Creatine kinase compactness and thiol accessibility during sodium dodecyl sulfate denaturation estimated by resonance energy transfer and 2-nitro-5-thiocyanobenzoic acid cleavage.
We have investigated the effect of increasing sodium dodecyl sulfate (SDS) concentrations on rabbit muscle cytosolic creatine kinase structure by two methods. We have first determined the variation of accessibility of the thiol groups of the enzyme during SDS denaturation by a technique which involves an irreversible chemical modification of CK accessible thiol groups, followed by NTCB cleavage before the unmodified cysteines in 8 M urea (pH 9) and analysis of the peptides obtained by resolutive gel electrophoresis, without sequencing. We have determined that the order of accessibility of CK MM cysteine residues during SDS denaturation is Cys-282, Cys-145 and then Cys-253. The fourth cysteine residue, Cys-73, is never titrated even at high SDS/CK molar ratio. In contrast, the three last residues are simultaneously titrated when CK is denatured in guanidinium chloride. Thus, SDS-denatured CK seems to retain some residual organized structure. In order to confirm this hypothesis, compactness of the molecule was estimated by fluorescence energy transfer between CK tryptophans and AEDANS, an extrinsic fluorophore. The location of this fluorophore on the accessible thiol of Cys-282 was verified by the previous technique. The results of these experiments do indicate that SDS-denatured CK is more compact than CK completely unfolded in guanidinium chloride.
Clottes
E
E
Biomembranes et Enzymes Associés, URA 1535, CNRS-LYON I, Villeurbanne, France.
Couthon
F
F
Denoroy
L
L
Vial
C
C
D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry*; D004735:Energy Transfer; D009132:Muscles / Q000201:enzymology; D011489:Protein Denaturation; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents; D013861:Thiocyanates
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013861:Thiocyanates; D012967:Sodium Dodecyl Sulfate; D003402:Creatine Kinase; C008658:2-nitro-5-thiocyanobenzoic acid