EDTA-kinetic procedure

The combination of kinetic single and steady-state sequential extraction methods has proved suitable for monitoring the fate of arsenic in slightly polluted soils through irrigation (Cornu et al., 2004). The EDTA-kinetic procedure confirmed that anthropogenic arsenic is fixed in soils mainly under labile forms and is easily... 

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

Relaxation Kinetics. The details of the experimental procedure have been described earlier (14). 0.1 M phosphate buffer, pH 7.0, containing 2 X 10"5 M EDTA was used in all relaxation experiments. These were performed with solutions of different initial reagent composition— either ferrocyanide was added to oxidized azurin or ferricyanide to reduced azurin. Temperature jumps of 2.9° or 4.7° were applied to the reaction solution. The subsequent transmission changes were monitored at 625 nm (absorption of oxidized azurin) or 420 nm (absorption of ferricyanide ). Each plotted value of the relaxation time or amplitude represents the average of at least four measurements. 

All ingredients are present in the reaction mixture, which is added to a lipid film, and liposomes are prepared containing all macromolecules (enzymes and DNA or RNA templates) as well as all substrate molecules (nucleotides, for example). Consequently, this procedure has to be performed very quickly otherwise, the enzymatic reaction would mainly occur outside the liposomes and a distinction between product molecules synthesized inside from those produced outside and entrapped later would be difficult to draw. After the formation of liposomes, the enzymes outside the liposomes have to be inhibited by potent inhibitors (inhibitors that do their job even in the presence of substantial amounts of phospholipids) or the liposomal dispersion has to be treated by digestive enzymes. This strategy has basically been applied in the case of the RNA replication by QP replicase inside oleic acid/oleate liposomes" and in the case of the polymerase chain reaction (PCR) inside POPC or POPC/PS liposomes." In the former case, EDTA was added after the formation of the liposomes to inhibit the non-entrapped enzymes (and the kinetics was followed after addition of the EDTA molecules), in the latter case, the non-entrapped DNA template molecules were digested by DNase I before the temperature was raised to 95°C and the polymerization started. 

It is important to note that the kinetics of lanthanide complexation reactions in general involve rapid association and dissociation reactions, except for structurally complex ligands like edta. Generally, lanthanide complexation kinetics in aqueous media can be considered sufficiently rapid as to have minimal effect on separations. Phase-transfer rates may be important in some systems, and should be considered in the optimization of an analytical separation procedure. The kinetics of lanthanide complexation reactions has been discussed in a previous report (Nash and Sulhvan 1991). There has been some consideration of kinetics-based separations for f-elements (Nash 1994, Merciny et al. 1986), but no useful analytical applications based solely on differences in lanthanide kinetics are known. 

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