Tryptophanase catalyzes the conversion of tryptophan to indole and acetic acid. Pyridoxal phosphate is a required cofactor. The HPLC method developed to assay this activity involves the separation of the tryptophan from the indole.
The separation was carried out by reversed-phase HPLC (C18 /xBondapak) using a mobile phase of anhydrous methanol-water (1:1, v/v). The column was
Figure 9.45 Chromatography of enzyme assay media. Peaks: 1, aspartate; 2, glutamate; 3, asparagine; 4, glutamine; 5, Tris-HCl buffer. Elution profile of the assay medium incubated for (A) zero time and (B) 30 minutes. (From Unnithan et al., 1984.)
eluted isocratically and detected fluorometrically with excitation and emission wavelengths of 285 and 320 nm, respectively. The separation obtained is shown in Figure 9.47.
The reaction mixture contained potassium dihydrogen phosphate buffered to pH 7.0 and bacterial cells. The cells were sonicated and preincubated with pyridoxal phosphate, and the reaction was started by the addition of
Figure 9.47 Separation of a synthetic mixture of tryptophan (Trp) and indole (I). Chromatographic conditions: column, Qg /xBondapak; eluent, anhydrous methanol-water (1:1 v/v); flow rate, 1.0 mL/min; temperature, ambient; detection, fluorescence, 285 nm excitation, 320 nm emission cutoff filter. (From Krstulovic and Matzura, 1979.)
tryptophan. At intervals, reactions were terminated with TCA and the solution clarified by centrifugation, filtered, and then analyzed. Several chromatograms showing the time-dependent increase in indole formation are illustrated in Figure 9.48.
The tryptophanase was obtained from E. coli.
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