Covalent Binding Determinations

The value of EM for a cooperative self-assembled structure provides a measure of the monomer concentration at which trivial polymeric structures start to compete, and therefore EM represents the upper limit of the concentration range within which the cooperative structure is stable (Scheme 2). The lower limit of this range is called the critical self-assembly concentration (csac) and is determined by the stoichiometry of the assembly and the strength of the non-covalent binding interactions weaker interactions and larger numbers of components raise the csac and narrow the stability window of the assembly (8). Theoretical treatments of the thermodynamics of the self-assembly process have been reported by Hunter (8), Sanders (9), and Mandolini (10). The value of EM is lowered by enthalpic contributions associated with... 

NU(C) base atoms (5) The stereoselectivity of the BPDEs during intercalative covalent binding in kinked DNA and (6) The possible reorientation of the complex to yield an externally bound adduct. The energetics for each of these processes will be presented to identify the important steps that influence the binding of specific isomers. It will be shown that the orientation of each diastereoisomer of BPDE about specific base atoms in kinked receptor sites in the duplex DNA during covalent bond formation is the determining factor in stereoselectivity. 

Immunochemical methods are valuable because of their sensitivity and specificity. The sensitivity depends on the method used to determine an end point. One of the reaction components may be tagged with radioactivity, or tagged by covalent binding of an enzyme capable of being detected, or by covalent binding of a totally unrelated species (i.e., fluorescein). 

Cytochrome P-450 spectra (reduced + CO) show the 2-nm shift to the blue as a result of 3-MC induction. No such shift is observed in the trout, control rat, and PB-induced rat liver microsomes. Cytochrome P-450 EtNC spectra were recorded at pH 7.4, 2 min after the samples were reduced with dithionite. The absorption peaks are at 430 and 455 nm. For pH curves the A Absorbance represents A A (430—490 nm) and A A (455— 490 nm). The number of (+) signifies only the relative activity or inhibition with respect to BP hydroxylation and covalent binding of BP to DNA ( ++-(-) signifies 2-4 times and (+-H—h) signifies 10-36 times the control rat microsomal activity (n.d.), not determined. 

In a study in which liver microsomes, prepared from male Wistar rats (200-250 g), were incubated with 5 x 10 mol/L2,3-dibromopH]propan-l-olandanNADPH-gene-rating system, covalent binding to protein was determined. Addition of the epoxide hydrolase inhibitor l,l,l-trichloropropene-2,3-epoxide led to an increase in the proteinbinding rate of 2,3-dibromopropan-l-ol (Soderlund etal., 1981). 

This nonextractable radioactivity was probably the result of covalent binding of the furazolidone intermediates to endogenous macromolecules. The bioavailability of these bound tissue residues from the above pig residue depletion study was determined by feeding rats lyophilized samples of liver and muscle tissues from animals sacrificed at 0 and 45 days after the last treatment (132). Results showed that the fraction of the bound residues bioavailable to rats was in the range 16-41%. The toxicological impact of these bioavailable bound residues has not been yet determined. 

If necessary, subsequent experiments can then determine irreversibility and potency (e.g., Ki and jnact) f°r metabolism-dependent inhibitors, as well as the potential for covalent binding. This general experimental strategy, including follow-up experiments, is illustrated in Figure 5. The design of experiments to determine Kt and /clnact will be covered in more detail in section II.C.7.C. 

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