We have constructed an experimental station (beamline) at the National Synchrotron Light Source to measure circular dichroism (CD) using soft x-rays (250 less than or equal to hv less than or equal to 900 eV) from a time modulated elliptically polarizing wiggler. The polarization of the soft x-ray beam switches periodically between two opposite polarizations, hence permitting the use of phase-sensitive (lock-in) detection. While the wiggler can be modulated at frequencies up to 100 Hz, switching transients limit the actual practical frequency to approximately equals 25 Hz. With analog detection, switching transients are blocked by a chopper synchronized to the frequency and phase of the wiggler. The CD is obtained from the ratio of the signal recovered at the frequency of polarization modulation, f, to the average beam intensity, which is recovered by synchronous detection at frequency 2f.

Sensitive optical methods developed over the past decade make it possible to monitor the dynamics of structural changes in proteins -- changes which can, for instance, modulate function in allosteric proteins or mark self-assembly toward the functional, native structure in protein folding -- by using linearly or elliptically polarized light to perform nanosecond spectral measurements. The ellipsometric approach to time- resolved circular dichroism (TRCD) spectroscopy, initially limited to near-UV-visible single-wavelength measurements with 50 - 100 ns time resolution, has been extended to allow for multichannel spectral measurements, expansion of the spectral range into the far UV, and refinement of the time resolution to 1 ns. The ellipsometric technique has also been extended to magnetically induced TRCD (TRMCD) measurements, while the development of closely related polarimetric techniques has made available nanosecond time-resolved linear dichroism (TRLD) and optical rotatory dispersion (TRORD) spectral measurements with high sensitivity. The sensitivity of the polarimetric TRLD method, for instance, is about two orders of magnitude greater than that of standard approaches. Applications of these techniques to the study of function in a number of proteins, including myoglobin, hemoglobin, and cytochrome c oxidase, and applications to the study of folding kinetics in peptides and proteins are discussed.

The increasing availability of fs lasers has resulted in expanded interest in multi-photon excitation of fluorescence. In this overview paper, we describe recent developments in multi-photon induced fluorescence spectroscopy and anisotropy. Experimental results are presented for three-photon excitation of tyrosine and tryptophan, and for the single tryptophan protein, troponin C Mutant F22. Three-photon excitation has been also applied to study DPH-labeled membranes and DNA stained with DAPI. The enhancement in spatial resolution resulting from three-photon excitation in spectroscopic and microscopic conditions is described for BBO scintillator in solution. We also describe two-photon excitation with using two-photon at different wavelengths.

We demonstrated that fluorescence anisotropy can be effectively decreased or increased in the presence of light quenching, depending on relative polarizations of excitation and quenching pulses. For parallel light quenching anisotropy decreases to 0.103 and z-axis symmetry is being preserved. In the presence of perpendicular light quenching, the steady- state anisotropy of pyridine 2 glycerol solution increases from 0.368 for unquenched sample to 0.484, for quenched one. We show that angular distribution of transition moments loses the z-axis symmetry in the presence of perpendicular light quenching. In these cases we used more general definitions of anisotropy. Induced by light quenching anisotropy can be applied in both, steady-state and time-resolved measurements. In particular, the systems with low or none anisotropy can be investigated with proposed technique.

Recently, we have shown that metal ions free in solution may be determined at low levels by fluorescence anisotropy (polarization) measurements. Anisotropy measurements enjoy the advantages of wavelength ratiometric techniques for determining metal ions such as calcium, because anisotropy measurements are ratiometric as well. Furthermore, fluorescence anisotropy may be imaged in the microscope. An advantage of anisotropy not demonstrated for wavelength ratiometric approaches using indicators such as Fura-2 and Indo-1 is that under favorable circumstances anisotropy-based determinations exhibit a much broader dynamic range in metal ion concentration. Determinations of free Zn(II) in the picomolar range are demonstrated.

