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 placenta was further purified using subsequent ion-exchange chromatography (IEC), affinity chromatography (AC) and centrifugal membrane concentration (CMC). During the process, the PLAP samples from the different stages of purification were characterized regarding purity and activity. This was accomplished by combining Lowry analysis, enzymatic activity assay, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and matrix-assisted 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. Interestingly, CZE of the pure PLAP revealed a cluster of peaks, which probably reflects the presence of various glycoforms and/or oligomers. The same analytical approach was used to characterize commercially available PLAP. This sample showed a moderate specific activity (15 U/mg) and appeared to be highly impure containing various other proteins.
Eriksson
H J
HJ
Department of Pharmaceutical Analysis, University of Groningen, The Netherlands.
Somsen
G W
GW
Hinrichs
W L
WL
Frijlink
H W
HW
de Jong
G J
GJ
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010920:Placenta / Q000201:enzymology*; D011786:Quality Control; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000469:Alkaline Phosphatase