Detection and quantitation of heme-containing proteins by chemiluminescence.
A commercial assay for chemiluminescence (CL) has recently been developed for visualizing horseradish peroxidase-conjugated probes for antibodies and nucleic acids. To assess the utility of CL for detecting the peroxidase activity of other heme-containing proteins, the sensitivity of CL and a standard chromogenic stain for visualizing heme-proteins in SDS-polyacrylamide electrophoretic gels were compared. The ability of these systems to visualize heme-proteins on electroblots and dot blots was also examined. The chromogenic stain, which uses 3,3',5,5'-tetramethylbenzidine for a dye, and CL had equal sensitivity in electrophoretic gels. Both assays were affected by 2-mercaptoethanol in the solubilization buffer. In blotting assays, CL was 10- to 10,000-fold more sensitive for detecting samples including cytochrome C and blood. Quantities of protein requiring 18 h to detect by staining were visualized in minutes by CL. Scintillation spectroscopy of CL emitted by blood, urine containing supplemental blood, or urine from a patient with hematuria resulted in a linear relationship between peroxidase activity and concentration, allowing for quantitation of blood over a broad range of concentrations. These results indicate that CL can rapidly detect and quantitate heme-proteins and may facilitate both basic studies of heme-proteins and clinical and forensic analyses of blood.
Dorward
D W
DW
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
D004591:Electrophoresis, Polyacrylamide Gel; D006417:Hematuria / Q000097:blood / Q000652:urine; D006420:Hemeproteins / Q000032:analysis* / Q000737:chemistry / Q000652:urine; D006801:Humans; D015151:Immunoblotting; D008163:Luminescent Measurements; D012680:Sensitivity and Specificity
D006420:Hemeproteins