Ultramicro assay of the cholinesterases

1. 1. A method is described for the simultaneous determination of cholinesterase activity and protein content in minute quantities of tissue, e.g., 50 μg. of nervous tissue or 0.2 μl. of human serum, having activities ranging from 0.01 to 0.1 μ M AcCh/hr. 2. 2. The method for cholinesterase is based on the formation of hydroxamic acid from the remaining choline ester after incubation with the enzyme; the color developed with acid ferric chloride is measured. 3. 3. Advantages of the method are: (a) It is simple and quick, and therefore suitable for routine work; (b) enzyme activity and protein content can be determined in the same sample; (c) a variety of substrates can be employed, allowing the study of the enzyme specificity; (d) the pH of incubation can be varied from pH 5.5 to 10.0; (e) the same procedure can be used to determine choline esters in biological samples. 4. 4. The reproducibility of the choline ester determination in a range of 0.01–0.1 μ M is ±.0008 μ M and that of the cholinesterase assay is ±0.0024 μ M/hr. in a range of 0.01–0.1 μ M/hr. 5. 5. The effects of the buffer system and the preparation of the sample have been studied in some detail.