Identification and partial purification of the major aspirin hydrolyzing enzyme in human blood

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Abstract
In normal whole human blood in vitro, the source of the enzyme controlling the hydrolysis of aspirin (ASA) was found to be the erythrocyte (RBC). Experiments were carried out to determine whether this enzyme was membrane-bound or free in the lysate. The mean rates of ASA hydrolysis in comparable concentrations of intact RBC (1.61 ± 0.20 γmole/liter/minute, 12 = n and 1.23 ± 0.17 γmole/liter/minute, n = 5) were much faster than that measured in isolated RBC membranes (0.23 ± 0.08 γmole/liter/minute, n = 6, P = ≤0.001). Detailed study showed that the RBC-related ASA esterase is located intracellularly and is not related to membrane acetylcholinesterase. The ASA esterase from crude lysate was purified 900-fold by means of DEAE Sephacel chromatography of active enzyme recovered from a 50% saturated ammonium sulfate fraction. Non-SDS polyacrylamide gel electrophoresis (pH 8.3 and 9.0) resulted in one major band and one or more small minor protein bands. When esterase activity was assayed in a non-stained gel, ASA depeletion and salicylate production corresponded exactly to the major stained band. This band was eluted from another unstained gel, concentrated, and applied to an SDS gel. This yielded a single band with a molecular weight of approximately 95,000. The partially purified enzyme had a mean Km of 66.6 ± 3.5 γM and a mean Vmax of 4.0 ± 0.9 γmole/liter/minute/mg under the assay conditions. The results of inhibition studies suggested that this enzyme'S activity is sulfhydryl group dependent, does not require divalent cations for activity, and is different from the RBC type D “nonspecific” esterases.