Purification of Antigonadotropic Sera by Enzymatic Digestion. ∗

Abstract

It has been shown by Coghill et al.. 1 that antitoxin in horse serum can be purified to a considerable extent without much loss of activity by digestion with the enzyme Taka-Diastase. Since the antitoxins as well as the antigonadotropic substances produced in the blood of animals over a long period with gonadotropic extracts are found in the globulin fraction of the serum, it was considered probable that the antigonadotropic serum could be purified by the same method. The results of our preliminary work support this view.

Antigonadotropic serum of the horse and the goat were used in this study. The horse serum was obtained from a mare pony which had been injected 3 to 6 times a week for 3 years with a crude sheep pituitary extract (SAP). The goat serum was obtained from an adult female goat which had been injected 5 to 6 times a week for 2 years with a purified pregnancy urine preparation (PU).

Both sera were digested with Taka-Diastase (obtained from Parke, Davis and Company, Detroit, Michigan) in a manner similar to that employed by Coghill et al. 1 One volume of serum was diluted with 2 volumes of distilled water and the pH adjusted to 4 by the addition of crystalline citric acid. Taka-Diastase in the proportion of 5 mg to each ml of undiluted serum was added together with a few drops of toluene. The mixture was shaken well and incubated for 96 hr at 37°C. During the digestion a large, flocculent precipitate was formed. At the end of 96 hr the mixture was filtered. The precipitate was removed from the filter paper, taken up in a volume of distilled water equal to that of the undiluted serum, and shaken well with glass beads to make a homogeneous suspension. This was filtered as before.