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

Abstract

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.

Facile detection of proteins on a solid-phase membrane by direct binding of dextran-based luminol–biotin chemiluminescent polymer

“…It utilizes the fact that the native enzymes such as horse radish peroxidase could catalyze the CL reaction between luminol and hydrogen peroxide [6,7]. In this technique, for example, the electrophoresis should be conducted with the usage of non-denatured gel and running at low temperature to keep the proteins active; otherwise CL signal cannot be seen.…”

A novel probe for chemiluminescent image detection of proteins in two‐dimensional gel electrophoresis

“…However, the experimental procedures for the CL immunodetection are rather complicated due to the immunoprocessing requirements. Previous stud-ies carried out in our laboratories have indicated that direct CL imaging detection of serum proteins on nondenaturing 1-D PAGE can offer fast, convenient, and inexpensive procedures [20][21][22][23]. Proteins such as hemoglobin (Hb) [22] and haptoglobin (Hp) with different phenotypes [21] have been well detected.…”

Copper(II)−Alizarin Red S Complex as an Efficient Chemiluminescent Probe for the Detection of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

“…In recent years, a direct CL imaging technique was developed for determining human serum proteins after PAGE, wherein several CL probes have been reported: using hemoglobin (Hb) or ammonium persulfate as probes to catalyze the luminol−H 2 O 2 CL reaction, some proteins in human serum and metalloenzymes in rat liver cytosol could be detected. − Sensitivity of the detection technique was improved. However, only the proteins containing a metal ion or metal complexes could generate CL emission under these conditions, hence more investigation was devoted to extend the detection range: [Ag(NH 3 ) 2 ] + has been reported as a probe for CL imaging, catalyzing the luminol−K 2 S 2 O 8 CL system .…”