Spectrometric and Fluorimetric Assays

Kinetic constants are obtained from the second part of the two-slope curve treatment of the data as a pseudo first-order reaction gives while interpolation of the slope the ratio Kds/trans. Although it may be marred by additive errors, K- t can be deduced from the equation Kds/trans = 

A protease-free assay using Suc-Ala-Ala-Pro-Phe-DFA (DFA is 2,4-difluoroani-line) was also developed, but its sensitivity at 246 nm is low ( 0.006 absorbance unity) for a typical peptide concentration of 21 pmol L-1 [56], The same problem limits the applicability of the assay reported by Fischer and coworkers based on the pH sensitivity of the secondary amide bonds. A pH jump (i.e. from 2.0 to 7.3) led to a slight variation of absorbance at 220 nm which reflects the pH-dependent CTI (Fig. 8.8). However, this assay cannot be used routinely with oligopeptides, although it gives kinetic data that cannot be easily obtained by other methods [21]. 

The fluorescence of the ortho-aminobenzoate motif was utilized to elaborate a continuous fluorimetric assay based on intramolecular fluorescence quenching through isomer-differential collision of the fluorophore and a quencher, i.e. nitro-phenylalanine or a para-nitrophenyl moiety at the C-terminus [57]. Relative fluorescence recorded at 410 nm (2ex = 317 nm) is cleared from the participation of the preexisting trans isomer, but this assay is limited to peptides bearing fluoro-phores and properly positioned fluorescence quencher moieties. 

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