Fig. 2

Optimization of reaction conditions of HERV-K(HML-2) Protease. Purified HML-2 Pro was incubated with a fluorescent anthranilyl-substrate and fluorescence emission was measured for the indicated time periods. Influence of different buffer compositions (top), pH values (middle), and Pepstatin A concentrations (bottom) on HML-2 Pro activity are depicted. Buffer compositions were as follows: Buffer 1: 20 mM PIPES, 100 mM NaCl, 1 mM DTT, 10% [v/v] Glycerol, pH 6.5; Buffer 2: 50 mM MES, 1 M NaCl, 20% [v/v] Glycerol, 1 mM EDTA, pH 5.0; Buffer 3: 50 mM MES, 1 M NaCl, 1 mM EDTA, pH 5.0; Buffer 4: 100 mM MES-TRIS, 1.25 M NaCl, pH 6.0. Effects of pH were measured in a buffer consisting of 100 mM MES, 1 M NaCl. Note the differing glycerol concentrations of buffers 2 and 3 (see the text). Also note that reactions at pH 5.5 and pH 6 depleted the substrate after approximately 110 min due to high HML-2 Pro activity. Effects of Pepstatin A at 200 μM were measured with and without pre-incubation of protease with Pepstatin A