When the fluorescence intensity and fluorescence anisotropy decays can be described as sums of exponentials, a simplifying assumption is often made: each intensity decay lifetime associates with each rotational correlation time. Numerous biological systems exist where this assumption is invalid. We have been evaluating a general kinetic scheme applicable to all possible associations between lifetimes and rotational correlation times. For the simple case of two lifetimes and two rotational correlation times, nine association models exist. We have been testing the ability of these different association models to discriminate against one another. Using a Monte Carlo algorithm, synthetic anisotropy data sets were generated according to each association model. Each data set was then analyzed by all models. To deduce which association model was used to generate a data set, we found that a global analysis of a family of anisotropy data sets differing in an independent parameter(s) is required; an example would be variable intensity decay amplitudes from decays collected at several emission wavelengths. Anisotropy decays of the two tryptophans per subunit of liver alcohol dehydrogenase were also analyzed by all of the two-lifetime, two-correlation-time association models to determine if one or both tryptophans experience local depolarizing motions.

Fluorescent analogs of nucleic acid bases are useful probes for observation of DNA structure, interactions, and dynamics. 2-aminopurine (2AP) and 3-methylisoxanthopterin (3MI) are analogs of adenine and guanine, respectively, which have single exponential fluorescence intensity decay kinetics when free in buffer at neutral pH but complex multi-exponential decays when incorporated into oligonucleotides. We have investigated the mechanisms underlying the complexity of the emission kinetics of these probes in DNA by observing decays as a function of local nucleic acid sequence and emission wavelength. For both probes, the intensity-averaged lifetime increases smoothly with increasing emission wavelength. Analysis of these data as a time-resolved emission spectrum (TRES) demonstrates that the complex decay law can be described as resulting from dipolar relaxation of the local environment of the probe on the same timescale as emission. While the mean fluorescence lifetime shows no dependence on nucleic acid sequence 5' or 3' of the probe, the mean time constant for dipolar relaxation is correlated with the identity of the neighboring bases. These results suggest that 2AP and 3MI may be sensitive probes of the local dynamics of nucleic acids, bound water, and counterions.

Many neurotransmitters and hormones convey their signals into cells via transmembrane receptors that activate heterotrimeric G proteins which in turn active phospholipase C-(beta) (PLC- (beta) ). Activation of PLC-(beta) by the (alpha) _q and (beta) (gamma) subunits of G proteins results in activation of protein kinase C and release of Ca²⁺ from intracellular stores, which in turn results in a multitude cellular changes. We have recently found that activation of PLC-(beta) by G proteins occurs by lateral association on the membrane surface. Here, we have measured the affinity of the membrane- bound species by fluorescence energy transfer and have conducted time-resolved studies to assess the lifetime of the PLC-G protein complexes in order to understand PLC signaling inside the cell. To better interpret these results, we outline methods to convert the two-dimensional dissociation constant measured for the membrane-bound proteins to a three dimensional one. We also detail calculations to determine the concentrations at which non-specific protein-protein interactions occurs due to membrane crowding. To differentiate between the physical association of PLC-G complexes and activation, we have developed a real-time fluorescence-based PLC-(beta) activity to determine the length of time that PLC- (beta) remains active after G protein dissociation. A model for activation will be presented.

Binding/unbinding kinetic rates of cytoskeletal proteins at the cytofacial surface of the plasma membrane of smooth muscle cells in culture are measured and imaged by total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) microscopy. Cells are first injected with either rhodamine monomeric actin or rhodamine phalloidin, which binds to polymeric actin. A TIR beam, which illuminates approximately 80 nm deep into the cell, is then used to preferentially observe the labeled actin in the vicinity of the membrane. Fluorophores at cell-substrate contact regions within the evanescent field illumination are then photobleached by a prolonged flash. The subsequent recovery, excited by a series of briefer flashes at 1 or 6 frames per minute, is recorded by a cooled CCD. The resulting stack of images can be curve-fit pixel-by-pixel to produce a spatially resolved image of the unbinding rate (the reciprocal of residency time) of protein reversibly adsorbed at the submembrane surface. For rhodamine actin and for rhodamine phalloidin, the two ways of marking actin in the cell, the average characteristic unbinding times are somewhat different: 308 plus or minus 142 sec and 833 plus or minus 140 sec, respectively. The spatially resolved images of rhodamine phalloidin, pseudo-colorized to show average unbinding rates at each pixel, reveal a considerable variation over the cell, ranging over an order of magnitude.

