Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Light-induced electron transfer in porphyrin-calixarene conjugates.

The fluorescence from a set of porphyrin-calixarene complexes is quenched upon addition of benzo-1,4-quinone (BQ) in fluid solution. In N,N-dimethylformamide solution, fluorescence quenching involves both static and dynamic interactions but there are no obvious differences between porphyrins with or without the appended calixarene. Under such conditions, the static quenching behaviour is attributed to pi-complexation between the reactants and it is concluded that the calixarene cavity does not bind BQ. An additional static component is apparent in dichloromethane solution. This latter effect involves partial fluorescence quenching, for which the intramolecular rate constant can be obtained by time-resolved fluorescence spectroscopy. The derived rate constants depend on molecular structure in a manner consistent with fluorescence quenching being due to electron transfer. In all cases, however, the dominant quenching step involves diffusional contact between the porphyrin nucleus and a non-bound molecule of BQ.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Kinetics of the rearrangement of the solvation shell of an excited fluorescent probe 4″-dimethylaminochalcone

A study was made of the processes associated with the quenching of 4″-dimethylaminochalcone (DMAC) fluorescence by proton-donor solvent (1-butanol). The kinetics of deactivation of the DMAC excited state was assessed by transient absorption spectra with a time resolution about 50 fs and by fluorescence decay with ～30-ps resolution. The following sequence of events could thus be envisaged: (i) the DMAC molecule in the ground state (prior to excitation) makes a hydrogen bond with an alcohol molecule; (ii) absorption of a light quantum causes a corresponding increase of the DMAC dipole moment; the H-bond is retained; (iii) the solvation shell formed by alcohol dipoles is reorganized in response to the raise of the DMAC dipole moment, with an energy expenditure about 24 kJ/mol and a time constant about 40 ps; the initial H-bond is still retained; (iv) processes leading to fluorescence quenching occur with an effective time constant of nearly 200 ps. Since quenching is far slower than solvate rearrangement, one can suppose that it is not a direct consequence of shell relaxation or prior H-bonding. Thus, DMAC fluorescence quenching may involve different processes observed with other aromatic molecules: H-bond rearrangement from a nonquenching to a more ‘efficient’ conformation, charge transfer between the excited molecule and alcohol, or solvent-induced out-of-plane twist of the DMAC amino group.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.     

Molecular interaction of some cardiovascular drugs with human serum albumin at physiological‐like conditions: A new approach

In the present study, the interaction of human serum albumin (HSA) with some cardiovascular drugs (CARs) under physiological conditions was investigated via the fluorescence spectroscopic and Fourier transform infrared spectroscopy. The CAR included Captopril, Timolol, Propranolol, Atenolol, and Amiodarone. Cardiovascular drugs can effectively quench the endogenous fluorescence of HSA by static quenching mechanism. The fluorescence quenching of HSA is mainly caused by complex formation of HSA with CAR. The binding reaction of CAR with HSA can be concluded that hydrophobic and electrostatic interactions are the main binding forces in the CAR‐HSA system. The results showed that CAR strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and nonradiation energy transfer happened within molecules. Fourier transform infrared spectroscopy absorption studies showed that the secondary structure was changed according to the interaction of HSA and CAR. The binding reaction of CAR with HSA can be concluded that hydrophobic and electrostatic interactions are the main binding forces in the CAR‐HSA system. The results obtained herein will be of biological significance in pharmacology and clinical medicines.     

Kinetics of rearrangement of solvation sheath of an excited molecule of the fluorescent probe 4"-dimethylaminochalcone

