Chemistry Reference
In-Depth Information
Figure 5.2 (A) Procedure for the attachment of 1,10-phenanthroline
to proteins via lysine derivatization. (B) 5-(iodoacetamido)-1,10-phe-
nanthroline (IOP, 1) and 5-(iodoacetylglycylamido)-1,10-phenanthro-
line (IAOP, 2).
To conjugate E. coli trp repressor with OP-copper, the four lysine amino groups were
first converted into sulfhydryl groups by reaction with 2-iminothiolane hydrochloride
in the presence of the corepressor L-tryptophan (Figure 5.2) [21, 24, 25]. The thiols
were then alkylated with 5-(iodoacetamido)-1,10-phenanthroline (IOP, Figure 5.2-
1
), yielding the TrpR-OP conjugate. DNase I footprinting experiments indicated
that the DNA-binding ability of TrpR was not jeopardized after modification with
OP. Early experiments involved the incubation of trpEDCBA operator with OP-
TrpR copper complex for 20 h, which resulted in double-strand scission as well as
single-strand nicks in 50% of the labeled strands [21].
