrose in buffered aqueous solutions and for detection of glucose in serum samples. A sensor response with improved sensitivity and reproducibility is obtained. With the on-line cleaning of the electrode surfaces by application of the triple-step potential waveform, uniform electrode activity is maintained even when the glucose sensors are operated in serum samples. The sensor response, obtained with continuous amperometric detection, proves to be limited by adsorption processes of H2O2 at the surface of the thick-film gold electrodes. On the other hand, the sensor response obtained with application of the triple-step potential waveform indicates limitation by diffusion processes and the upper limit of the linear range is extended from 5 to 12 mM for glucose and from 4 to 15 mM for sucrose in aqueous solutions.