Purification and determination of glutamine synthetase by high-performance immunoaffinity chromatography.
High-performance immunoaffinity chromatography (HPIAC) with anti-glutamine synthetase polyclonal antibodies bound to epoxy-activated silica was used to purify and determine this enzyme from the cyanobacterium Synechocystis. A single-step HPIAC procedure with cell-free extracts yielded electroporetically homogeneous glutamine synthetase. In the determination of glutamine synthetase by HPIAC a linear response in the range 10-60 micrograms of enzyme was observed. Recoveries of 70% of the loaded enzymatic activity and 100% of protein were obtained. The determination of glutamine synthetase protein by HPIAC was compared with that obtained by rocket immunoelectrophoresis. The chromatographic method is proposed as a possible alternative to other immunochemical quantitative techniques, particularly when non-limiting amounts of samples are available.
Alhama
J
J
Departamento de Bioquímica y Biología, Facultad de Veterinaria, Universidad de Córdoba, Spain.
López-Barea
J
J
Toribio
F
F
Roldán
J M
JM
D002846:Chromatography, Affinity / Q000379:methods*; D000458:Cyanobacteria / Q000737:chemistry*; D005974:Glutamate-Ammonia Ligase / Q000032:analysis / Q000302:isolation & purification*; D007122:Immunoelectrophoresis
D005974:Glutamate-Ammonia Ligase