Purification and Partial Characterization of the Alkaline Phosphatase of Swine Kidney*

In 1957, we reported (1) the purification of alkaline phosphatase of swine kidney to a specific activity of about 150,000 units on a basis of total nitrogen content; the units were those as defined by Roche and Bouchilloux (2). At that time, this was the highest activity reported for any alkaline phosphatase, but one or two other workers have since appeared to achieve a similar order of activity. However, certain of the claims of higher activity must be questioned. Mathies (3) reported a specific activity of 10,500 units according to the assay of King et al. (4), and Schales and Arai (5) reported an activity of 11,600 King units; in both cases, the nitrogen content was measured by indirect methods, and the specific activities are to be considered as reflecting only the order of magnitude of activity. Schramm and Armbruster (6) have proposed a factor of about 7 to convert King units to Roche units; we have confirmed this factor, and, since Mathies performed his assays at 25’, have determined the coefficient for conversion of results at 25” to those at 37” to be about 1.4. Thus, the preparation of Mathies would have a specific activity of 10,500 x 7 X 1.4, or 102,900 Roche units, and the preparation of Schales and Arai would have a specific activity of 11,600 x 7, or 81,200 Roche units. Preparations somewhat more comparable to ours have been reported by Portmann (7) and by Alvarez and Lora Tamayo (8); in their procedures, however, the yields were quite low. Since 1957, we have investigated a number of methods for further purification and have settled upon gradient elution from Ecteola cellulose (9) as the most useful method; with this method and with a variety of less suitable methods, it has been possible to achieve a purification to about 300,000 units per mg of total nitrogen. The particular value of the methods as developed is the excellent yield. Another problem, common to all studies of alkaline phosphatase, that of testing of homogeneity, has not been entirely overcome. Practically all workers with preparations of 50,000 units or greater have claimed homogeneity and have presented electrophoretic and other data to document their claims. As we have pointed out (l), electrophoretic methods are incapable of separating materials as unrelated as the peptidases and the alkaline phosphatases of swine kidney; claims of homogeneity on the basis of electrophoresis would seem to be worthless. The material prepared by us is homogeneous in electrophoresis, in paper chromatography, by rechromatography on substituted cellulose and by countercurrent distribution, but, perhaps more to the point, we have not been able to exceed 300,-