Titration methods, cholinesterase activities

Effect of substrate concentration. In the following experiments the cholinesterase activities were measured by a continuous titration method. The digest of acetylcholine and horse-serum cholinesterase (total vol. 10 ml.), containing bromothymol blue and 0-0002 m phosphate, was titrated with 0-01 n NaOH to maintain the pH at 7-4. The titrations, which were carried out at 20°, were linear over a period of 10-15 min. The velocity was expressed as ml. 0-01 n NaOH/5 min. under the conditions used, it was proportional to the enzyme concentration. When an inhibitor was added, this was equilibrated with the enzyme, etc., for 5 min. at 20° before adding the substrate contained in a volume of 1 ml. 

True and pseudo-cholinesterase. The above serum preparations contained both the true and pseudo- cholinesterases of Mendel and Rudney.1 The effect of di-isopropyl phosphorofluoridate on these components was examined separately by means of the specific substrates described by Mendel, Mundel and Rudney,2 using the titration method described above. Phosphorofluoridate (5 x 10 8m) gave an inhibition of 57 per cent of the activity towards 00045m acetylcholine, 30 per cent of the activity towards 0-0005 m acetyl-/ methyl-choline, and 40 per cent of that towards 0-005 m benzoylcholine, after incubating the enzyme with the poison for 5 min. Thus in these experiments there appeared to be no appreciable difference in sensitivity of the true and pseudo-cholinesterases of horse serum to phosphorofluoridates. 

Cholinesterase Activity. Cholinesterase activity was assayed by automatic, continuous, alkali titration of acid released from the substrate, a method previously utilized by Wilson (12), Main and Dauterman (6), Shellenberger et al. (8), and others. 

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See also in sourсe #XX -- [ ]

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