Analysis of human bone alkaline phosphatase isoforms: Comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography

Abstract

Background Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). Methods We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5–9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. Results Whilst the isoforms B1x (pI = 4.48), B1 (pI = 4.32) and B2 (pI = 4.12) resolved as well-defined bands, B/I resolved as a complex (pI = 4.85–6.84). Neuraminidase altered the migration of all BALP isoforms to pI = 6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11–20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54–73%. IEF resolved the commercial BALP into 2 major bands (pI = 4.41 and 4.55). Conclusions Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI = 3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.