Activity staining of halophilic enzymes: substitution of salt with a zwitterion in non-denaturing electrophoresis.
Several NAD(P)(+)-dependent dehydrogenases were partially purified from Halobacterium halobium. When salt (2M (NH4)2SO4) was replaced with glycine betaine (4M or 6M), the zwitterion stabilised activity less completely than the salt. Nevertheless most of the enzyme activity still remained after 90h, e.g. 70% for malate dehydrogenase. This level of stabilisation permitted non-denaturing gel electrophoresis in 4M betaine after dialysis to replace salt. Coomassie Blue staining showed good separation of the proteins, and activity staining, hitherto impossible for halophilic enzymes, readily identified the individual dehydrogenase bands. Transfer of activity-stained gels to Coomassie staining solution halted background formazan staining and showed up activity and other protein bands in contrasting colours.
Cadenas
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Department of Biochemistry, University of Alicante, Spain.
Engel
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PC
D000645:Ammonium Sulfate; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001622:Betaine / Q000494:pharmacology*; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D019617:Halobacterium salinarum / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D012965:Sodium Chloride
D001426:Bacterial Proteins; D001622:Betaine; D012965:Sodium Chloride; D010088:Oxidoreductases; D000645:Ammonium Sulfate