Location of Alpha-2-Globulin by Demonstration of Alkaline Phosphatase during Paper Electrophoresis

Abstract

ON electropapergrams, the alpha-2-globulin protein peak has been shown to coincide exactly with the locus of maximum non-specific alkaline phosphatase activity¹. This enzyme may be detected during the run by an adaptation of the Huggins and Talalay sodium phenolphthalein phosphate method, which determines alkaline phosphatase by ascertaining the colour due to free phenolphthalein produced at pH. 9.2 by incubating a serum sample with the colourless substrate (a blank correction is made for non-enzymatic substrate hydrolysis). Because of its broad optimum pH range, alkaline phosphatase has considerable activity at pH. 8.6, which is most often used for paper electrophoresis, and a significant proportion of free phenolphthalein is coloured at this pH. To detect alkaline phosphatase during paper electrophoresis, 0.1–1.0 per cent of sodium phenolphthalein phosphate is included in the buffer; the region of enzymatic activity is seen as a well-localized red band. At pH 9.2 the band is considerably more impressive in colour.