P-Nitrophenyl acetate, and HSA

Figure 2. Inhibition of the reaction between p-nitrophenyl acetate and HSA by carboxylate ions. Relative reactivities are shown as a function of the mole ratio of decanoate (O), octanoate (A), hexanoate (0), and butyrate (M) as determined at 25°C in pH 7.5, 0.02M triethanolamine/HCl (29).

Figure 3. Inhibition of the reaction between p-nitrophenyl acetate and HSA by drugs. Data for oxazepam (0), fiufenamic acid (D), and naproxen (O) obtained at pH 7.4 in 0.03M triethanolamine/HCl at 25°C with 3.5 X 10 5M HSA. Dissociation constants are given in Table II.

Figure 9. Inhibition of the reaction between p-nitrophenyl acetate and HSA by phenylbutazone and by phenylbutazone plus warfarin. Increasing amounts of phenylbutazone were added to a solution of HSA as described in Figure 8(0) and after the addition of 10 4M warfarin (M).

The interactions of many important drugs, metabolites, and structurally related compounds with HSA have been char-acterized on the basis of their inhibition of its acetylation by p-nitrophenyl acetate. Dissociation constants of 10 5M or lower have been determined for tryptophan, phenylpyru-vate, several pharmacologically important benzodiazepines, several types of small, apolar, anionic drugs and related compounds that interact specifically with the inhibitory site. Two other classes of inhibitory compounds have been identified and similarly characterized, the members of which interact either to a comparable extent or preferentially with one other site. The inhibitory site appears to be elongated, apolar, and has a monoanion binding site near one end. 

To help characterize the interactions of some drugs and metabolites with HSA and their competition for its limited number of binding sites, we and others have studied the influence of those substrates on its acetylation by p-nitrophenyl acetate (29,30,31,32,33). This reaction is very fast and results in the irreversible acetylation of a particular tyrosine residue (i.e. 411) which is located in a major apolar binding site of that protein (34). Because this reaction is so fast under most conditions, competing reactions are not significant and formation of p-nitrophenolate is a convenient reflection of the reaction rate (35). 

Small fatty acids, for example, bind strongly to HSA and, as shown in Figure 2, strongly inhibit its reaction with p-nitrophenyl acetate (29). In each case, and as is particularly obvious with decanoate ion, complete inhibition appears to require a single carboxylate ion, each, however, having a different affinity in proportion to its hydrophobicity. 

Dissociation constants were determined from their inhibition of the reaction between HSA and p-nitrophenyl acetate as determined in pH 7.4, 0.03M triethanolamine / HCl at 25° C. Nonpolar surface areas were calculated according to Hermann (48/ The carboxylate ions are as follows (1) butyrate (2) valerate (3) hexanoate (4) heptanoate (5) octanoate (6) nonanoate (7) decanoate (8) undecanoate (9) dodecanoate (10) cyclohexanecarboxylate (11) 1-methyl-1-cyclohexanecarboxylate (12) cyclohexylacetate (13) 2-n-propylvalerate and... 

The affinity of this site for anions presumably reflects its content of one or more cationic groups. To characterize this cationic center we have compared the inhibitory effects of a number of different kinds of anions. Figure 15 shows the effect of two closely related anions, phenylphosphate, a dianion at pH 8.0, and phenylmethylphosphonate, a monoanion under the same conditions, on the reaction between HSA and p-nitrophenyl acetate. Only the latter compound is inhibitory which along with other results suggests that the site is specific for monoanions. 

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