Processes accompanying the quenching of the fluorescent probe 4"-dimethylaminochalcone by hydroxyl groups of the proton-donor solvent 1-butanol have been studied. The kinetics of the deactivation of the excited state of 4"-dimethylaminochalcone has been monitored from the transition absorption spectra at a time resolution of 50 fs and fluorescence decay at a time resolution of 30 ps. The data obtained allow thinking that the next picture occurs in 1-butanol. At first stage, the 4"-dimethylaminochalcone molecule in its ground state forms a hydrogen bond with an alcohol molecule. At the second stage, the absorption of light quantum and corresponding rise of the dipole moment of 4"-dimethylaminochalcone take place, the initially existing hydrogen bond is retained. The third stage consists in the rearrangement of the 4"-dimethylaminochalcone solvation shell formed by alcohol dipole molecules due to an increase of the dipole of moment 4"-dimethylaminochalcone; this rearrangement takes an energy of about 24 kJ/mol, the arrangement time constant is close to 40 ps; the initial hydrogen bond is retained. The fourth stage involves processes that lead to fluorescence quenching; their time constant is about 200 ps. Taking into account that the quenching is a much slower process than the relaxation of the solvation shell, it was supposed that the quenching is not a direct consequence of the solvation shell relaxation or the existence of the hydrogen bond formed prior to excitation. Then the fluorescence quenching of 4"-dimethylaminochalcone can be accomplished through some other processes that are observed in other fluorescent molecules: (a) rearrangement of the initial hydrogen bond from a conformation that cannot quench the fluorescence of 4"-dimethylaminochalcone to a more "effective" conformation, (b) charge transfer between the excited of molecule 4"-dimethylaminochalcone and alcohol, or (c) solvent-induced twist of the 4"-dimethylaminochalcone amino group (its withdrawal from the molecule plane) by the action of the solvent.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Interaction of carbon nanotubes with curcumin: Effect of temperature and pH on simultaneous static and dynamic fluorescence quenching of curcumin using carbon nanotubes

Curcumin (Cur) has medicinal properties, undergoes hydrolysis, and has low water solubility that limits its bioavailability and industrial usage. Different host molecules such as carbon nanotubes (CNT) can be useful in improving solubility and stabilizing Cur, therefore understanding the interaction of Cur with host molecules such as CNT is crucial. In this study, UV–visible light absorption and fluorescence spectroscopic techniques have been applied to reveal the interaction of Cur with CNT. Visible light absorption of Cur increases with CNT concentration, whereas fluorescence intensity of Cur is quenched in the presence of CNT. The obtained results confirm that fluorescence reduction is due to both static and dynamic quenching and is a result of the ground state and excited‐state complex formation. The pH environment influences the quenching rate due to deprotonation of Cur at higher pH; excess OH‐ ion concentration in the solution further discourages electrostatic interaction between the deprotonated form of Cur with CNT. It is found that at lower temperatures (<35°C) dynamic quenching is much more dominant and at higher temperatures (45°C) static quenching is more dominant. The interaction is further supported using X‐ray diffraction patterns and Fourier transform infrared spectra in the solid state, and suggests encapsulation of curcumin within the CNT. It is further evident that fluorescence quenching of Cur using CNT is further enhanced in the presence of several salts, as increase in ionic strength of the solution pushes the hydrophobic Cur molecule towards the CNT core by increasing the proximity between them.     

Use of oxonol V as a probe of membrane potential in proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Absorbance changes in the anionic dye bis[3-phenyl-5-oxoisoxazol-4-yl]pentamethineoxonol (oxonol V) can be used to monitor the membrane potential of liposomes and cytochrome c containing cytochrome oxidase proteoliposomes (c-loaded COV). Diffusion potentials (positive inside the vesicles) cause an increase in the dye extinction, with a maximum at 640 nm. A similar increase is seen upon energization of internally facing cytochrome oxidase molecules in c-loaded COV. Both "passive" and "active" responses are only seen when the dye is fully bound to the vesicle membrane. Calibration curves using potassium or n-butyltriphenylphosphonium ion (BTPP+) diffusion potentials are linear up to 100 mV and pass through the origin. Diffusion potentials (positive inside) also cause an increase and red shift in the oxonol V fluorescence emission spectrum. However, potentials of the same sign induced by cytochrome oxidase turnover induce a large fluorescence quenching in c-loaded COV. A similar anomaly has been observed with submitochondrial particles [Smith, J. C., Russ, P., Cooperman, B. S., & Chance, B. (1976) Biochemistry 15, 5094-5105]. A model is proposed consistent with these responses. It is suggested that the dye molecules move further into the membrane phase upon energization, causing the absorbance increase. In the presence of active enzyme, anionic dye molecules are attracted to a positive dipole on each enzyme molecule, causing self-quenching of the fluorescence.     

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.     

Interactions of sulindac and its metabolites with phospholipid membranes: An explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO?) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of Sulindac and its metabolites was tested and related with their membrane interactions. Drug–membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.     

Interactions of sulindac and its metabolites with phospholipid membranes: an explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO*) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of sulindac and its metabolites was tested and related with their membrane interactions. Drug-membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.     

Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol

Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.     