We wish to develop a biophysical understanding of the structure-function relationship of Tissue Factor (TF), the membrane-bound protein that triggers hemostasis and arterial thrombosis by essential activation of the enzyme Factor VIIa (VIIa). Catalysis by TF-bound VIIa occurs in the blood stream, and flow-dependent shear affects its enzyme kinetics. From the known structure of the TF:VIIa complex, the catalytic domain of VIIa could be as close as 1 nm or as far as 12 nm from the membrane surface depending on geometry under the influence of shear. As models of blood vessels, we use glass capillary tubes coated on the inside surface with a lipid bilayer containing TF. This setup permits two types of measurements as a function of flow: enzyme kinetics of TF:VIIa by a colorimetric assay; and analysis of the spatial and dynamic relationship of TF:VIIa with respect to the membrane surface. Time-resolved depolarization and resonance energy transfer are measured via a microscope using either direct or evanescent wave excitation. We demonstrate the feasibility of these experiments by using rhodamine-labelled phospholipids at probe densities down to approximately 300 molecules/micrometer² in the presence or absence of the acceptor malachite green and by the emission anisotropy decay of probes in glycerol.

Flow cytometry is uniquely capable of making sensitive and quantitative multiparameter fluorescence measurements with discrimination of free from particle-bound fluorophore. Recent advances in mixing and sample delivery have extended these capabilities into the sub-second time domain. Access to these time scales has enabled us to use flow cytometry to measure molecular interactions. Using the general approach of immobilizing one molecule on a microsphere and fluorescently labeling another, we have been able to make real-time measurements of ligand-receptor and enzyme-substrate interactions involving proteins, nucleic acids, carbohydrates, and lipids. We are developing schemes for immobilizing active biological molecules in defined and homogeneous orientations relative to the surface. We are also developing approaches for homogeneous fluorescent labeling of active biomolecules and calibration schemes for quantitative measurements by flow cytometry. We will present several examples of applications of this new technology, including DNA- and protein-protein interactions, nucleic acid hybridization, and interactions on artificial membrane surfaces. These approaches should have wide applications for mechanistic analysis, diagnostics, and drug development.

We report here a novel application of flow cytometry for the quantitative analysis of the high affinity interaction between membrane proteins both in detergent solutions and when reconstituted into lipid vesicles. The approach is further advanced to permit the analysis of binding to expressed protein complexes in native cell membranes. The G protein heterotrimer signal transduction function links the extracellularly activated transmembrane receptors and intracellular effectors. Upon activation, (alpha) and (beta) (gamma) subunits of G protein undergo a dissociation/association cycle on the cell membrane interface. The binding parameters of solubilized G protein (alpha) and (beta) (gamma) subunits have been defined but little is known quantitatively about their interactions in the membrane. Using a novel flow cytometry approach, the binding of low nanomolar concentrations of fluorescein-labeled G(alpha) _i1 (F- (alpha) ) to (beta) (gamma) both in detergent solution and in a lipid environment was quantitatively compared. Unlabeled (beta) $gama reconstituted in biotinylated phospholipid vesicles bound F-(alpha) tightly (K_d 6 - 12 nM) while the affinity for biotinylated-(beta) (gamma) in Lubrol was even higher (K_d of 2.9 nM). The application of this approach to proteins expressed in native cell membranes will advance our understanding of G protein function in context of receptor and effector interaction. More generally, this approach can be applied to study the interaction of any fluorescently labeled protein with a membrane protein which can be expressed in Sf9 plasma membranes.

