In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction.
Two methods for measurement of urease activity are described and demonstrated on extracts from several crop plants. With an in-gel staining method based on the principle of Fishbein's noninhibitory stain for urease as little as 25 microU of jackbean urease can be detected within 2 h following electrophoresis. A comparison with published in-gel staining methods shows that the sensitivity is improved by at least two orders of magnitude. The second method allows quantification of urease activity from small amounts of plant material without the need for special laboratory equipment. It employs the detection of ammonium by the indophenol reaction. To eliminate reducing agents which are often necessary to maintain urease activity during extraction, but which interfere with ammonium detection, a simple spin-column procedure is used. The quantification of less than 5 mU/ml extract is possible.
Witte
C P
CP
Unit of Plant Biochemistry, Scottish Crop Research Institute, Invergowrie, DD2 5DA, United Kingdom. cwitte@scri.sari.ac.uk
Medina-Escobar
N
N
D004586:Electrophoresis; D004795:Enzyme Stability; D005782:Gels; D007215:Indophenol / Q000737:chemistry*; D009580:Nitroblue Tetrazolium; D010084:Oxidation-Reduction; D018515:Plant Leaves / Q000201:enzymology*; D013194:Staining and Labeling / Q000379:methods; D014510:Urease / Q000032:analysis*
D005782:Gels; D009580:Nitroblue Tetrazolium; D007215:Indophenol; D014510:Urease