Extinction coefficient protein

A problem associated with the use of UV-Vis detection with SEC is the relatively low or missing response for such DOM/HM moieties, which possess no UV chromophore or widely differing low extinction coefficients (proteins, sugars, amino sugars, aliphatic acids, etc.) which, however, are more or less ubiquitous components of aquatic NOM. Different kinds of "homemade" online DOC detectors have been constructed [2,23,46] to improve the accuracy of the SEC methods in the water research. An advantage of the DOC detection is that the signal is directly proportional, irrespective of functionality, to the concentration of different organic carbon species. The DOC detector can be coupled in tandem with other detection systems, such as UVA and fluorescence, to record a multidimensional data set for characterization of the EXDM/HM distribution in different environmental systems [47]. 

Extinction Coefficients and Ellipticities of the Rieske Protein from Bovine Heart bc Complex (ISF) and of the Rieske-Type Ferredoxin from Benzene Dioxygenase (FiIbed)... 

D. gigas AOR was the first Mo-pterin-containing protein whose 3D structure was solved. From D. desulfuricans, a homologous AOR (MOD) was purified, characterized, and crystallized. Both proteins are homodimers with-100 kDa subunits and contain one Mo-pterin site (MCD-cofactor) and two [2Fe-2S] clusters. Flavin moieties are not found. The visible absorption spectrum of the proteins (absorption wavelengths, extinction coefficients, and optical ratios at characteristic wavelengths) are similar to those observed for the deflavo-forms of... 

A bright cyan-green fluorescent protein was isolated from Clavu-laria coral [86]. Since one of the intermediates displayed fast bleaching, a screen for more photostable variants was performed. The optimized monomeric variant was named teal fluorescent protein 1 (mTFPl). It has an excitation and emission maximum at 462 and 492 nm, respectively, so this protein is spectrally located in between CFP and GFP. With an extinction coefficient of 64,000 M 1 cm-1 and a quantum yield of 0.85 mTFPl is a very bright fluorescent protein. 

Fluorescein-5-maleimide is slighdy soluble in aqueous solutions above pH 6 ( lmM concentration). It may be dissolved in DMF at higher concentrations and a small addition of this solution made to an aqueous reaction mixture to initiate labeling. Do not exceed 10 percent DMF in the reaction buffer to avoid protein precipitation. At pH 8, the reagent has an extinction coefficient at 490nm of about 78,000M 1 cm-1. 

Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar extinction coefficient for the chromogenic group (e = 29,000 M-1cm-1) to determine the concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide the molar concentration of biotin by the molar concentration of protein present (which may be determined by using the Coomassie assay or the BCA assay methods). 

Cytochrome Determinations. Microsomal suspensions (1-5 mg protein/ ml) were assayed for cytochromes bs and P-A50 (12) using a Cary 15 spectrophotometer operated at room temperature (20-23°C). Suspensions in 0.05M phosphate buffer, pH 7.A, were contained in 3 ml cuvettes with a 1 cm path length. Sodium dithlo-nite was the reductant. The extinction coefficient of 171 mM 1 cm 1 was applied to the A28-A90 nm absorbance increment. 

The aromatic amino acids each have two major absorption bands in the wavelength region between 200 and 300 nm (see reviews by Beaven and Holiday(13) and Wetlaufer(14). The lower energy band occurs near 280 nm for tryptophan, 277 nm for tyrosine, and 258 nm for phenylalanine, and the extinction coefficients at these wavelengths are in the ratio 27 7 l.(14) As a result of the spectral distributions and relative extinction coefficients of the aromatic amino acids, tryptophan generally dominates the absorption, fluorescence, and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids. 

The direct absorbance methods require only a protein-specific extinction coefficient to deliver an accurate protein concentration. These methods typically require minutes to perform and require only a spectrophotometer and a good quantitative... 

Determination of the extinction coefficient is a relatively straightforward task. The target protein is diluted to give live different concentrations. These samples are then divided into two aliquots. Amino acid analysis (AAA) accurately determines the protein concentration of one set of samples at the live concentrations, and the absorbance at 280 nm (A280) is measured for the other set of samples. The slope of a plot of A2s0 vs. protein concentration by AAA yields the extinction coefficient. 

As soon as the protein is activated with the heterobifunctional crosslinker, the extinction coefficient determined for pure Amb a 1 no longer applies because the heterobifunctional crosslinker absorbs at 280 nm. At this step in the production of AIC, the manufacturing overhead cost requires the use of a fast protein assay, whereas the exact stoichiometry of the subsequent reaction dictates the use of an accurate and precise method. Hence we developed a new extinction coefficient for the activated protein based on experimental data and demonstrated that within the normal activation range of 9 to 12 crosslinkers per Amb a 1, the new extinction coefficient remained constant. The concentration of the purified activated Amb a 1 determined by this direct absorbance A280 method is more precise and accurate than could be assigned by a colorimetric assay. Consequently, the activated Amb a 1 concentration allows for the accurate addition of 1018 ISS required to consistently produce AIC with optimal activity. 

A number of investigators have studied the effect of ozone on the ultraviolet absorption spectra of proteins and amino acids. A decrease in the absorption of 280-nm light in a number of proteins was originally reported ly Giese et aV to be a consequence of ozone exposure they suggested that this was due to an interaction of ozone with the ring structures of tyrosine and tryptophan. Exposure of a solution of tryptophan to ozone resulted in a decrease in 280-nm absorption, whereas the extinction coefficient of tyrosine increased. Similar results with tyrosine were reported by Scheel et who also noted alterations in the ultraviolet spectra of egg albumen, perhaps representing denaturation by ozone. 

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