Acetylation of human serum albumin by p-nitrophenyl acetate

Abstract

Human serum albumin reacts very rapidly with p-nitrophenyl acetate (NphOAc). Rapid acetylation of the protein accompanies and largely accounts for the easily observed rapid formation of of p-nitrophenolate ion. One group is acetylated much faster than all others. It appears to be located in a high affinity binding site for small fatty acid anions, to have a pKa of 8.7, and a limiting bimolecular rate of reaction with NphOAc of approximately 3 X 10(4) M-1 sec-1 at alkaline pH values. Rapid reversible binding appears to be a major contributor to the high reaction velocity.

Aryl acylamidase activity of human serum albumin witho-nitrotrifluoroacetanilide as the substrate

“…It is concluded that the rate of formation of the acylated albumin adduct is fast (k 2 ), and that the slow step in hydrolysis of the p-NPA ester is deacylation (k 3 ). This provides direct evidence that deacylation is the rate-limiting step (k 3 , k 2 ) in albumin-catalyzed hydrolysis of p-NPA, confirming results obtained from burst kinetics [2]. The reaction of o-NTFNAC with albumin rapidly produced a yellow color (o-nitroaniline), demonstrating that the acetanilide substrate was being hydrolyzed.…”

“…Albumin is capable of binding numerous compounds and displays several enzymatic activities [1], including esterase activity with p-nitrophenyl acetate and other aryl-esters, including drugs and prodrugs [2][3][4]. The esteratic site of human albumin was found to be the binding site for several drugs, organophosphates and fatty acids.…”

Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps

“…Moreover, data here reported indicate that the deacylation process is rate limiting in the HSA-catalyzed hydrolysis of NphOAc (i.e., k +3 << k +2 ). Lastly, values of K s and k +2 here obtained are in agreement with those previously reported [12,13].…”

“…HSA (from Sigma-Aldrich, St. Louis, MO, USA) was essentially fatty acid free, according to the charcoal delipidation protocol [9][10][11] .0°C, were analyzed in the framework of the minimum three step-mechanism reported in Scheme 1 [8,[12][13][14][15], where HSA is the substrate-free protein, NphOAc is the substrate, HSA:NphOAc is the reversible protein-substrate complex, HSA-OAc is considered to be an ester formed between the acyl moiety of the substrate and the O atom of the Tyr411 phenoxyl group [8], AcOH is acetic acid, k +l is the second-order rate constant for the formation of the HSA:NphOAc complex starting from HSA and NphOAc, k À1 is the first-order rate constant for the dissociation of the HSA:NphOAc complex to HSA and NphOAc, K s (=k À1 /k +1 ) is the pre-equilibrium constant, k +2 is the first-order acylation rate constant, k À2 is the first-order rate constant for the conversion of HSA-OAc to HSA:NphOAc, k +3 is the first-order deacylation rate constant, and k À3 is the second-order rate constant for the formation of the HSA-OAc adduct starting from HSA and AcOH.…”

Ammonium sulfate precipitation combined with liquid chromatography is sufficient for purification of bovine serum albumin that is suitable for most routine laboratory applications

“…Thermocoagulation removes contaminating proteins such as nucleases and proteases; however, prolonged exposure to heat can cause BSA molecules to form hydrophobic aggregates which may not revert to monomers upon cooling [16][17][18][19][20]. In some preparations, nucleases are inactivated by acetylation using chemical agents such as p-nitrophenyl acetate [21,22]. Acetylation of tyrosine residues in BSA limits its laboratory use by interfering with color development in assays such as the Lowry's.…”