The activity of carboxylic esterases was demonstrated by incubation in the following substrates: 1-naphthyl acetate (?-naphthyl; B.D.H., Poole) with o-dianisidin tetrazotized (diazo blue B; E. Gurr, London) in phosphate buffer 0.1 M, pH. 7.5; 2-naphthyl acetate (?-naphthyl; Mann Lab., New York) in phosphate buffer 0.1 M pH. 7.5, diazo blue B; carbonaphthoxycholine iodide (Serlabo, Paris) in phosphate buffer 0.1 M, pH 7.5, diazo blue B; indoxyl acetate (Mann Lab., New York) in veronal buffer 0.05 M, pH 8.2. Fig. 1. Esterase activity (?-naphthyl acetate) and proteins after electrophoresis on starch gel of horse, donkey, mule and hinny sera. On the left, amido-black staining only; on the right, amido-black after esterase revelation; albumin does not take the stain intenselyFig. 2. Esterase activity (a-naphthyl acetate) after electrophoresis on agar gel: 2 horse 2 donkey, 1 hinny and 2 mule sera The inhibitors used were: 'Prostigmine' (Roche) (dimethyl carbamic ester of trimethyl-3-hydroxyphenyl ammonium, methyl sulphate) M 10?5 and M 10?4, DFP (L. Light, Colnbrook) (diisopropylfluorophosphate) M 10?5 and M 10?4, copper acetate M 10?3, heating to 60 for 1 h.On starch gel, using either ?- or ?-naphthyl acetates or the indoxyl acetate, the sera of horse, mule and hinny give an identical pattern of two intensely stained bands (with the first substrate some samples show also two faint zones of activity slower than the transferrin) (Fig. 1). The faster migrating zone, called E3, is the most intensely stained. These zones form well-defined spots, although in a few samples the slower zone, E1 was double. Their localization was achieved by cutting two parallel incisions in the slice of gel to delimit the zone of esterase activity and then staining the gel with amido-black for proteins. The slow esterase zone E1 is somewhat faster than the slow ? 2, and the fast esterase (E3) is localized just behind the albumin (Fig. 1). The donkey sera contain only the slow esterase (Fig. 1).With carbonaphthoxycholine as substrate, the sera of all four groups of animals exhibit only the slow esterase band, which can thus be identified with serum cholinesterase. The inhibition investigations show that both esterases are thermolabile; the slow esterase is partially inhibited by copper ion and completely by DFP M 10?5, prostigmine and p-chloro-mercuri-benzoate M 10?5 (with all substrates used). The fast esterase is not inhibited by prostigmine, even M 2 10?4 or p-chloro-mercuri-benzoate M 10?4 or by copper. It is, however, partially inhibited by DFP M 10?5 and totally inhibited by DFP M 10?4.In agar gel the two activity zones are only slightly separated and the region between them is coloured with naphthyl or indoxyl acetates. The faster migrating zone, situated just behind the albumin, is the more intensely stained. We did not observe any activity in components migrating faster than albumin. The difference between horse, donkey, mule and hinny serum esterases is, therefore, less marked than in starch; however, the donkey serum does not exhibit a fast zone, only a coloured tailing (Fig. 2).With the carbonaphthoxycholine all sera give the same pattern: a uniformly coloured tailing between the albumin and the origin. As the two activity zones are well separated in starch gel, we tried the identification of these components with the esterases as revealed on agar gel.The activity-containing zones of a starch-gel slice were cut off after superimposing one coloured slice on to another untreated one. The cuttings were inserted in an agar-gel plate and after 3 h of migration the plate was treated for esterase activity with a-naphthyl acetate. Another identically prepared plate was stained for proteins. No activity subsisted at the insertion region. The 'slow' esterase migrated as a slightly active tail; the staining spread over 3 cm; the 'fast' esterase forms a rather intensely stained spot with a little tail: this spot migrates definitely less than the E1.The horse, mule and hinny fast esterase spots were fairly similar in migration; the migration of the cholines-terases of four animals is also the same. Fig. 3. Schematic representation of the spots of esterase activity as revealed by carbonaphthoxycholine iodide (horizontal lines) and indoxyl acetate (vertical lines) and the outline of the albumin spot coloured only by amido-black. Cuttings from a starch-gel plateThe amido-black staining reveals that a part of the protein content of cuttings migrates toward the cathode, mainly from 'slow' esterases (cholinesterases); and that, further, all raservoirs are coloured. The spots of 'fast' esterase are intensely coloured and well defined, but the tailing is less important than when revealed by esterase activity reaction. The staining of 'slow' esterase is soma-what reduced when compared with the activity zone. The migration is shorter than for the 'fast' esterase, more likely resembling their original migration in starch. Furthermore, the spot is better denned, although it is always less intense than the 'fast' esterase spot. In another analogous experiment, the cuttings of E1 E3 and albumins of four animals were transferred to an agar plate. This was first incubated in carbonaphthoxy-choline and the purple staining appeared only for E1 the plate was then washed and incubated in indoxyl acetate, which coloured principally the E3 spots in blue. No reaction was observed for albumin (Fig. 3).The identification of E1 with serum choli nest erase seems thus confirmed. The comparison of the migration of four cholinesterases and three 'fast' esterases shows that the mobilities of those components in sera of horse, donkey, mule and hinny are the same.These results show that the donkey serum lacks an osterase present in horse serum and that both their hybrids resemble the horse, whether it is the father or the mother.