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Assay Method Information |
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| | CM Assay |
| Description: | Reaction volumes of 0.4 mL of chorismate (typically, 1 mM) in 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.1 mg/mL bovine serum albumin, and 10 mM β-mercaptoethanol were incubated at 37 °C for 5 min. The reaction was started with 5 pmol of MTB CM in a 10-μl volume (i.e. 185 ng of MTB CM equivalent toa 12.5nM final concentration of the dimer based on the molecular mass of 36 948 Da). The reaction was allowed to proceed at 37 °C and was terminated after 1 min to 5 min with 0.4 ml of 1N HCl. After a further incubation at 37 °C for 10 min to convert prephenate, which is formed in the enzymatic reaction, tophenylpyruvate, 0.8 ml of 2.5N NaOH was added. The absorbance of the phenylpyruvate chromophore was read at 320 nm. We set up a blank with no enzyme for every reaction to account for the non-enzymatic conversion of chorismate to prephenate and added the enzyme after NaOH addition. The A320 for the blank varied from 0.1 to 0.3, depending on the concentration of chorismate and duration of the reaction. Each reaction was carried out in triplicates. |
| Affinity data for this assay | |
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