Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.     

Comparison of fluorescence characteristics of products of peroxidation of membrane phospholipids with those of products derived from reaction of malonaldehyde with glycine as a model of lipofuscin fluorescent substances

The fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to product (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorescent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.     

Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide

Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500-530 nm; emission, 590-620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed.     

The correction of reaction rates in continuous fluorometric assays of enzymes

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D-Arg-NH(2), was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at microM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10(-4) x s(-1) were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH(2), were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was -2.96 x 10(-4) a.u microM(-1) x s(-1) that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate]/(final-initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K(m) and overestimation of V(max).     

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.     

Flow cytofluorometric analysis of enzyme reactions based on quenching of fluorescence by the final reaction product: detection of glucose-6-phosphate dehydrogenase deficiency in human erythrocytes

We developed a method for accurate cytofluorometric analysis of the final reaction product of enzyme reactions in individual cells. Glucose-6-phosphate dehydrogenase (G6PD) activity in human erythrocytes was demonstrated cytochemically, and the amount of final reaction product (formazan) per cell was detected indirectly by quenching of autofluorescence generated by glutaraldehyde fixation. Formazan quenches fluorescence in a dose-dependent manner. The method has been used for detection of G6PD deficiency. Heterozygous and homo(hemi)zygous deficiency could easily be established, even in cases of extreme "Lyonization" where microscopic inspection failed to discriminate between either normal individuals and heterozygously deficient patients or heterozygously and homozygously deficient patients. The principle of quenching of fluorescence by final reaction products of enzymes can be applied to flow cytofluorometric analysis of enzyme activity in individual cells in general.     

Spectrofluorometric determination of lipase activity.

A fluorescent triglyceride, 1(3)-pyrenylbutanoyl-2,3(1,2)-dipalmitoyl-sn-glycerol, was synthesised, characterised by NMR and mass spectrometry, incorporated into a lipid emulsion and used as a fluorescent substrate for pancreatic lipase. It is shown that the product of the reaction, pyrene butyric acid, diffuses into the aqueous phase resulting in a decrease in the excimer fluorescence of the pyrene fluorophore in the emulsion and an increase in its monomer fluorescence. The phenomenon can be used to assay the enzyme thereby cirumventing the need to extract the fatty acid product.     

Dual-enzyme assay of glutamate in single cells based on capillary electrophoresis

A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Glutamate dehydrogenase and glutamic pyruvic transaminase were used to catalyze the glutamate reaction. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. Glutamate dehydrogenase catalyzed the formation of NADH, and glutamic pyruvic transaminase drives the glutamate dehydrogenase reaction by removing a reaction product and regenerating glutamate. The detection limit of glutamate is down to the 10⁻⁸ M level, which is 1 order of magnitude lower than previously reported detection limits based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most amino acids. The glutamate content in single human erythrocytes and baby rat brain neurons were determined with this method and the results agreed well with literature values.     

Investigation of catalytic reaction with spectrofluorimetric method

Numerous derivatives of nicotinic acid hydrazids are suitable for sensitive fluorescence determination of metal ions. The reaction proceeds upon ultraviolet illumination in the presence of an oxidizing agent and catalyst forming a fluorescent product. Therefore, the catalyst metal ion can be quantitatively determined by fluorescence methods. We have studied the reaction of nicotinic acid (di-pyridin-2-yl-methylene)-hydrazide (NADPMH), catalyzed by Mn(II)ion, and determined the optimal parameters of metal ion method.     

Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase

When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATP-Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001-1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high-performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min-1 at pH 8.3, 25 degrees C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.     

Fluorescence detection of enzymatically formed hydrogen peroxide in aqueous solution and in reversed micelles

A sensitive enzymatic assay for oxidase reactions both in aqueous solution and in hexadecyltrimethylammoniumbromide (CTAB) reversed micelles has been developed. The assay is based on the fluorescence detection of dichlorofluorescein, which is formed by hydrogen peroxide oxidation of the nonfluorescent precursor dichlorofluorescin. Hydrogen peroxide as product of the reaction catalyzed by glucose oxidase served to select the reaction conditions. The reaction rate is distinctly enhanced in CTAB reversed micelles as compared to the rate in aqueous solution. This effect, combined with the high sensitivity owing to the strong fluorescence of dichlorofluorescein, makes the assay attractive for the detection of low enzyme, substrate, or peroxide concentrations.     

Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis

The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the double-rolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C(75)) with a pK(a) close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5'-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G(76) of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP(76) is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP(76) becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C(75) becomes positioned for reaction chemistry only along the trajectory from precursor to product.     

Spectroscopic studies of 1,2-diaminoanthraquinone (DAQ) as a fluorescent probe for the imaging of nitric oxide in living cells.

Spectroscopic analysis of the fluorescent probe 1,2-diaminoanthraquinone () provides information about the mechanism of nitric oxide imaging in living cells. Fluorescent aggregates of a reaction product of are thought to be responsible for the images obtained with confocal fluorescence microscopy.     

The use of steady-state treatment in the rapid kinetics of horse liver alcohol dehydrogenase. The effect of addition of product on the "burst" reaction.

The effect of addition of product on the amplitude of the “burst” reaction of horse liver alcohol dehydrogenase was studied using a stopped-flow spectrophotofluorimeter. The amplitude of the “burst” formation of enzyme-bound NADH fluorescence was completely diminished by the addition of excess acetaldehyde or benzaldehyde in the reaction with NAD⁺ and ethanol or NAD⁺ and benzylalcohol, respectively. The results indicate that a significant concentration of the ternary enzyme-coenzyme-substrate complex was formed during the steady-state in the presence of product, and this ternary complex did not exhibit NADH fluorescence. The dissociation constants for the ternary complex were determined from the amplitudes of the “burst” reactions. The “active site” titration of the enzyme with NAD⁺ in the presence of ethanol and iso-butyramide is also described.     

