Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.
Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.
D003851:Deoxyribonucleases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D011119:Polynucleotides
D011119:Polynucleotides; D003851:Deoxyribonucleases; D006026:Glycoside Hydrolases