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Figure 2
(a) 10% Tris–tricine SDS–PAGE analysis of ADC site-directed mutants after purification. Bands corresponding to the zymogen (π, ∼15.5 kDa) and the activated α-chains (∼11 kDa) and β-chains (∼4.5 kDa) are evident for all site-directed mutants other than T57V. (b) MALDI–TOF analysis of the Y22F (black) and Y58F (grey) site-directed mutants show two peaks at ∼11 kDa corresponding to the α- and α′-chains, indicating that pyruvoyl cofactor formation occurs in both cases. (c) MALDI–TOF analysis of T57V after prolonged incubation at 70°C reveals trace cleavage of the protein to generate only an α′-chain with an N-terminal serine (11 015 Da; grey trace). The proportion of α′-chain relative to π-chain (∼15.5 kDa) is very low compared with other site-directed mutants, which are completely activated after incubation at 37°C (e.g. the black trace for Y58F)

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ISSN: 2059-7983
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