Characterization of human placental alkaline phosphatase by activity and protein assays, capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Placental alkaline phosphatase (PLAP) that had been isolated from human pla centa was further purified using subsequent ion-exchange chromatography (IE C), affinity chromatography (AC) and centrifugal membrane concentration (CM C). During the process, the PLAP samples from the different stages of purif ication were characterized regarding purity and activity. This was accompli shed by combining Lowry analysis, enzymatic activity assay, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and matrix-assis ted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF -MS). The sample obtained after IEC had a rather low specific activity (6.8 U/mg) and appeared to contain several major contaminants, among which was human serum albumin (HSA). AC followed by CMC yielded PLAP with a specific activity of 128 U/mg. The purity and identity of the protein was indicated by MALDI-TOF-MS yielding a spectrum with one major peak at m/z 58 101, Inte restingly, CZE of the pure PLAP revealed a cluster of peaks, which probably reflects the presence of various glycoforms and/or oligomers. The same ana lytical. approach was used to characterize commercially available PLAP. Thi s sample showed a moderate specific activity (15 U/mg) and appeared to be h ighly impure containing various other proteins. (C) 2001 Elsevier Science B .V. All rights reserved.