Many cellular responses, particularly in the immune system, are triggered by ligand binding to a cell-surface receptor. However, as indicated by bell-shaped dose-response curves, ligand binding alone is sometimes insufficient to trigger a response. Often, ligand binding must also induce the aggregation of cell-surface receptors through crosslinking, which occurs when a ligand binds simultaneously to two or more receptors. Thus, an important goal in cell biology has been to establish quantitative relationships between the amount of ligand present on a cell surface and the number of crosslinked ligand-specific cell-surface receptors. To better understand ligand-induced receptor aggregation, we have been investigating the binding of a model multivalent antigen (DNP₂₅PE) to cell-surface anti-DNP FITC-labeled IgE (FITC- IgE). To determine the kinetic and equilibrium parameters that characterize crosslinking in this system, we have developed a combined theoretical and experimental approach that is based on multiparameter flow cytometry. With this approach, we can measure both the average number of ligand molecules that are bound per cell and the average number of receptor binding sites that are bound per cell. The average number of DNP₂₅PE per cell is determined by measuring the fluorescence of phycoerythrin. The average number of occupied IgE sites per cell is determined by measuring the fluorescence of FITC, which is quenched upon ligand binding. This novel approach, together with conventional methods for changes in intracellular calcium, allows us to correlate for the first time the dynamics of IgE crosslinking with cell activation.

Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. We have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, we used fast valves to overcome the disparity between mixing 100 (mu) ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 (mu) l/sec. Particles were detected within 100 msec after mixing, but unstable flow was created which lasted for 1 sec after injection of the sample into the flow cytometer. We used optical criteria to discriminate particles which were out of alignment due to the unstable flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system we were able to automate sample handling and delivery with intervals of a few seconds. We used a fluidic approach to defeat the instability caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of instability was reduced to approximately 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.

We report new and novel electronics to quantify lifetimes of free (fluorophore solution) and cell/particle-bound fluorophores. This technology combines flow cytometry and frequency-domain fluorescence lifetime spectroscopy measurement principles to provide unique features for making excited-state lifetime measurements of free fluorophore and cell/particle-bound fluorophore on a cell-by-cell basis in real time. Cells labeled with fluorophore and suspended in fluorophore solution are analyzed as they intersect a high- frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence pulse (cells) and steady-state (fluorophore solution) signals are processed by separate phase-sensitive detection channels to quantify lifetimes. The cell-bound fluorophore measurement channel employs a phase comparator to provide two output signals (proportional to the sine and cosine of the phase difference between the signal pulse input and a steady-state reference signal), whereas, the free (solution) fluorophore lifetime measurement channel employs a second phase comparator to provide steady-state sine and cosine outputs which can be gated externally (pulse generator) or by a cell being analyzed. The phase comparator outputs are input to ratio modules for determining the respective lifetimes. Examples of solution and cell/particle lifetime measurements using common fluorophores are described.

Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

We present a demonstration of an optical microfluidic system for rapid and simultaneous measurement of several fluid analytes in a microscale volume that is based on fluorescent indicators immobilized on polymer microbeads. The fluorescence properties of such 'reporter beads' can be a function of the concentration of an analyte, and can be monitored in a silicon microfabricated flow channel. The sizes of microbeads can be determined by the scattering signal, so this system can be used to analyze several analytes at the same time by using beads of different sizes that are sensitive to different analytes. The fluorescent indicator Carboxy-SNAFL 1 was immobilized on amino-functionalized polystyrene beads of 5 micrometer diameter. The intensities of two fluorescent peaks (at 560 nm and at 620 nm) were measured and their ratio was dependent on the pH value of the analyte. A 488 nm air-cooled argon laser was used to excite the fluorescent beads flowing through a microfabricated V-groove channel. Two miniature photomultiplier modules and interference filters were used to simultaneously measure both fluorescence intensities.

