Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing

We have developed an isoelectric focusing procedure for resolving alkaline phosphatase (EC 3.1.3.1) isoenzymes and isoforms in serum. We use a thin-layer agarose gel film containing synthetic carrier ampholytes and a "separator" to flatten the pH gradient in the region of the isoenzyme and isoform isoelectric points. Sharp, highly resolved zones of enzyme activity are obtained by limiting diffusion; for this we rapidly couple the released product, 1-naphthol, to a diazonium salt, which forms a colored precipitate at the site of activity. We have resolved and identified 12 zones of alkaline phosphatase activity in the serum of ostensibly healthy persons within a wide age range. Theoretically, three basic isoenzymes are produced from independent gene loci: intestinal, placental, and nonspecific tissue alkaline phosphatase. The other zones of activity may be isoforms.