Method for the detection of pyruvate kinase, aldolase, and other pyridine nucleotide linked enzyme activities after electrophoresis

Abstract

A method is presented for the detection of pyruvate kinase (PyK) activity after electrophoresis on cellulose polyacetate strips. The method is sensitive and selective and has the advantage of producing a permanent photographic record. The method has been extended to aldolase and is adaptable to other systems involving oxidation or reduction of pyridine nucleotides. PyK is electrophoresed in the presence of stabilizing reagents and fructose diphosphate, which enables the separation of bands of PyK A and PyK C activities. Regions of enzymic activity are detected by placing the strips on the surface of a thin agar film containing the assay reagents. After an appropriate incubation period, the changes in the oxidation state of the pyridine nucleotide are observed visually by fluorescence, and recorded by contact-printing on photographic paper with UV light.