Extinction coefficient nitrophenol

Another proton transfer studied by the E-jump technique in acetonitrile (42) is that between p-nitrophenol (AH) and tri-ethylamine (B). The extinction coefficients for each of the species in the following equilibrium have been measured by Kree-voy and Liang (3) ... 

Determine the absorbance of each dilution at 420 nm in a 1-cm path-length cell, with a suitable spectrophotometer, using water as the blank. For each dilution, plot absorbance against pmol of o-nitrophenol (this must result in a straight line through the origin). Divide the absorbance of each dilution by pmol of o-nitrophenol to obtain the extinction coefficient (M) at that dilution (the slope of the line is the extinction coefficient). Average the seven values thus calculated (this should result in a value of 4.60 0.05). 

Using an extinction coefficient for p-nitrophenol of 18.8 x 10 cm /mole, convert all the absorbance readings to p.moles p-nitrophenol formed. 

Procedure Take 0.5 ml. of o-nitrophenyl fl-D-galactoside solution, 0.125 ml. of the salt solution, 0.1-0.5 ml. of the enzyme solution, and make to 2.5 ml. with buffer. The buffer, sodium chloride, and substrate solutions are measured into a photometer cell (d — 1 cm.), and brought to the required temperature (20°). The reaction is started with enzyme and is followed by noting the optical density at 405 m/i (against water) every 30 seconds. The increase of optical density (AE) in 2 minutes is taken for calculation of the turnover number. The molecular extinction coefficient of o-nitrophenol at 405 mp and pH 7.6 is 3.1 X 10 cm /mmole. ... 

M) in pyridine (7 mL) for 24 h at 25°C. The carboxylic derivative is then washed with methanol (four times) and ether. Analysis for the extent of carboxylation involves a two-step procedure. An accurately weighed aliquot is treated with DCC and p-nitrophenol in pyridine. After several washings with THF to remove uiu eacted p-nitrophenol, piperidine-pyridine (1 9) is added to the silica gel, and the amount of p-nitrophenol released is measured at A = 410 nm by using 1.57 X lO" cm as the extinction coefficient of p-nitrophenoxide. The incorporation of carboxylic acid is 200 /u-mol/g. 

A portion of the organic by-products was dissolved in carbon tetrachloride or chloroform (spectroscopic grade) for infra-red cuialysis. The absorption peaks corresponded to nitrophenols, nitrocresols, dinitrophenols, dinitrocresols, traces of trinitro conpounds euid nitro-hydroxy carboxylic acids. The presence of individual compounds wais confirmed by comparison with the absorption spectrum of the pure compounds euid by thin-layer chromotography. Quantitative cuialyses of the concentrations of several components of the by-products were made. If a component did not have an absorption bcuid free from interference by euiother constituent of the mixture, queuititative cUialysis could still be achieved by mectsurement of the extinction coefficients of the interfering components at severcLL different frequencies, together with the absorbance of the mixture at these frequencies. Characteristic absorption bands used are shown in Tetble I. 

An additional complication is that many metabolities/cofactors have temperature-dependent extinction coefficients those for NADH and for potassium ferri-cyanide, for example, are about 10% lower at 80° than at 20°. Fourage et al. point out that the effect of temperature on the absorbance and Xmax of p-nitrophenol can lead to substantial errors in fccat values measured by continuous release of p-nitrophenol, if the calibration curve is not determined at the same temperature as the assay. 

Determination of Alkaline Phosphatase Activity. Vomeronasal and olfactory epithelia were removed and transferred to 0.9% NaCl solution where they were maintained at 4°C. All soluble forms of enzymes were washed out from the surface of the receptor tissue (see Chukhrai et al., 1992 for details). Using procedures that are described in detail in Chukhrai et al. (1992), alkaline phosphatase activity in olfactory and vomeronasal epithelia was determined at pH=8.3 in 0.9% bicarbonate buffer. Disodium p-ni-trophenol phosphate (Sigma) was used as a substrate its initial concentration was 8.1 x 10" M in the buffer. The increase of p-nitrophenol (the product of p-nitrophenylphosphate hydrolysis) was measured with a double-beam spectrophotometer at 400 nm every 30 sec. The velocity of the reaction was determined by estimating the angle of the slope (optical units [OU]/min). The obtained values were divided into the extinction coefficient (C ) under corresponding pH values to obtain the reaction velocity values in pM/min. Effective parameters of the Michaelis-Menten equation were compared using standard procedures (Chukhrai et al., 1992). 

The hydrolysis of paraoxon (a P-O bond breakage) was monitored by incubating each cotton sample in five milliliters of 1 mM paraoxon in 20 mM CHES, pH 9.0, at 25°C with constant shaking at 150 rpm on a Lab-Line Junior Orbit Shaker. Aliquots were removed at various time points and the amount of p-nitrophenol released as a cleavage product was quantitated at 400 nm and converted to concentration using Beer s law and an extinction coefficient of 17,000 M cm ... 

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