The determination of human-serum-cholinesterase activity with o-nitrophenyl butyrate

Abstract

Human-serum and horse-serum cholinesterases hydrolysed o-nitrophenyl butyrate about twice as rapidly as acetylcholine. Human-serum cholinesterase had a greater affinity (Km 0.11 mM), and greater activity towards o-nitrophenyl butyrate than toward any other nitrophenyl carboxylic ester tested. Human-serum cholinesterase hydrolysed ortho-nitrophenyl carboxylic esters more rapidly than the para esters. A-Esterases had the reverse specificity. In human serum, the cholinesterase was responsible for not less than 96% of the enzymic hydrolysis of o-nitrophenyl butyrate. A-Ester-ases accounted for the remaining activity. In the presence of 5% (v/v) butan-1-ol, cholinesterase-catalyzed hydrolysis of o-nitrophenyl butyrate was activated threefold, whereas the A-esterase activity was completely inhibited. A rapid, sensitive colorimetric method, suitable for routine analysis was developed as a consequence of these investigations. Over conveniently wide ranges, the activity was independent of both the pH and substrate concentration. The o-nitrophenol liberated by hydrolysis was measured directly in the reaction medium. The colour was stable. The precision of the method was [plus or minus]2.5%; each determination required 2-3 min. manipulation. A method for the synthesis and purification of o-nitrophenyl butyrate is described.