Spectroscopic assays

In this paper we review the fluorescence and spectroscopic assays commonly used in our lab to study neutrophil activation. The advantages of these assays are threefold. (1) These assays are very sensitive. (2) These are kinetic assays which can be measured with a time resolution of 1 s or less. This is important since many of these responses occur within seconds of stimulation and are... 

We will first summarize the fluorescence and spectroscopic assays that have been developed for the fluorometer and then describe their applications using flow cytometry. We will summarize research which exemplifies the utility of simultaneous measurement of responses and shows how these methods have provided Information about the signal transduction pathways and activation in neutrophils. 

The use of the perchlorate anion to precipitate the cobalt complex to determine the yield [1] is deprecated on the grounds of potential hazard [2], though it was not found possible to cause the salt to detonate by pounding, but it will bum if ignited. A spectroscopic assay method is suggested as an alternative to precipitation [3],... 

All three enzymes have the advantage of being subject to convenient spectroscopic assays. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

To begin with, we should remind the reader that the lack of 7t-conjugation of the mefa-substituted triads turned out to be an advantage in the subsequent spectroscopic assays. In other words, the fact that the only significant absorption stems from Cgo and exTTF opened the opportunity to test a large range of concentrations to study and follow the evolution of (exTTF-oMPE -Cgo) hybrids by spectroscopic means. 

Spectrophotometric methods are, of course, not restricted to assays based on the NAD(P)+/NAD(P)H pair of coenzymes, and there are many natural and synthetic substrates whose reactions can be assayed in this way. The p-nitrophenyl group forms the basis of many convenient spectroscopic assays, for example for the enzyme glucuronidase ... 

The techniques to follow aggregation of amyloidogenic proteins and to determine their molecular conformation range from spectrometric assays (thiohavin T, Congo red) over spectroscopic assays (FTIR and CD) and visualization techniques (AFM and TEM) to methods that provide detailed insight into the atomic coordinates (solid-state NMR and X-ray diffraction). 

Spectroscopic assay obscured by high concentrations of acetic acid. 

The DNA lesion 8,5 -cyclo-2 -dG, formed by attack of hydroxyl radicals, contains damage to both base and sugar, and is therefore repaired by nucleotide excision repair enzymes, and is involved in diseases with defective nucleotide excision repair. A mass spectroscopic assay has been developed for the quantitation of the lesion after enzymatic separation of the 5 (R) and 5 (S) isomers. The thermodynamic stability of ODNs containing the oxidative lesion, 2-hydroxy-dA has been examined. It was shown that when the lesion was in the middle of a DNA duplex it behaved as a universal base, in that there was no dilference in Tm when opposite any of the canonical bases. On the other hand, when it was near the termini, there was a preference for base pairing with thymidine, but it also formed base pairs with other nucleotides which was sequence dependent. The extent of oxoprenylation by malondialdehyde or adenine propenal has been investigated in DNA, see (139). ssDNA was found to be more sensitive to oxoprenylation, and supercoiled-DNA more susceptible than linearised plasmid DNA. A variety of intercalators were used, some of which inhibit oxoprenylation, e.g. netropsin, whilst others, like ethidium bromide, caused enhanced oxoprenylation. 

Authentic Materials Authentic materials (AM) are reference standards, which are qualified for identity and approximate purity. The chromatographic purity of an AM need only be 80% or greater. They are not used in quantitative assays but are generally used to establish chromatographic system suitability and as identity comparators for spectroscopic assays. Authentic materials are usually available in small amounts and are frequently obtained by preparative chromatography. 

G. E. Ritchie, E. W. Ciurczak, H. L. Mark, Validation of a Near-Infrared Transmission (NIT) Spectroscopic Assay of Sustained Release Prescription Analgesic Tablets, Proc. PittCon, New Orleans, March, 2000. 

The two phases are thoroughly mixed to give buffer-saturated octanol in the top phase and octanol-saturated buffer in the bottom. Once the two phases have separated (this can take a while), the drug is added and the whole flask is shaken mechanically for at least an hour. The two phases are allowed to separate (or centrifuged, if you are in a hurry) and the concentration of drug in the aqueous phase is then determined. This may be done by titration if the drug is sufficiently acidic or basic or, more usually, spec-trophotometrically The concentration in the octanol phase is found by subtraction and the value of P is calculated. This method works perfectly well if there is sufficient sample and the drug possesses a chromophore to allow spectroscopic assay of the aqueous phase. 

The most important part of any spectroscopic assay is not the performance of the spectrophotometer (although the accuracy of the instrument is checked periodically). The crucial part of any experiment is the accurate preparation of the test and standard solutions. This often involves the accurate dilution of a stock solution using the volumetric glassware introduced in Chapter 6, namely the pipette and the volumetric flask. 

Ritchie, G. E., Ciurczak, E. W, and Mark, H. Validation of a near-infrared transmission (NIT) spectroscopic assay of sustained-release prescription analgesic tablets. In Pittsburgh Conference of Analytical Chemistry and Applied Spectroscopy, March 2000, New Orleans, LA. 

Some of the spectroscopic assays used to quantitate lipid transfer provide a means of continuously measuring the transfer without destroying the incubation mixture. In addition, the rate of lipid transfer between two nearly identical membranes can be measured. 

Competition-in-solution assays can quantify the effect of library components on the interaction between components of a protein complex. These assays include traditional biochemical assays that measure interactions through antibody- or tag-mediated pull-down or label displacement assays, along with biophysical methods that measure changes in the property of a component upon binding to its partner protein, such as fluorescence spectroscopic assays and SPR. It is essential for assay development that the requirements (affinity and binding site) for assembly of the interaction partners be at least partially defined such that a tractable model system for the target interaction can be developed, as was the case for MDM2/p53, IL-2/IL2-aR and Bcl/Bak [138-140],... 

Calculate the mean and standard deviation for each set of data using =AVERAGE(range) and =STDEV(range). For the spectroscopic assay the values of the mean and standard deviation are 1.852 and 0.085 mM, respectively, and for the enzyme electrode they are 1.550 and 0.212mM, respectively. These are plotted for comparison in figure 3.8. 

Determine the concentration of glucose in a wine sample and its associated uncertainty as a 95% confidence interval by the standard addition method using the enzyme spectroscopic assay of example 5.1. An aliquot of 200 pL of wine is added to a 5mL volumetric flask to which the reagents for the enzyme assay are added and then the flask is filled to the mark with buffer. The... 

Once the transient species has been formed, it has to be monitored by some form of kinetic spectroscopy, typically with ultraviolet-visible absorption or emission, infrared (time-resolved infrared or TRIR) (74), or resonance Raman (time-resolved resonance Raman or TR3) (80) methods of detection. The transient is usually tracked by a probe beam at a single characteristic frequency, thereby giving direct access to the kinetic dimension. Spectra can then be built up point by point, if necessary, with an appropriate change of probe frequency for each point, although improvements in the sensitivity of multichannel detectors may be expected to lead increasingly to the replacement of the laborious point-by-point method by full two-dimensional methods of spectroscopic assay (that is, with both spectral and kinetic dimensions). 

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