An alternative, effective substrate for erythrocyte acid phosphatase phenotype determinations.
A method is described for obtaining nondiffusing, nonfading fluorogenic zymograms for erythrocyte acid phosphatase variants separated by isoelectric focusing. The synthetic substrate 4-trifluoromethylcoumarin phosphate was impregnated into cellulose diacetate membranes and air-dried overnight. After isoelectric focusing, the substrate overlay membrane was rehydrated in 0.05M citrate buffer, pH 4.0, lightly blotted, and overlayed on the gel. A 5- to 10-min incubation at 37 degrees C produced intensely fluorescing, light-blue bands on the diacetate membrane. Interaction of the trifluoromethyl group on the substrate with hydrophobic regions of the diacetate membrane impeded diffusion, yielding a permanent zymogram. There were no discrepancies in phenotype determinations using this method when compared with the 4-methylumbelliferyl phosphate assay approach. Further, an increased number of conclusive calls were obtained (91.8 versus 79.5% and 54.1 versus 34.9%) with this new assay when compared with the 4-methylumbelliferyl phosphate substrate on known liquid bloods and questioned dried bloodstains, respectively.
Budowle
B
B
Forensic Science Research and Training Center, Laboratory Division, FBI Academy, Quantico, VA.
Gambel
A M
AM
D000135:Acid Phosphatase / Q000097:blood / Q000235:genetics*; D003374:Coumarins; D004912:Erythrocytes / Q000201:enzymology*; D005456:Fluorescent Dyes; D005554:Forensic Medicine / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D010641:Phenotype
D003374:Coumarins; D005456:Fluorescent Dyes; C057457:4-trifluoromethylcoumarin phosphate; D000135:Acid Phosphatase