The recombinant azurin mutant Y72F/Y108F
from Pseudomonas aeruginosa was expressed and purified
as described previously9 (link),10 (link) with modifications.13 (link) The single-tryptophan apoprotein mutant was
generated from the holoprotein using a cyanide procedure29 (link) and stored in 50 mM acetate buffer at pH 4.5.
The apoprotein is referred to as apoAzW48. The ratio of absorbance
at 630 to 280 nm of the purified apoprotein was less than 0.003. All
experiments were performed in 20 mM phosphate buffer at pH 7.3. An
aliquot of stock 10.0 mM aqueous solution of the exogenous electron
acceptor [Co(NH3)5Cl]2+ was added
to azurin samples when appropriate. N-Acetyl-l -tryptophanamide (NATA) was prepared as a 0.1 mM aqueous, buffered
(phosphate, pH 7.2) stock solution for fluorescence quantum yield
measurements. The reagents were obtained from the following commercial
sources and used without purification: K2HPO4 and KH2PO4 salts from Fisher Chemical; [Co(NH3)5Cl]Cl2 (98%) from Sigma-Aldrich; KCN
(96%) from Spectrum Chemical; NaCH3COO (99%) and CH3COOH (99%) from Fisher Chemical; CuSO4 (99%) from
Alfa Aesar; and NATA (98%) from Sigma-Aldrich.
from Pseudomonas aeruginosa was expressed and purified
as described previously9 (link),10 (link) with modifications.13 (link) The single-tryptophan apoprotein mutant was
generated from the holoprotein using a cyanide procedure29 (link) and stored in 50 mM acetate buffer at pH 4.5.
The apoprotein is referred to as apoAzW48. The ratio of absorbance
at 630 to 280 nm of the purified apoprotein was less than 0.003. All
experiments were performed in 20 mM phosphate buffer at pH 7.3. An
aliquot of stock 10.0 mM aqueous solution of the exogenous electron
acceptor [Co(NH3)5Cl]2+ was added
to azurin samples when appropriate. N-Acetyl-
(phosphate, pH 7.2) stock solution for fluorescence quantum yield
measurements. The reagents were obtained from the following commercial
sources and used without purification: K2HPO4 and KH2PO4 salts from Fisher Chemical; [Co(NH3)5Cl]Cl2 (98%) from Sigma-Aldrich; KCN
(96%) from Spectrum Chemical; NaCH3COO (99%) and CH3COOH (99%) from Fisher Chemical; CuSO4 (99%) from
Alfa Aesar; and NATA (98%) from Sigma-Aldrich.
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