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

• 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. © 2007 Elsevier B.V. All rights reserved.

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