The Reaction of Creatine Kinase With Dithiobisnitrobenzoic Acid

Two derivatives of creatine kinase (CrK) have been formed by reaction with the sul‐phydryl group reagent, dithiobisnitrobenzoic acid (DTNB). In the first derivative, TNB(2) ‐CrK, the two essential ‐SH groups of the active dimer form were blocked. The inactivated enzyme had similar physico‐chemical properties to the native enzyme, including the ability to bind manganese‐nucleotide substrates. Full activity could be restored by treatment with dithiothreitol. It is suggested that TNB(2)‐CrK could serve as a convenient means of protecting the essential ‐SH groups of CrK, while the effects of other group‐specific reagents are explored. A second derivative, TNB(8)‐CrK, was formed by carrying out the reaction in the presence of 8 M urea. Under these conditions, the CrK dimer is dissociated into monomers with exposure of a further six ‐SH groups. After removal of the urea, a significant concentration of the protein remained in solution in the monomer form. This derivative bound Mn 2+ strongly but the nucleotide binding site was destroyed. A role for the “buried” sulphydryl groups in the formation of the stable dimer form of creatine kinase is indicated.