pH gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: detection and inhibition of enzyme activity in the gel.
A simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8-10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory-type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as substrate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory-type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental-type RNase inhibitor, can be performed.
Department of Legal Medicine, Fukui Medical School, Japan.
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Nadano
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Yasuda
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Sawazaki
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Takeshita
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Kishi
D000468:Alkalies / Q000032:analysis*; D002021:Buffers; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012016:Reference Values; D012259:Ribonuclease, Pancreatic / Q000032:analysis / Q000037:antagonists & inhibitors; D012260:Ribonucleases / Q000032:analysis* / Q000037:antagonists & inhibitors; D012680:Sensitivity and Specificity
D000468:Alkalies; D002021:Buffers; D004791:Enzyme Inhibitors; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic