TAILIEUCHUNG - Báo cáo Y học: A comparison of the urea-induced unfolding of apo¯avodoxin and ¯avodoxin from Desulfovibrio vulgaris

The kinetics and thermodynamics of the urea-induced unfolding of ¯avodoxin and apo¯avodoxin fromDesulfo-vibrio vulgariswere investigated by measuring changes in ¯avin and protein ¯uorescence. The reaction of urea with ¯avodoxin is up to 5000 times slower than the reaction with the apoprotein ( s )1 in 3 Murea in 25 mMsodium phosphate at 25°C), and it results in the dissociation of FMN. | Eur. J. Biochem. 269 212-223 2002 FEBS 2002 A comparison of the urea-induced unfolding of apoflavodoxin and favodoxin from Desulfovibrio vulgaris Brian O Nuallain and Stephen G. Mayhew Department of Biochemistry University College Dublin Belfield Dublin Ireland The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein s-1 in 3 M urea in 25 mM sodium phosphate at 25 C and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding but they do not exclude the possibility of intermediates. Calculation suggests that 21 and 30 of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein. Keywords apoflavodoxin urea unfolding Desulfovibrio vulgaris.

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