Novel fluorescent methods for biotechnological and biomedical sensoring: assessing antioxidants, reactive radicals, NO dynamics, immunoassay, and biomembranes fluidity.

We proposed and developed a series of fluorescent methods for analysis and investigation of biological systems with a view of future biotechnological and biomedical applications. The methods we describe have been built upon several photochemical and photophysical phenomena including fluorescent quenching, photochrome photoisomerization, and energy transfer. Three new types of molecular probes have been developed and employed for such studies: (1) dual fluorophore-nitroxide compounds, (2) fluorescence-photochrome molecules, and (3) super molecules containing both fluorescence and fluorescent quenching segments. The fluorescent properties of the new probes were intensively exploited for several practical applications including a real-time analysis of antioxidants, nitric oxide, superoxide, reactive radicals, trinitrotoluene, and metal ions, investigation of molecular dynamics of biomembranes in a wide range characteristic times, detection of protein conformational transition, and characterization of surface system. Owning high sensitivity, simplicity, and availability of fluorescent techniques, these methods can be widely employed and are adaptable to fibrooptic sensoring. A general survey of the physical principles and application of the new fluorescent methods has been provided.

Novel fluorescence method for real-time monitoring of nitric oxide dynamics in nanoscale concentration.

A novel assay was developed for the measurement of nitric oxide. The proposed method is based on fluorescence, using a fluorophore-heme dual functionality probe (FHP). The heme group can serve as an effective NO-trap, due to its very fast reaction with NO and the high stability of the resulting complex. Since the heme is connected with a fluorophore as a part of the FHP dual-functionality probe, the heme can quench the fluorophore fluorescence, under certain conditions, by a singlet-singlet energy transfer mechanism. The proposed method was tested using myoglobin covalently modified by a stilbene label. The change in emission intensity of the stilbene fragment, versus an increasing concentration of NO precursors, clearly demonstrated the spectral sensitivity required to monitor the formation of a heme-NO complex in a concentration range of 10 nM-2 microM. Furthermore, the new methodology for NO measurement was also found to be an effective assay using tissues from rabbit and porcine trachea epithelium. The measured NO flux (in an initial time interval) in tissue sample from rabbit trachea epithelia and porcine trachea epithelia is approximately 7.9x10(-12) mol/sxg and approximately 3.0x10(-12) mol/sxg respectively.

Neonatal blood is more resistant to oxidative stress induced by stable nitroxide radicals than adult blood.

Neonate erythrocytes are more susceptible to oxidizing drugs than adults; however, there are controversial reports in the literature regarding the total antioxidant capacity of neonate blood. Stable nitroxide radicals (NRs) are reduced by blood and some other biological materials to the corresponding hydroxylamines. The kinetics of the nitroxide's disappearance using electron paramagnetic resonance (EPR) spectroscopy, provides useful biochemical and biophysical information about the antioxidant properties of biological systems. In order to investigate the antioxidant properties in the newborn's blood, we applied this novel method on 38 umbilical vein blood samples and 40 healthy adults. The NR, 5-Dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6,-tetramethyl-piperidine-oxyl (R), was used for this purpose. Ascorbate is the only known natural antioxidant that reduces R. We found that the reduction rates of R in neonate's whole blood are significantly higher (P < 0.001) than the reduction rates of R in adult's whole blood. However, there were no significant differences in the antioxidant capacity between the two groups. Newborn's blood has significantly higher ability to deal with oxidative stress, caused by R, in comparison with adult blood. We suggest that the system that responds to the recycling of ascorbate is more efficient in neonate blood than in adult's blood.

The novel FluoroChrome ImmunoAssay -- (FCIA). The role of molecular environment upon molecular structure exemplified by constriction of a flourescence-photochrome flanked by two proteins.

A rapid, sensitive, and quantitative novel immunoassay [FluoroChrome ImmunoAssay, FCIA] technique was developed which auspiciously combines both the high sensitivity of fluorescence measurements with the high specificity of an antibody. As opposed to existing immunoassays, FCIA is performed without separation of antibody-bound haptens from those that are free, and utilizes fluorescence measurements from widely available standard commercial fluorimeters. FCIA is based on the hypothesis that an appropriately designed stilbene-antigen analogue probe will suffer considerable steric hindrance to trans-cis photoisomerization when bound within the combined constraints of both an antibody binding site and a second globular protein. Specifically, an appropriately designed 2,4-dinitrophenyl-hapten derivative of fluorescent trans-4,4'-diaminostilbene (DAS), was squeezed between two large globular proteins: lysozyme (Lys) from one side, and anti-2,4,6-trinitrophenyl antibody (antiTNP) from the other side, in order to provide the desired constricted environment to restrict trans/cis-stibene isomerization within the antiTNP-DNP-DAS-Lys adduct. As was theoretically predicted and then experimentally verified, the trans-cis photoisomerization rate for the bound probe was found to be markedly inhibited, compared to that expected for the free probe in solution. The fluorescence-photochrome labeled probe was competitively displaced from the antiTNP binding site in the presence of the picric acid hapten, and photoisomerization then commenced to produce the fluorescence-silent cis-stilbene diastereomer. The process of association and dissociation of a hapten-antibody complex was readily monitored by the fluorescence technique in the presence of both antibody-bound and free haptens.

