Determination by fluorescent assay

Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

Analytical Procedure. Method for Determination of AA. Residual AA was determined immediately after irradiation by spectrophoto-fluorometry. In general the solutions were acidified to pH = 2.8 and analyzed using AeXit = 342 and Aemit = 428 m/x. To avoid quenching of the fluorescence because of AA, the irradiated solutions were diluted to an initial concentration of 2 X 10"5M AA. In experiments with nitrate, residual AA was determined by two different spectrophotofluorometric procedures. In the first method, carried out as described above, it was necessary to correct for a decrease in fluorescence caused by radiation induced nitrite which slowly diazotized AA. This reaction was followed with time and AA at the time of acidification was determined by extrapolation. In the second method, AA was determined by fluorescence assay of the diluted neutral solution (AeXit = 290 m/x and Aemit = 340 m/x). The radiation products (aniline 3-hydroxy- and 5-hydroxyanthranilic acid) did not interfere in either method, and both gave the same value for residual AA in the irradiated solutions (33). 

Figure 4. X-ray induced formation of o-, m- and p-hydroxybenzoic acid as a function of dose. Samples were flushed with either N2, 02, or N20, before and during irradiation. The hydroxybenzoic acids formed were determined by fluorescence assay (10, 33). In the case of p-hydroxybenzoic acid, an internal standard was added to the irradiated sample. In the experiments with H202 (4.4 X 10 3M) the solution was flushed with N2 the yields were corrected for ultraviolet induced hydroxylation as described in the text...

After extraction, the urethanated films were subjected to alkaline hydrolysis of urethanes to liberate the corresponding amines, while the adipoylated films were hydrolyzed after having reacted with 7-hydroxycoumarin. Amounts of the released amines and coumarin were determined by fluorescence spectroscopy as described in the Experimental section. Since aniline as well as butylamine has no appreciable fluorescence by themselves, their fluorescence assay was made after reacting with o-phthalaldehyde in the presence of mercaptoethanol. In Figure 3, where relative fluorescence intensities are plotted as a function of concentrations of amines and hydroxycoumarin, one can see that the fluorescence intensities vary linearly with their concentration to permit us the quantitative determination of extremely small amounts of amines and hydroxycoumarin. 

To estimate the conformations of (I) and (II) at the enzyme active site of fungi or plants, IR and H-NMR spectra of the azole compounds in solutions were measured. From the results of mode of action and binding assay, (I) and (II) are considered to locate in the close proximity to the prosthetic porphyrin group of cytochrome P-450 enzymes. The polarity of macromolecules close to the porphyrin moiety of apohemog1obin has been determined by fluorescence study to be similar to that of n-octanol (1 0)> In our study, carbon tetrachloride and deuterioch1 oroform of which polarities were similar to that of n-octanol were used. 

Table I describes several of the fluorescent assays that have been used in our lab to study neutrophil activation. Fluorescein-labeled W-formylhexapeptide (FLPEP) has been used to characterize the ki- netics of ligand binding, dissociation, and internalization at 37°C (7,8). FLPEP is added to a suspension of cells, then receptor-bound and free FLPEP in solution are distinguished by adding antibody to fluorescein. This is a high-affinity antibody which binds free FLPEP within 1 s hut does not bind cell-bound FLPEP. When it binds the FLPEP, it quenches the fluorescein fluorescence. Hence the residual fluorescence after antibody addition represents FLPEP that is bound to the cell. Nonspecific binding is determined in cell suspensions that contain an excess of nonfluorescent peptide.

Figure 8. Simultaneous measurement of intracellular Ca and oxidant production in neutrophils. Cells were labeled with Quin-2 and suspended at 2 x lo cells/mL buffer. At time zero, 1 nJf FLPEP was added (upper trace in each panel). In addition, the receptor blocker tBOC was added (3 x 10" M) after 30 s to stop further binding of the stimulus (lower trace in each panel). The excitation wavelength was 3A0 nm. Top panel Quin-2 fluorescence determined on channel B (of Figure 1) using a Corion A90-nm interference filter. The crossover from the superoxide assay has been subtracted. Middle panel Oxidant production (superoxide equivalents) determined by the para-hydroxyphenylacetate assay. Fluorescence was observed at AOO nm (on channel A of Figure 1).

