Affinity complex

Ferguson, K.M., et al. Structure of the high affinity complex of inositol triphosphate with a phospholipase C pleckstrin homology domain. Celt 83 1037-1046, 1995. 

For the enzyme isomerization mechanism illustrated in scheme C of Figure 6.3, there are two steps involved in formation of the final enzyme-inhibitor complex an initial encounter complex that forms under rapid equilibrium conditions and the slower subsequent isomerization of the enzyme leading to the high-affinity complex. The value of kohs for this mechanism is a saturable function of [/], conforming to the following equation ... 

Fig. 8. Model for the high affinity complex between horse Cc and CcO determined by Roberts and Pique (34). The backbone of horse Cc and CcO subunit II are shown with the side chains of selected lysines and acidic residues colored blue and red, respectively. The residue numbers on subunit II are for R. sphaeroides CcO. Van der Waals surfaces are shown for Cc heme and subunit II Trp143 and Met263. The CuA coppers are represented by green Corey-Pauling-Koltun models. Reprinted with permission from Ref. (18). Copyright 1999, American Society of Biochemistry and Molecular Biology.

Compound complexation — One of the most challenging aspects of dealing with complexation is that several different complexation mechanisms may be operating simultaneously. Affinity complexation is one example, as discussed in the... 

Affinity complexation — Many proteins have affinities for other molecules that can be exploited to alter their retention characteristics in IEC. For example, some enzymes may be combined with synthetic substrates, cofactors, or products.1315 The same principle can be applied to other protein/receptor systems. One well-characterized example is the change in chromatographic behavior of fructose 1,6-diphosphatase in the presence of its negatively charged substrate... 

Any detectable effect on the reaction or behavior of a particular system by the interior wall of the container or reaction vessel. Because proteins can form high-affinity complexes with glass and plastic surfaces, one must exercise caution in the choice of reaction kinetic conditions. Wall effects can be discerned if one determines catalytic activity under different conditions that minimize or maximize contact of the solution with the container. In principle, an enzyme-catalyzed reaction should proceed at the same rate if placed in a capillary or a culture tube however, contact with the wall is maximized in a capillary, and wall effects should be more prominent. Some investigators add bovine serum albumin to prevent adsorption of their enzyme onto the container s walls. 

The synthesis of DNA analogues with a backbone of poly(/V-(2-amino-ethyl)glycine) (peptide nucleic acids = PNA) instead of phosphate-ribose, which were introduced into affinity labeling by Egholm et al. (28), opened up several new applications for affinity separations (23,28-42). Since this backbone is by far less polar, the behavior of the monomer as well as that of DNA/PNA hybrids is largely different, allowing the complete separation of affinity complexes. In addition, it seems that the stability of duplexes is... 

Overall, the affinity complexes were readily distinguished from the unbound molecules, although the relative increase in fluorescence polarization upon complex formation varied with the molecular size of the binding pairs. 

The detection of HIV-related proteins is one of the most challenging tasks. This is especially true because AIDS should be diagnosed as early as possible to enable an early and effective therapy of this infection. Pavski and Le (57) used the aptamer strategy to detect reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1). A direct and specific ACE method was proposed using laser-induced fluorescence (ACE/LIF) as detection principle. Single-stranded DNA aptamers as probes fluorescently labeled were synthesized. The resulting aptamer is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-l RT was stable, with calibration curves linear up to 50 nM (6 /xg/mL) HIV-1 RT concentration. Both... 

X 107 M 11 for a 1 1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1 1 stoichiometric ratio (complex 1). The other complex (complex 2) is proposed to have a stoichiometry with an excess of anti-BSA to BSA, ... 

Macromolecular ligands (e.g., proteins) [224], polysaccharides [225], small ligands (e.g., triazine dyes) [226], metal chelates [227], and inhibitors [228] can provide unparalleled selectivity in forming Hgand-Hgate affinity complexes. 

The structure determination confirmed the importance of the spacing of the two hexamers as a discrimination factor in an impressive maimer. A spacing of only 3 nucleotides between the two hexamers would lead to steric overlap of both receptors a high affinity, cooperative binding would not be possible. With a spacing of more than 4 nucleotides a high affinity complex could also not be formed due to the relative rigidity of the two monomers. 

The affinity for Ca Vcahnodulin is increased by close to three orders of magnitude. Ca /calmodulin only dissociates very slowly from this high affinity complex. The activated state is thus preserved over a longer period of time. Even when the Ca signal has died away and the Ca concentration has fallen to a level of lO M, the enzyme... 

Cyclic voltammograms of the COx/Cyt.c electrode corresponding to the bioelectrocatalyzed reduction of 02 (i), and to the reference system, where 02 is excluded (ii). (c) Assembly of an integrated LDH electrode for the bioelectrocatalyzed oxidation of lactate by the surface cross-linking of an affinity complex formed between LDH and different structures of a boronate-linked PQQ-NAD monolayer. Parts (a) and (b) Reproduced from Ref. 27 by permission of the Royal Society of Chemistry (RSC). Part (c) Reproduced with permission from Ref. 25. Copyright 2002 American Chemical Society. 

Scheme 13 Electronic and optical transduction of the formation of antigen-antibody affinity complexes on transducers (A) ampero-metric transduction at an electrode (R+/Ris a redox label in the electrolyte solution), (B)...

Mei H, Wang D, McKee S, et al. Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site. Biochemistry 1996 35 15800-6. 

The elaborate mechanism by which blue oxidases react with dioxygen to produce water was tackled by studying the possible role of H202. We have observed the formation of a stable and high affinity complex between tree laccase and H202. Moreover, the finding that the oxidation of the reduced enzyme with H202 follows a pattern which is different from that operative in the reduction of the oxidized enzyme may have important implications for the mechanism of action of laccase. 

Depending on the nature of the target, either Ag or Ab is added in excess. The excess is needed to ensure that all the analyte of interest is being complexed. A large excess facilitates a shift of equilibrium toward complex formation and in some cases can compensate for reduced affinity of the system. After incubation, the mixture is analyzed by CE. A peak of an affinity complex or a peak(s) of unbound reagent(s), resolved from each other and compared with that of calibration standards, is then used for analyte quantification. 

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