Structural features of transiently modified beta-lactoglobulin relevant to the stable binding of large hydrophobic molecules.

Binding sites for hydrophobic molecules on bovine beta-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to beta-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein-probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially-and reversibly-modified form of beta-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to beta-lactoglobulin may be modulated by simple physical treatment of the protein.

Study of rare encounters in a membrane using quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals.

Measurements of active encounters between molecules in native membranes containing ingredients, including proteins, are of prime importance. To estimate rare encounters in a high range of rate constants (rate coefficients) and distances between interacting molecules in membranes, a cascade of photochemical reactions for molecules diffusing in multilamellar liposomes was investigated. The sensitised cascade triplet cis-trans photoisomerisation of the excited stilbene involves the use of a triplet sensitiser (Erythrosin B), a photochrome stilbene-derivative probe (4-dimethylamino-4'-aminostilbene) exhibiting the phenomenon of trans-cis photoisomerisation, and nitroxide radicals (5-doxyl stearic acid) to quench the excited triplet state of the sensitiser. Measurement of the phosphorescence lifetime of Erythrosin B and the fluorescence enhancement of the stilbene-derivative photochrome probe, at various concentrations of the nitroxide probe, made it possible to calculate the quenching rate constant k(q)= 1.1 x 10(15) cm2 M(-1) s(-1) and the rate constant of the triplet-triplet energy transfer between the sensitiser and stilbene probe k(T)= 1.0 x 10(12) cm2 M(-1) s(-1). These values, together with the data on diffusion rate constant, obtained by methods utilising various theoretical characteristic times of about seven orders of magnitude and the experimental rate constants of about five orders of magnitude, were found to be in good agreement with the advanced theory of diffusion-controlled reactions in two dimensions. Because the characteristic time of the proposed cascade method is relatively large (0.1 s), it is possible to follow rare collisions between molecules and free radicals in model and biological membranes with a very sensitive fluorescence spectroscopy technique, using a relatively low concentration of probes.

Detection of nitric oxide from pig trachea by a fluorescence method.

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO(2). Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2 nmol/g x min, and NO-synthase stimulated by extracellular adenosine 5'-triphosphate produced NO flux of 0.9 nmol/g x min.

Molecular dynamics investigation of an antibody binding site by the fluorescence-photochrome method.

A combined fluorescence-photochrome approach was used for investigation of the molecular dynamics antiDNP antibody binding site and its cavity. A 4-(N-2,4-dinitrophenylamino)-4'-(N,N'-dimethylamino)stilbene (StDNP) fluorescence DNP analog was incorporated into the antibody binding site. This was followed by measurements of fluorescence and photochrome parameters such as the StDNP excitation and emission spectra, fluorescence lifetime, steady-state and time-resolved fluorescence polarization, kinetics of trans-cis and cis-trans photoisomerization, and fluorescence quenching by nitroxide radicals freely diffused in solution. In parallel, computational modeling studies on the location and dynamics of DNP/TEMPO spin-label (NslDNP) and StDNP guests within a model of the binding site were performed. When all the experimental evidence is considered (including data from the antibody X-ray study), one can conclude that wobbling of the Trp 91 L/Trp 96 H binding-site.bound-hapten moiety (StDNP), can be responsible for the label's nanosecond dynamics monitored by fluorescence polarization techniques. A similar conclusion may be reached as a result of data analysis on NslDNP mobility within the antibody binding site. The mobility of Trp 91 L and Trp 96 H moieties provides the induced fit needed for effective stacking and release of the DNP epitope. Analysis of the above-mentioned data allows one to explore the mechanism of the probe's movement within the binding site and enables one to discuss the local dynamics of the binding site region. The combined fluorescence-photochrome approach can be used for investigation of local medium molecular dynamics in the immediate vicinity of specific sites of proteins and nucleic acids, as well as for other biologically important structures and synthetic analogues.

Heterogeneity in the structural dynamics of Sulfolobus solfataricus beta-glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry.

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.

Effect of ionic strength on the binding of ascorbate to albumin.

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.

A fluorescent-photochrome method for the quantitative characterization of solid phase antibody orientation.

A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis-trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site. Furthermore, a theoretical method for the determination of the depth of immersion of the fluorescent label in a two-phase system was developed. The model exploits the concept of dynamic interactions and is based on the empirical dependence of parameters of static exchange interactions on distances between exchangeable centers. In the present work, anti-dinitrophenyl (DNP) antibodies and stilbene-labeled DNP were used to investigate three different protein immobilization methods: physical adsorption, covalent binding, and the Langmuir-Blodgett technique.