Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis.
The cytosolic enzymes extracted from rat hepatocytes were separated by native porous gradient-PAGE (PG-PAGE) and were detected with a sensitive and fast chemiluminescence (CL) imaging method. Several peroxidases including glutathione peroxidase, Cu/Zn-superoxidase dismutase, and some other metallo-enzymes such as catalase, carbonic anhydrase-III (CA-III) present in the cytosol of rat hepatocytes have been selectively and sensitively detected by the direct CL imaging method using the luminol-H(2)O(2) chemiluminescent reagents. All detections after PG-PAGE were completed within 9 min. The linear range for the typical metallo-enzyme, e.g., CA-III is 0.75-4.9 microg/mL, with a detection limit of 0.25 microg/mL. In comparison with the traditional CBB-R250 staining method, the detection period decreased about 70 times and the detection sensitivity improved over ten times. Furthermore, two enzymes present in rat liver cytosol were identified employing MALDI-MS analysis of the tryptic digest after PG-PAGE.
Chen
Huaying
H
Department of Chemistry, Beijing Normal University, PR China.
Zhao
Heping
H
Huang
Lingyun
L
Baeyens
Willy R G
WR
Delanghe
Joris R
JR
He
Dacheng
D
Ouyang
Jin
J
D000818:Animals; D024403:Carbonic Anhydrase III / Q000032:analysis*; D002374:Catalase / Q000032:analysis*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005979:Glutathione Peroxidase / Q000032:analysis*; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000032:analysis*
D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D024403:Carbonic Anhydrase III