SITE-SPECIFIC MODIFICATION OF RABBIT MUSCLE CREATINE-KINASE WITH SULFHYDRYL-SPECIFIC FLUORESCENCE PROBE BY USE OF HYDROSTATIC-PRESSURE

We investigated the effect of pressure on the reactivity of cysteine r esidues of rabbit muscle creatine kinase (CK). Performing the fluoresc ent modification under high pressure, a unique sulfhydryl group (Cys-2 53) of CK was labeled, in addition to Cys-282, which is known as a sin gle reactive sulfhydryl under ambient conditions. CK is composed of tw o identical subunits, containing four cysteine residues in each subuni t. Cys-282 plays an important role in enzymatic activity. In the press ure range from 0.1 MPa to 300 MPa, only one sulfhydryl group for each subunit of CK reacted with the reagents. However, at 400 MPa 2 sulfhyd ryl groups were modified. The 2-nitro-5-thiocyanobenzoic acid (NTCB) c leavage method revealed that both Cys-282 and Cys-253 were modified at 400 MPa. The chemical modification of Cys-282 induced a loss of enzym atic activity. By taking advantage of the modification under high pres sure, selective modification of Cys-253 with (iodoacetamidoethyl)amino ]-naphthalene-1-sulfonate (IAEDANS) was performed. PI reversible block ing of Cys-282 at atmospheric pressure was followed by the reaction of Cys-253 with the fluorescent probe at 400 MPa. After the decompressio n, Cys-282 was unblocked, and obtained Cys-253-modified CK retained up to 64% of the catalytic activity of the intact CK. The fluorescent pr operties of IAEDANS covalently bound at Cys-253 were not significantly different from those of IAEDANS covalently bound at Cys-282.