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Saturation may become apparent at low temperature.
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Calculation of the difference spectrum required precise knowledge of the concentration of both native and inactivated enzyme cofactor. Whereas this could be calculated in the former case from the known extinction coefficient of enzyme-bound TPQ, this was estimated for the BPAO-14g TPQ derivative by assuming that the shoulder at 370 nm represented the proposed pyrrole and that the displacement of the shoulder over the background rise in absorbance in this region could be converted by use of the same extinction coefficient as exhibited by model 16b at 370 nm. The concentration estimated in this way agreed reasonably well with that based on dilution of the sample obtained following gel filtration.
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