This paper presents theoretical modeling and experimental results of light scattering studies on latex (polystyrene) particle dimers and colloidal gold particles coupled to individual latex particles. The latex particle size range is 1 - 10 micrometer (in diameter) and that of the gold particles is 80 - 100 nm. In general, the latex particles are spherical, while the colloidal gold particles have varied shapes. The theoretical model is based on exact solution of the problem of light scattering by interacting spheres. Calculations based on interacting and noninteracting particles will be compared to experimental results. The experimental results were obtained with high resolution light scattering measurements of particle coupling in a flow particle analyzer. This analyzer, Copalis^TM (Coupled Particle Light Scattering), exploits particle coupling induced by antigen-antibody, receptor- hormone, or nucleic acid interactions in medical diagnostic assays. Although latex particle coupling produces a range of aggregate sips, only dimer light scattering results are relevant. Coupling of gold to latex particles in the Copalis^TM diagnostic system produces from one to many gold particles per latex particle. Coherent addition of fields scattered by many gold-latex pairs has been used to simulate the gold-based assay. The comparison of theoretical simulations and experimental results for the coupling will be discussed. The results of this study are being used to refine the particle coupling results in the Copalis^TM diagnostic system.

Lanthanide chelates, due to their unique luminescence properties, offer a potential for improvement and simplification of fluorescence resonance energy transfer distance measurements. In this report we present a procedure for incorporation of highly-luminescent europium chelate into internal sites of ds DNA. Using this labeling strategy donor- acceptor labeled 20 bp DNA fragments were prepared containing a stretch of six A residues (A-tract) in the middle of the fragment. Distance measurement between the donor (europium chelate) and acceptor (Cy5) were performed and compared to measurements with a DNA fragment lacking the A-tract. Only small changes in measured distances were observed between these two DNA molecules. The implications of these results for existing models of A-tract induced DNA bending are discussed.

A luminescent rhenium(I) metal-ligand complex, [Re(bcp)(CO)₃(4-COOHPy)](ClO₄), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been synthesized and characterized. This complex displays high quantum yields and long excited- state lifetimes (0.3 - 10 microseconds) in fluid solution at room temperature. The metal-to-ligand charge transfer (MLCT) emission from this compound exhibits sensitivity to micro- environment. [Re(bcp)(CO)₃(4-COOHPy)]⁺ displays highly polarized emission with a maximum anisotropy near 0.3 in the absence of rotational diffusion. This Re(I) MLCT complex was conjugated to several biomolecules, including the proteins human serum albumin (HSA) and bovine immunoglobulin G(IgG) as well as an amine containing lipid. When bound to a protein or lipid, the decay time is near 3 microseconds and the quantum yield is approximately 0.12 in aqueous air-equilibrated solution at room temperature. The unique spectral properties and reactive carboxylic acid functionality of [Re(bcp)(CO)₃(4-COOHPy)]⁺, allowed utilization of this probe in numerous biophysical and biomedical applications.

The use of tryptophan phosphorescence measurements in probing the structural flexibility of globular proteins in solution at room temperature has been limited to date by a number of factors which include: identification of the emitting residues, the low occurrence of tryptophan side chains in sufficiently rigid domains in globular proteins, as well as the absence of a molecular basis for the empirical correlation between phosphorescence lifetimes that are observed at room temperature, and the local viscosity. A review is presented here of our recent work in which we have attempted to address these concerns. A model for the relation between the long- lived triplet lifetimes from buried tryptophan residues and protein flexibility is described along with experimental evidence consistent with the model. Employing site-directed mutagenesis with E.coli alkaline phosphatase, we have both identified the residue responsible for the long-lived emission with the wild-type protein, as well as to reintroduce the indole side chain into alternate relatively inflexible domains resulting in the appearance of RTP.

