Crystallization isosbestic point

If the absorption at an isosbestic point suddenly starts changing in the course of a reaction, this indicates an unexpected process. An example is given by Thurston et al. The absorbances at two isosbestic points were constant during a clean, single-step reaction. By observing a sudden change in absorbance after some time, the crystallization of the product was detected. 

Complex IV (cytochrome c oxidase) activity is measured by following at 550 nm (e 19,100-MAcnr1 isosbestic point 540 nm) the oxidation of cytochrome c (Fig. 3.8.5). It could be calculated as a first-order rate constant, or by estimating the pseudolinear initial rate of the reaction. Noticeably as a check, this initial rate should represent about double the rate measurable when 50% of the added cytochrome c has been oxidized (Fig. 3.8.5). Indeed, the affinity constant (Km) for reduced cytochrome c is about equivalent to the K, for oxidized cytochrome c. Reduced cytochrome c is easily prepared by adding a few crystals of dithionite to a solution of oxidized cytochrome c. After the immediate color change (from deep red to light orange-red), the solution should be carefully stirred to eliminate any trace of dithionite and should be totally odorless. 

By analogy with the studies of Johansen and Vallee, it could be that the positive charge influencing the ionization behavior of nitrotyrosine-248 in nitrocarboxypeptidase is the active site zinc ion. To test this possibility, the effect of physical state on the nitrotyrosyl pK was examined. Titration of nitrocarboxypeptidase crystals from pH 6.5 to 9.5 increases absorbance at 428 nm and decreases it at 381 nm with an isosbestic point at 381 nm. However, in contrast to the enzyme in solution, the midpoint of this titration occurs at pH 8.2 rather than at pH 6.3 (Figure 8). This dramatic shift in titration behavior, brought about solely by a change in physical state of this enzyme, indicates that in nitrocarboxypeptidase as in arsanilazocarboxypeptidase, the conformations of tyrosine-248 in solution and in the crystalline state are different. 

Photodimerization of the Methyl q-cyano-4-[2-(2-pyridyl)ethenyl]-cinnamate III (2-pyridyl, X CN, Y COOMe) Crystal (13c). In the course of the photoreaction of the crystal of III (2-pyridyl, X CN, Y COOMe), the UV absorption band at 365 nm decreased and a new absorption peak appeared at 313 nm. In addition, clear isosbestic points were seen in the spectra at 258 and 329 nm. The photoreaction was fast, and, finally, the TLC of the photoproduct showed a single spot. These spectral data and the results of TLC analysis indicate the progress of a single reaction on photoirradiation of the crystal. 

In both liquid water and ice, H2O molecules interact extensively via O— bonds. However, there are marked differences between the two phases. In the latter, H2O molecules are tetrahedrally hydrogen-bonded, and this local structure is repeated throughout the crystal. In liquid water, however, the O—H- O bond distance and angle vary locally, and the bond is sometimes broken. Thus, its vibrations cannot be described simply by using the three normal modes of the isolated H2O molecule. According to Walrafen et al. [444] an isosbestic point exists at 3403 cm in the Raman spectrum of liquid water obtained as a function of temperature, and the bands above and below this frequency are mainly due to non-hydrogen-bonded and hydrogen-bonded species, respectively. In addition, liquid water exhibits librational and restricted translational modes that correspond to rotational and translational motions of the isolated molecule, respectively. The librations yield a broad contour at 1000-330 cm while the restricted translations appear at 170 and 60 cm [445]. For more details, see the review by Walrafen [446]. 

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