Analysis of intra- and intermolecular interactions in some oxidases using an analytical ultracentrifuge
The molecular mass of glutamate oxidase from Streptomyces endus was determined by means of sedimentation equilibrium experiments, sedimentation velocity and diffusion studies using an analytical ultracentrifuge. The values obtained vary between 73000 and 98000 in the concentration range of 0.05–0.65 mg/ml. By fitting the concentration-dependent point molecular masses to different models, the best approximation to the experimental data was obtained assuming a monomer-dimer equilibrium of the enzyme. The monomer molecular mass was estimated to be 57000 ± 1800 and the equilibrium constant amounts, K d = 3.0 · 10−6 M. — Glucose oxidase from Penicillium notatum and horseradish peroxidase in an equimolar mixture at pH 5.5 are able to form a 1:1 complex with an association constant K a = 1.0 · 105 M−1. This behavior can be deduced from sedimentation equilibrium runs. In the presence of (NH4)2SO4 or other lyotropic salts the values of the association constant can increase slightly. This fact should be important for preparing bi- or multienzymes of soluble proteins.
Key wordsAnalytical ultracentrifugation enzyme d issociation equilibrium constants bienzyme formation lyotropic salts
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