Proteins are fundamentally dynamic entities, undergoing conformational changes in response to interaction with ligands during the performance of their biological function, or during the process of folding into their final, biologically active, structures. Advanced transient laser spectroscopy techniques based on intrinsic chromophores provides a powerful means to study these changes. Specifically, time-resolved phosphorescence of tryptophan (Trp) provides a means to observe the dynamics associated with different regions of the protein surrounding the emitting Trp residue. Using these methodologies, we have been able to demonstrate intrinsic equilibrium conformational heterogeneity in proteins, and have been able to study, in real time, slow events in the unfolding and refolding of these macromolecules. In addition, we have used circularly polarized phosphorescence to report on the chirality of the excited triplet state of Trp, which enables us to resolve two or more phosphorescing Trps with similar lifetimes. We briefly summarize some of these results, and present new room temperature phosphorescence (RTP) data characterizing the trifluoroethanol (TFE) induced (beta) -sheet to (alpha) -helix transition in the bovine milk protein, (beta) -lactoglobulin A.

Traditional methods, such as stopped flow, used to study early events in protein folding are limited, by instrument dead times, to investigating events which occur milliseconds after the initiation of folding. We have developed a laser-based temperature jump apparatus capable of measuring changes in the fluorescence of proteins undergoing folding or unfolding on the microsecond timescale following a laser-induced pH, or temperature, jump. The development of the system is discussed and the results for experiments with RNase T1 and Apomyoglobin are summarized.

Tryptophan phosphorescence provides a long-lived optical signal whose lifetime and quantum yield are sensitive to the local environment of the protein The phosphorescence from tryptophan analogs, however, has not been studied. We report here data on the room temperature phosphorescence of tryptophan, 4-, 5-, and 6-fluorotryptophan, and 5- bromotryptophan embedded in sucrose glasses. The absorption of the analogs was either blue-shifted (4-F-trp), markedly red- shifted (5-F-trp and 5-Br-trp), or not shifted (6-F-trp) with respect to tryptophan. The phosphorescence emission spectra of all analogs were red-shifted compared to trp (442 nm) with maxima at 446 nm (5-F), 451 nm (6-F), 452 nm (5-Br), and 469 nm (4-F). The 5-F and 6-F analogs had emission intensities similar to tryptophan (relative quantum yields of 0.68 and 0.91, respectively), while the emission intensities of the 4-F and 5-Br analogs were much lower (relative quantum yields of 0.039 and 0.022, respectively). All analogs exhibited complex decay behavior which required several exponentials for an adequate fit. The average lifetimes were all significantly lower than that of trp (2109 ms). The average lifetimes of the fluorinated analogs (5-F: 1496 ms, 6-F: 1004 ms, and 4-F: 87 ms) scaled approximately with the relative quantum yields while that of 5-Br (1.03 ms) was significantly lower.

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Rotational and lateral dynamics of the MHC class II antigen I-A^d on A20 cells fixed with various concentrations of paraformaldehyde were examined by time- resolved phosphorescence anisotropy and fluorescence photobleaching recovery, respectively. Probes were erythrosin and tetramethylrhodamine conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde progressively increased I-A^d's limiting anisotropy at 4 degrees Celsius above the value of 0.042 seen in untreated cells while leaving the rotational correlation time of 22 microsecond unchanged. On the other hand, translational diffusion coefficients decreased from about 2 X 10^-10 cm² sec^-1 while recovery remained unchanged at 40 - 50%. Together these results suggest that fixation crosslinks class II molecules with each other or with other membrane proteins into structures large enough (greater than 500,000 kDa) to appear rotationally immobile but small enough to diffuse translationally with size-dependent rates. Fixation effects on both class II rotation and lateral diffusion are half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between biology of class II effector functions and physical sizes of fixation-induced aggregates are discussed.

