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Globinexpressing E. coli cultures were grown in shake flasks at 37¡ãC and at 200 rpm for 4 h followed by a period of 10 h at 100 rpm, on Luria-Bertani (LB) medium  supplemented with 100 mg L-1 ampicillin. Cells from total of 3 L of culture were harvested, resuspended in 100 mM potassium phosphate buffer, pH 7.0, containing 1M phenylmethanesulfonyl fluoride (PMSF) to reach an OD600 ) 600, and lysed in a French press (SLM-Aminco)
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£¨1£© protein purification steps were performed at 4 ¡ãC. Lysates were clarified by centrifugation
and filtration (0.45m). His-tagged Hb and flavoHb proteins were purified on an affinity column, Sephadex HisTrap chelating column (Amersham Pharmacia Biotech),and eluted stepwise in imidazole buffer (40 and 300 mM imidazole in 100 mM potassium phosphate buffer, pH 7.0) followed by a desalting column (Sephadex G25).£¨2£©mple purity was estimated to be >95% for bacterial Hbs and
>90% for flavoHbs from SDS-12% polyacrylamide gels stained in Coomassie Brillant Blue
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FAD content was assayed by releasing FAD from flavoHb by boiling the purified protein sample for 3
min and determining the fluorescence at 520 nm with excitation at 460 nm, by comparison with pure FAD as a standard.
¡°determining the fluorescence at 520 nm with excitation at 460 nm,¡±Ôõô»áÓÐÁ½¸ö²¨³¤ÄØ£¿²»Ì«¶®Õâ¸ö

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