Analysis of activity of esterase captured onto an immunoaffinity membrane.
BACKGROUND
Specific proteins in biological fluids can be captured on an immunoaffinity membrane after polyclonal anti-porcine liver esterase antibodies are separated by non-denaturing 2-dimensional electrophoresis (2-DE) and transferred onto the membrane. The enzymatic activities of these captured proteins can then be monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).


METHODS
Polyclonal anti-porcine liver esterase antibody was separated by non-denaturing 2-DE, transferred onto a polyvinylidene difluoride membrane and stained with Ponceau S. Esterase activity was examined by enzyme activity staining and MALDI-TOF MS after antigens, including purified carboxylesterase from porcine liver and cytosolic esterase from porcine retina, were captured on the immunoaffinity membrane.


RESULTS
Esterase activity was detected on the immunoaffinity membrane after the enzyme was captured. Phosphatidylcholine hydrolysis by the esterase was monitored after the esterase was captured onto the membrane and attached to the target plate for MALDI-TOF MS.


CONCLUSIONS
This method could be used to analyze changes in enzymatic activity under biological conditions such as health and disease conditions using immunoaffinity membranes and MALDI-TOF MS.
Graduate School of Science and Engineering (Science section), Ehime University, Matsuyama, Japan. shimazaki.yoji.my@ehime-u.ac.jp
Youji
Y
Shimazaki
Takahiro
T
Sakikawa
Ayaka
A
Kimura
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004950:Esterases / Q000378:metabolism*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008567:Membranes, Artificial; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008567:Membranes, Artificial; D004950:Esterases