JBC, Vol. 252, Issue 12, 4293-4297, Jun, 1977
Photolabeling reagent for thiol enzymes. Studies on rabbit muscle creatine kinase
J. Henkin
A mixed disulfide reagent for photolabeling is described which reacts
stoichiometrically with a cysteine sulfhydryl group of rabbit muscle
creatine kinase to form a new mixed disulfide between enzyme and
2-thiobenzyl[14C]diazoacetate. When irradiated at 254 nm for 5 s in a
photoreactor, the enzyme-bound diazo group is destroyed, presumably via a
carbene intermediate. After photolysis, the enzyme can only be 69+/-2%
reactivated by dithiothreitol, and only 67+/-1% of the radiolabel can then
be removed from the protein by dialysis in the presence of dithiothreitol.
Less than 3% of this permanent labeling occurs if photolysis is carried out
in 6 N guanidine hydrochloride, which shows that the native enzyme
structure is required for photolabeling. Identification of the tagged
products after acid hydrolysis indicates that 30% of the carbene produced
on photolysis reacts with the hydroxyl groups of threonine andserine with
O-[14C]carboxymethylthreonine as the major product. Photochemical Wolff
rearrangement is estimated to occur to less than 30%, and no
S-carboxymethylcysteine was detected. The reagent employed and its isomers
are proposed as bifunctional photolabeling probes to "scan" the amino acid
residues near the active sites of thiol enzymes.
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