新定量PCR数据处理方法的理论探讨

日新月异的生命科学技术的发展及临床医学科学研究的需求,一般的PCR技术已远远不能满足工作的需要。PE公司在进行了大量的PCR动力学研究的基础上,发现了利用荧光标记探针在PCR循环过程中积累的荧光强度达到仪器捡出阈值时,系统的初始模板数量与循环次数之间有线性关系,据此建立了目前的PE 7700 、PE 5700仪器的定量PCR技术,开创了PCR技术的新局面。但是由于这一技术的误差较大,尚不能满足生命科学及临床医学科学研究的需求,因此需要继续研究新的定量PCR技术。PCR动力学数学模型是根据PCR 技术的原理提出的,能够准确描述PCR反应产物分子数量积累规律的动力学方程,给出了PCR产物数量或者荧光强度与初始模板数量及其他反应条件间的函数关系。利用这一关系,根据PCR反应已积累的产物数量,可以实现准确的定量PCR分析,得到初始模板数量达到定量PCR的目的。使用动力学数学模型做定量PCR分析,其结果的误差仅与使用的荧光强度数值的精确度相关。使用精确到6位数的荧光强度数据,模板数自100~1 000 000区间定量结果的准确性可达99%以上。本文根据模拟实验数据进行了初步的定量PCR分析,结果提示,目前的定量PCR仪器使用PCR动力学模型理论处理分析数据,定量分析的结果会比目前的CT值方法在准确性方面提高几十倍以上,可以满足各方面研究工作误差水平的需要。 Abstract:Today standard PCR can't satisfy the need of biotechnique development and clinical research any more.After numerous dynamic research,PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700.But the error of this technique is too great to satisfy the need of biotechnique development and clinical research.A better quantitative PCR technique is needed.The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system.This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions,and can reflect the accumulating rule of PCR product molecule accurately.Accurate quantitative PCR analysis can be made use this function relation.Accumulated PCR product quantity can be obtained from initial template number.Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used.For an example,when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1 000 000,the quantitative result accuracy will be more than 99%.The difference of result error is distinct using same condition,same instrument but different analysis method.Moreover,if the PCR quantitative analysis system is used to process data,it will get result 80 times of accuracy than using CT method.     

Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain

In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The solid reaction product has significant fluorescence that enables the use of confocal microscopy to generate high-resolution three-dimensional (3-D) imaging of gene expression.     

Biogenesis of phycobiliproteins: II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 OF beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002

The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcS-I copurified with His(6)-tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apophycocyanin, the reaction product had an absorbance maximum of 622 nm and was highly fluorescent (lambda(max) = 643 nm). In control reactions with PCB and apophycocyanin, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). Tryptic peptide mapping showed that the CpcSU-dependent reaction product had one major PCB-containing peptide that contained the PCB binding site Cys-82. The CpcSU lyase was also tested with recombinant apoHT-allophycocyanin (aporHT-AP) and PCB in vitro. AporHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of approximately 27 kDa. When aporHT-AP was incubated with PCB and CpcSU, the product had an absorbance maximum of 614 nm and a fluorescence emission maximum at 636 nm, the expected maxima for monomeric holo-AP. When no enzyme or CpcS-I or CpcU was added alone, the products had absorbance maxima between 645 and 647 nm and were not fluorescent. When these reaction products were analyzed by gel electrophoresis and zinc-enhanced fluorescence emission, only the reaction products from CpcSU had PCB attached to both AP subunits. Therefore, CpcSU is the bilin lyase-responsible for attachment of PCB to Cys-82 of CpcB and Cys-81 of ApcA and ApcB.     

Enhancement of the fluorescence and stability of o-phthalaldehyde-derived isoindoles of amino acids using hydroxypropyl-beta-cyclodextrin

Addition of hydroxypropyl-beta-cyclodextrin to o-phthalaldehyde (OPA)-amino acid-thiol reaction mixtures is shown to cause significant enhancement of the fluorescence of the isoindole product for a wide range of amino acids, with the largest effects observed in the cases of glycine and lysine. The largest enhancement observed was a factor of 2.67 in the case of the derivative of glycine. This fluorescence enhancement is the result of the formation of a 1:1 host:guest inclusion complex between the isoindole and the cyclodextrin. Relatively small association constants of 44 and 130 M(-1) were obtained for the inclusion of the derivatives of glycine and lysine, respectively. Inclusion of the isoindole derivative into hydroxypropyl-beta-cyclodextrin was also found to result in a significant stabilization of the isoindole derivatives, contrary to what has been previously reported for inclusion into beta-cyclodextrin. For example, the lifetime of the lysine derivative was found to increase from 42 to 222 min, a factor of 5.3. These results have potential applications in fluorescence-based HPLC and high-performance capillary electrophoresis amino acid analysis methods using OPA derivation. Addition of hydroxypropyl-beta-cyclodextrin to the reaction mixture results in an increase in both the fluorescence and the stability of the isoindole product, providing potentially significant improvements to the method.     

Mechanistic studies on the mononuclear ZnII-containing metallo-beta-lactamase ImiS from Aeromonas sobria

In an effort to understand the reaction mechanism of a B2 metallo-beta-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s(-)(1) and a slower rate of fluorescence regain of 98 s(-)(1). Stopped-flow UV-vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s(-)(1). These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 ms that is assigned to a reaction intermediate that has a five-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 ms. When these results are taken together, they allow for a reaction mechanism to be offered for the B2 metallo-beta-lactamases and demonstrate that the mono- and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.