Improved identification of antitrypsin phenotypes through isoelectric focusing with dithioerythritol.
Isoelectric focusing has replaced acid starch-gel electrophoresis for the routine determination of antitrypsin phenotypes in recent years. We observed increased sharpness of antitrypsin bands and decreased background stain following the addition of DTE to serum before isoelectric ofcusing in polyacrylamide gels. DTE and DTT were the only sulfhydryl reducing agents which produced this effect. Electrophoretic mobility of antitrypsin bands was increased very slightly. Decreased background stain resulted from the precipitation of albumin. Precipitation (coagulation) of albumin was complete in serum after 60 min incubation at 37 degrees C at concentrations of 30 mM DTE or DTT. Optimal pH for denaturation was 7.6 to 8.8. Ionic concentrations reduced the strength of the coagulum at 2.5M sodium chloride but had little effect at lower concentrations. Marked temperature effects were noted. As a result of these studies, we recommend examination of native and reduced (30 mM DTE) serum on isoelectric focusing in polyacrylamide gel for all samples submitted for routine antitrypsin phenotype determinations. It also seems possible that the nontoxic DTE (DTT) precipitation of albumin could prove useful for studies of serum proteins other than alpha 1-antitrypsin.
D000818:Animals; D002417:Cattle; D002700:Chloramines / Q000494:pharmacology; D004226:Dithioerythritol / Q000494:pharmacology*; D004229:Dithiothreitol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006898:Hydroxylamines / Q000494:pharmacology; D007525:Isoelectric Focusing; D010641:Phenotype; D012709:Serum Albumin / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics*
D002700:Chloramines; D006898:Hydroxylamines; D012709:Serum Albumin; D000515:alpha 1-Antitrypsin; D012710:Serum Albumin, Bovine; D004226:Dithioerythritol; D006861:Hydrogen Peroxide; D004229:Dithiothreitol