Potential of capillary electrophoresis for the monitoring of the stability of placental alkaline phosphatase.
Alkaline phosphatase (AP) is a potential therapeutic agent in the treatment of sepsis. In this paper the potential of capillary zone electrophoresis (CZE) for the monitoring of the degradation of placental alkaline phosphatase (PLAP) was investigated. To induce degradation PLAP samples were exposed to high temperatures, low and high pH and freeze-drying. The samples were then analyzed by CZE and enzymatic activity assay. Upon exposure to temperatures above 65 degrees C, PLAP lost its activity exponentially over time, while CZE revealed both a linear decrease of the area of the main peak and a rise of degradation products. At acidic pH the enzyme appeared to lose its activity. CZE revealed a decrease of the area of the main peak, but no degradation products could be detected. At pH 12 the enzymatic activity and the area of the main peak both decreased linearly over time and, in addition, formation of degradation products could be detected by CZE. Activity and CZE profile of PLAP remained unchanged upon freeze-drying in the presence of inulin. Prolonged storage of freeze-dried samples at room temperature caused a slight decrease of enzymatic activity, while the potential formation of oligomers was revealed by CZE analysis. The examples in this study show that, in combination with activity assays, CZE can provide useful complementary information, especially on the status of the protein and the presence of degradation products.
Eriksson
H J C
HJ
Department of Pharmaceutical Analysis, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. j.c.eriksson@farm.rug.nl
Wijngaard
M
M
Hinrichs
W L J
WL
Frijlink
H W
HW
Somsen
G W
GW
de Jong
G J
GJ
D000469:Alkaline Phosphatase / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004795:Enzyme Stability; D005612:Freeze Drying; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010920:Placenta / Q000201:enzymology*
D000469:Alkaline Phosphatase