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

Five anticonvulsants including valproic acid were determined by the Abbott TD x fluorescence polarization immunoassay automatic analyzer. Recoveries were 94.8-106% and the coefficients of variations were 1.0-9.7% [23], Fluorescence polarization immunoassay and enzyme immunoassay were compared for the determination of free valproic acid in serum [24], Good correlation (R = 0.9992) was obtained between the two assays. Higgins [25] reported on the determination of valproic acid in serum by enzyme immunoassay with use of EMIT reagents and the Abbot ABA-200 analyzer. Responses were rectilinear up to 150 mg/L. 

For biological assays, lipidated peptides embodying a fluorescent label like the bimanyl- and the NBD-group, are required for determining membrane binding or subcellular distribution by fluorescence spectroscopy and fluorescence microscopy. Also, attachment of a biotin group allows research-... 

Intensive effort has been devoted to the optimization of CCP structures for improved fluorescence output of CCP-based FRET assays. The inherent optoelectronic properties of CCPs make PET one of the most detrimental processes for FRET. Before considering the parameters in the Forster equation, it is of primary concern to reduce the probability of PET. As the competition between FRET and PET is mainly determined by the energy level alignment between donor and acceptor, it can be minimized by careful choice of CCP and C. A series of cationic poly(fluorene-co-phenylene) (PFP) derivatives (IBr, 9, 10 and 11, chemical structures in Scheme 8) was synthesized to fine-tune the donor/acceptor energy levels for improved FRET [70]. FI or Tex Red (TR) labeled ssDNAg (5 -ATC TTG ACT ATG TGG GTG CT-3 ) were chosen as the energy acceptor. The emission spectra of IBr, 9, 10 and 11 are similar in shape with emission maxima at 415, 410, 414 and 410 nm, respectively. The overlap between the emission of these polymers and the absorption of FI or TR is thus similar. Their electrochemical properties were determined by cyclic voltammetry experiments. The calculated HOMO and LUMO... 

Aboul-Enein and Al-Duraibi (1998) employed dansyl chloride in a fluorescence assay for PUT, SP, SPD, and their acetylated derivatives by ion-pair reverse-phase chromatography. This assay could be applied to the separation of free and acetylated polyamines in biological samples. Dansyl chloride has also been used as the fluorescence reagent in the determination of polyamines in urine by Molins-Legua and colleagues (1999). Derivatization was carried out within the C18 cartridges that were used during the SPE extraction procedure. Recoveries were 80-95% for all four polyamines analyzed and the hmit of detection was 10 ng/ml. 

Absolute values for transporter abundance (Bmax) have been obtained with this assay by using fluorescent calibration beads to convert fluorescence signals to MESF (Wiley et al., 1994), or by calibrating the assay with a standard cell population in which Bma was determined by radioligand binding (Gati et al., 1997). Figure 13-4 illustrates an extension of the SAENTA-fiuorescein assay in which the correlation of es transporter content with the expression of immunophenotypic markers was tested in a clinical sample. 

The initial mixture and each time point are then assayed for doxorubicin and lipid. Lipid concentrations can be quantified by the phosphate assay (see above) or by liquid scintillation counting of an appropriate radiolabel. Doxorubicin is quantified by an absorbance assay (see below). The percent uptake at any time point (e.g., t = 30 minutes) is determined by %-uptake = [(D/L), =30minutes] x 100/[(D/L) inuiai]. Doxorubicin can be assayed by both a fluorescence assay and an absorbance assay, but we find the latter to be more accurate. The standard curve consists of four to five cuvettes containing 0 to 150 nmol doxorubicin in a volume of 0.1 mL samples to be assayed are of the same volume. To each tube is added 0.9 mL of 1% (v/v) Triton X-100 (in water) solution. For saturated lipid systems such as DSPC/Chol, the tubes should be heated in a boiling water bath for 10 to 15 seconds, until the detergent turns cloudy. Samples are allowed to cool, and absorbance is read at 480 nm on a UV/Visible spectrophotometer. 

Recently, a SAMDI-MS assay was described by means of which endogenous caspase protease activities in cell lysates can be determined [26], Similar to the assay used to determine anthrax lethal factor inhibitors, peptide substrate SAMs for either caspase-3 or -8 were treated with cell lysates. In contrast to fluorescence assays, also longer peptide substrates could be used, thus enabling a better resolution of the two caspase activities. 

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