Activation spectra, measurement forms

The significance of vibrational optical activity becomes apparent when it is compared with conventional electronic optical activity in the form of optical rotatory dispersion (ORD) and circular dichroism (CD) of visible and near-ultraviolet radiation. These conventional techniques have proved most valuable in stereochemistry, but since the electronic transition frequencies of most structural units in a molecule occur in inaccessible regions of the far-ultraviolet, they are restricted to probing chromophores and their immediate intramolecular environments. On the other hand, a vibrational spectrum contains bands from most parts of a molecule, so the measurement of vibrational optical activity should provide much more information. 

NLO active molecules can be embedded in or chemically anchored to a sol-gel-matrix without changing the optical absorption spectrum. Disperse Red 1, a very efficient molecule for NLO applications, was embedded in a sol-gel-matrix, synthesized by hydrolysis and condensation of 3-glycidoxypropyltrimethoxysilane in the presence of N-methylimidazole. The dye-doped gel was applied to glass substrates and thermally cured to form a layer of perfect optical transparency. Currently, poling experiments and NLO measurements with these layers are being performed. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

In PLS, the variance is generated using the quantities generated by the referee analytical method. Therefore, the factors in a PLS analysis (especially the first) resemble the spectrum of the active ingredient (assuming a quantitative analysis of a constituent). When measuring a physical attribute (hardness, polymorphic form, elasticity), the PLS factor may not resemble any of the materials present in the mixture. 

There are at least three possibile ways in which the inhibitor can bind to the active site (1) formation of a sulfide bond to a cysteine residue, with elimination of hydrogen bromide [Eq. (10)], (2) formation of a thiol ester bond with a cysteine residue at the active site [Eq. (11)], and (3) formation of a salt between the carboxyl group of the inhibitor and some basic side chain of the enzyme [Eq. (12)]. To distinguish between these three possibilities, the mass numbers of the enzyme and enzyme-inhibitor complex were measured with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI). The mass number of the native AMDase was observed as 24766, which is in good agreement with the calculated value, 24734. An aqueous solution of a-bromo-phenylacetic acid was added to the enzyme solution, and the mass spectrum of the complex was measured after 10 minutes. The peak is observed at mass number 24967. If the inhibitor and the enzyme bind to form a sulfide with elimination of HBr, the mass number should be 24868, which is smaller by about one... 

The enzymatic activity of RNase A shows a bellshaped dependence on pH, with an optimum near pH 7 (fig. 8.14). The operation of the enzyme appears to require that a dissociable group with a pKa of about 6.3 be in the proton-ated form, and that a group with a pKa of about 8 be unpro-tonated. These groups have been identified as His 12 and His 119 by measuring the NMR spectrum of the enzyme as a function of pH. Spectral peaks due to histidine residues undergo characteristic shifts as the imidazole ring is proton-ated. 

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