An overall approach into the differential investigation of membrane/cytoplasm related metabolism and of cell-cycle of yeast cells after two color soft X-ray irradiation is presented; the soft X-rays being generated in trains of picosecond pulses by laser-plasma interaction. The two color X-ray differential technique is based on the generation of approximately 0.6 KeV X-rays which are deposited only in the membrane-wall complex switching off the anaerobic (fermentative) activity of yeast cells and on the generation of approximately 1.2 KeV X-rays which are mainly deposited in the cytoplasm, mitochondria and nucleus of yeast cells, mainly affecting the aerobic metabolism. A synergetic analysis of the metabolism is discussed, mainly founded on the recording of different correlated metabolic parameters, both on-line and delayed. Among the relevant access, pressure monitoring in batch samples acquire a dominant role allowing the identification of metabolic oscillation, that represent a marker of physical and chemical actions performed on the samples at different times. The experience acquired on yeast cells metabolism is being used to investigate lymphocytes metabolism and the related oscillatory properties of relevant enzymatic complexes. Actually even if it is not exactly the same as the mammalian situation, it should really propel the whole field forward.

The potential of surface enhanced spectroscopy consists in the detection of very small but specific structures of biomaterials. Surface enhanced infrared absorption (SEIRA) yields fingerprint information on the biomaterials. Electronic interactions between individual groups in the molecule are detected by means of surface enhanced fluorescence (SEF). During the investigation of biomembranes with SEIRA a tenfold intensity enhancement could be obtained. Enhancement factors greater than 100 can be achieved with SEF. Enhancement is considerably influenced by the properties of the metal cluster structure. Biomembranes formed from vesicles containing the nicotinic acetylcholine receptor were spectroscopically characterized. The adsorption of the vesicles on Ge- and Ag surfaces was investigated. The metal cluster structure was optimized in order to obtain high intensity enhancement factors.

Neuronal adhesion molecules expressed during embryogenesis are essential for axonal pathfinding and development of neural connections and can interact by homophilic or heterophilic adhesion. We used and further developed a simple flow cytometric (FCM) microbead method to assess these interactions. This method allows the detection of homo- or heterophilic interactions and gives estimates of the binding affinity. It is possible to determine binding domains of a molecule using site specific antibodies or protein fragments. Adhesion molecules of the immunoglobulin-superfamily or the tenascin (TN)-family isolated from chick brain were analyzed. Purified molecules were covalently or non-covalently coupled to microbeads. For investigation of heterophilic interactions different molecules were coupled to beads of different colors. Measurements were done on single laser FCM with UV or 488 nm excitation. From the measured particle numbers the numbers of beads in homo- and/or heterophilic aggregates were determined. From these values the percentage of beads in aggregates and the aggregate size was calculated. Homophilic interaction was found for the molecules NCAM, NgCAM and NrCAM. Heterophilic interaction were detected e.g. for: NrCAM/F11, TN-R/F11 and CALEB/TN-C and CALEB/TN-R. Using fragments of TN-R we found, that the third of 8 fibronectin type III domains bound to F11. The FCM results were confirmed by neurite outgrowth assays. Our data demonstrate that FCM analysis of microbead aggregation is an easy and reliable method to characterize protein interactions.

This work centers around developing methodologies to isolate the PAI-1 coding sequence of the DNA plasmid pET3a-PAI-1. Size Selective Capillary Electrophoresis (SSCE), using entangled polymer filled small i.d. capillaries, is used to develop digestion conditions (time and enzyme concentration) that provide single cuts (at variable positions) of the plasmid using BstYI restriction enzyme. After obtaining optimum partial digest conditions for this enzyme, digestion with Ndel will produce a mixture of fragments that includes the fragment (1354 bp) which contains the intact region of interest. Sensitive detection is achieved via laser induced fluorescence using running buffers containing intercalating dye. Using small i.d. capillary conditions as a starting point, the SSCE system is increased to the micro-preparative scale using various larger i.d. capillaries. The effects of capillary diameter, applied voltage, injection amount, and sample buffer concentration on separation performance are studied. Subsequently, single or limited numbers of injections of the single cut sample using a relatively large i.d. capillary should prove adequate material for digestion with Ndel prior to PCR amplification of the 1354 bp fragment.