Kinetic Theory Model for Ion Movement through Biological Membranes: I. Field-Dependent Conductances in the Presence of Solution Symmetry

A model for ion movement through specialized sites in the plasma membrane is presented and analyzed using techniques from nonequilibrium kinetic theory. It is assumed that ions traversing these specialized regions interact with membrane molecules through central conservative forces. The membrane molecules are approximated as massive spherical scattering centers so that ionic fractional energy losses per collision are much less than one. Equations for steady-state membrane ionic currents and conductances as functions of externally applied electric field strength are derived and numerically analyzed, under the restriction of identical solutions on each size of the membrane and constant electric fields within the membrane. The analysis is carried through for a number of idealized ion-membrane molecule central force interactions. For any interaction leading to a velocity-dependent ion-membrane molecule collision frequency, the membrane chord conductance is a function of the externally applied electric field. Interactions leading to a collision frequency that is an increasing (decreasing) function of ionic velocity are characterized by chord conductances that are decreasing (increasing) functions of field strength. For ion-neutral molecule interactions, the conductance is such a rapidly decreasing function of field strength that the slope conductance becomes negative for all field strengths above a certain value.     

Localization of adriamycin in model and natural membranes. Influence of lipid molecular packing

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster ovary cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2-APam, 7-ASte, 12-ASte and anthracene-phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Egg-yolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems. Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0-100 microM, the efficiency of energy transfer was 12-ASte greater than 7-ASte greater than 2-APam, with 100% energy transfer for 12-ASte above a drug concentration of 30 microM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7-ASTe greater than 12-ASte = 2-APam was observed. Measurements of fluorescence quenching using the non-permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer. Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains. In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of these cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments. These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.     

Detection of C677T mutation of MTHFR in subject with coronary heart disease by hairpin probe with enzymatic color on microarray

Molecular beacon (MB) is especially suited for detection of single nucleotide polymorphism (SNP), and the type of MB immobilized on the surface of microarray in particular, may detect multi-sample and multi-locus. However, the majority of MB needs to be labeled with fluorescence and quenching molecules on the two ends of the probe, and observed the reaction of fluorescence or complicated electrochemical signal produced hybridization of MB and target sequence by complex and expensive instruments. The "molecular beacon" and microarray designed appropriately in our study can produce visible light response signal induced by amplification effect of enzymatic color, and are avoided with the marker of fluorescence and quenching molecules and expensive instruments. The "molecular beacon" without fluorescence and quenching molecules is entitled as "hairpin DNA probe" by us for only the "hairpin" structure of traditional molecular beacon is adopted. The merits of two techniques, molecular beacon and amplification effect of enzymatic color, are successfully combined, and the technique is simple, sensitive and specific, to detect and compare the methylenetetrahydrofolate reductase (MTHFR) Gene C677T mutation of subjects between coronary heart disease (CHD) and control group. The results showed that MTHFR Gene C677T polymorphism is an independent risk factor for CHD.     

Three-dimensional molecular modeling with single molecule FRET

Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.     

The susceptibility of membrane vesicles to interaction with ethanol

The susceptibility of membranes to interaction with ethanol is an important consideration in the further understanding of the ethanol-membrane interaction. Interaction of membrane vesicles, including passive diffusion of ethanol across membranes, leakage of internal molecules out of membranes and membrane-membrane interaction, were examined systematically using two populations of fluorescent probe-encapsulated phospholipid bilayer vesicles, each prepared with 1,2-dimyristoyl phosphatidylcholine, cholesterol and a fluorescent probe. Fluorescence quenching experiments with these vesicles were performed in a medium containing a wide range of ethanol concentrations (0.30-3.5 M). In the presence of a lower concentration of ethanol in the external medium, passive diffusion of ethanol across membrane vesicles occurred. This was demonstrated by an interaction of ethanol with the encapsulated fluorescence probe molecules inside the vesicles, resulting in an increase in the fluorescence intensity and a shift of the fluorescence emission spectrum to a shorter wavelength. While, in the presence of a higher concentration of ethanol in the external medium, a strong perturbation of lipid bilayers by ethanol was found, leading to an over expansion of membranes and consequently causing the membrane leakage. As a result of this, the initially encapsulated probe molecules leaked out of the vesicles so as to interact with the other probe molecules in the external medium. Consequently, fluorescence quenching was observed. Moreover, studies of the mixture of two populations of fluorescence probe-encapsulated membrane vesicles revealed that ethanol acted on individual membranes and did not promote membrane-membrane interactions. The implication of the present results to the alcohol-mediated expansion of membranes is discussed.