Photolabeling reagent for thiol enzymes. Studies on rabbit muscle creatine kinase.
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.
D001665:Binding Sites; D003402:Creatine Kinase / Q000528:radiation effects*; D003979:Diazonium Compounds / Q000138:chemical synthesis; D010782:Photolysis; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000138:chemical synthesis / Q000528:radiation effects
D003979:Diazonium Compounds; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D003402:Creatine Kinase