Average values standard affinity

Fluorescence and affinity measurements - Peptide in 25 mM Tris, 100 mM KCl and 1 mM CaCl2 at pH 7.5 and 30 C was titrated with a stock solution of calmodulin in UV transmitting plastic cuvettes since the peptides appear to bind to glass. Fluorescence titration spectra were recorded using a SPEX FluoroMax fluorescence spectrometer with excitation at 280 nm and emission scanned from 310 to 390 nm. The value of fluorescence intensity at 330nm was plotted as a function of calmodulin concentration and fitted using standard non-linear least squares methods (6) to obtain optimal values of the dissociation constant (Kj) and the maximum fluorescence enhancement (F/F ). The detection limit under our experimental conditions was 50 nM peptide and all quoted Kj values are the average of at least 3 independent determinations. 

Antibody Characterization. Typical c-ELISA s for LLNL-Hept-2 using heptachlor as competitor (IS samples run over a 6-month interval) are presented in Figure 2. These data indicate that the average Iso tor heptachlor occured when 3.0 ng of analyte was added to the reaction. A standard deviation of 7% was observed causing the Iso values to range between 1.9 and 4.S ng/well. Similar variations were observed with other competitors. The LLNL-Hept-2 antibody used in the above experiments, and all subsequent experiments, was purified from ascites fluid by affinity chromatography on Sepharose Protein G (see methods section). 

This particular reaction has been chosen for the reason of its high value of standard chemical affinity for this reaction (j / = — 7.1 kcal/mole). As we noted above, due to this circumstance the system behavior can reveal the deviation from that prescribed by the classical Arrhenius mechanism. The conformational changes in malatdehydrogenase were tested by measuring the average life-time of intrinsic tryptophane fluorescence (ff). This parameter is known to be sensitive to the immediate surrounding of tryptophane residues. The chemical transformation of the substrate was detected from changes in the coenzyme redox state measured in terms of the sample optical density at 340 nm (NADH absorption maximum). 

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