Extinction coefficient, millimolar

Charon and Szabo17 demonstrated that chromophore 4 may be produced to a significant extent from a 5-O-substituted derivative of KDO (which does not contain a free diol grouping at C-4-C-5). These authors synthesized 3-deoxy-5-0-methyl-2-octulosonic acid (7 configuration at C-5-C-7, arabino at C-4, unknown) by the Comforth reaction18,19 from 4-0-formyl-2-0-methyl-D-arabinose (see Scheme 4 and Section IV,1). Compound 7 gave a millimolar extinction coefficient of 13 in the TBA assay (as compared to 92 5 for KDO). Based on this result, Charon and Szabo17 formulated for the TBA reaction of 5-0-... 

For each of the diluted Standard Solutions—1, 2, and 3— plot absorbance against p-nitroaniline mM concentration. The result is a straight line that passes through the origin. Calculate the millimolar extinction coefficient (e) of each Standard p-Nitroaniline Solution using the following formula ... 

The averaged millimolar extinction coefficient, M, should be approximately 18.2. 

Fig. 46. Comparison of the absorption spectra of wild-type and mutant (cys G-439 and cys 1-68) sulfite reductases from Salmonella typhimurium. Spectra of S. typhi-murium sulfite reductase, cys G-439 NADPH-cytochrome e reductase, and cys 1-68 NADPH-oytoohrome c reductase, each dissolved in 0D5 M potassium phosphate buffer, pH 7.7, containing 0.1 mM EDTA, were read against a blank containing only buffer. The spectrum of each enzyme is presented in terms of its millimolar extinction coefficients, assuming 8 moles of flavin per mole of enzyme. Light broken line, calculated difference spectrum between those of wild-type and cys G enzymes when both enzyme solutions contain equal concentrations of flavin. From Siegel et al. (394).

Lactoperoxidase was purified as previously described. A stock solution was prepared of 1 x 10 M. The concentration of the enzyme was determined by employing a millimolar extinction coefficient of 114 at 412 nm. Small aliquots of the enzyme were frozen at -30°. Samples were thawed just prior to use. 

The stock solution of peroxide was freshly prepared for each experiment. Peroxide (30%) was diluted approximately 1 to 1000 in 0.05 M phosphate buffer, pH 7.4, to produce a 1 x 10 M solution. The concentration was established using a millimolar extinction coefficient of 72 at 230 nm. 

The method described by Morrison and Bayse (1970) for the enzymic iodination of tyrosine can be readily adapted to the modification of proteins. The reaction mixture contains, in order of addition, L-tyrosine (8.1x10 M), KI (1.0 xlO M), lactoperoxidase (7.4 X 10 M), in 0.05 M K-phosphate buffer, containing 1 x 10 M EDTA, at pH 7.4. The iodination is initiated by the addition of H2O2 to a concentration of 1.0 x 10 M. The specific activity observed for lactoperoxidase under these conditions was 1.05 x 10 moles of L-3-iodotyrosine per min per mole of enzyme at 25°C. At pH 7.4, the rate of enzymatic conversion of L-3-iodotyrosine to L-3,5-diiodotyrosine was 0.34 that of monosubstitution (Morrison and Bayse 1970). The desired level of iodination can be attained by successive equimolar additions of KI and HjOj to the reaction mixture. In this manner, only a low concentration of H2O2 is maintained, minimizing oxidation reactions. The concentration of lactoperoxidase may be calculated from the millimolar extinction coefficient of 114 at 412 run, while the concentrations of stock H2O2 solutions may be determined from the absorbance at 230 nm and a molar extinction coefficient of 72.4 (Phillips and Morrison 1970). 

The fundamental quantity to be used in any standardized spectro-photometric determination of hemoglobin should be the quarter millimolar extinction coefficient because of the four Fe atoms per hemoglobin molecule. This quantity is independent of the molecular weight of hemoglobin. Consequently, a redetermination of the molecular weight will not change the value of the extinction coefficient to be used and will not affect hemoglobin concentrations when these are expressed in quarter millimoles per liter. 

Thus there now exists overwhelming experimental evidence that the quarter millimolar extinction coefficient of HiCN at X = 540 nm is very near 11.0. This value was therefore proposed by the standardization committee of the European Society of Hematology at its 1963 meeting in Lisbon (B2) and was definitely adopted at the next meeting of this committee (Stocldiolm, 1964, B3). 

Fia. 6. e/X curve of hemoglobin (Hb). Quarter millimolar extinction coefficients based on = 11.0. The solid part of the graph represents the mean value resulting from... 

In the determination of the quarter millimolar extinction coefficients of hemoglobin derivatives, the following sources of error are present ... 

One way to parameterize the absorbance spectrum e(v) is by calculation of the moments of the millimolar extinction coefficient distribution. The nth moment of a distribution is given by... 

Values given in parentheses are the millimolar extinction coefficients at the wavelength indicated. The buffer used was 0.05 M potassium phosphate, pH 7.4. 

In solutions normally used for absorption work (below ten millimolar), nucleosides generally follow the Lambert-Beer Law, i.e., the signal is linearly proportional to the concentration. Above these concentrations, deviations from linearity are observed (Fig. 6.16). From this hypochromism, it was deduced that base-base interactions (stacking) occur. Generally, the decrease in extinction coefficient is gradual over relatively wide concentrations, and no sharp breaks are seen (Fig. 6.16). It is thus difficult to extract precise information from such data. Two other techniques have here been largely employed vapor pressure osmometry and NMR. 

The wavelengths and millimolar extinction coefficients at the absorption peaks of several important hemoglobin compounds have been summarized by Lemberg and Legge (1949). A partial list of their values is given in Table I. 

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