Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence.
Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated from energy donor quenching of fluorescence by steady-state and phase-modulation lifetime measurements and determination of sensitized emission of the acceptor. Several sulfhydryl-specific donor fluorophores were used including 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, 7-(dimethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin, and 2-[4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS). Energy transfer acceptors included 5-(iodoacetamido)fluorescein and the nonfluorescent dye [4-[[4-(dimethylamino)phenyl]azo]phenyl]iodoacetamide. In order to prepare the necessary homodimer labeled with both donor and acceptor, advantage was taken of the biphasic reaction between creatine kinase and IAANS. In some instances, donor/acceptor hybrids were prepared by denaturation/renaturation procedures, and possible deviations from expected hybridization stoichiometry were considered. Disproportionation of singly labeled dimers (to unlabeled and doubly labeled dimers) was not observed when the brain isozyme of creatine kinase was used to trap dissociated dye-conjugated or unlabeled muscle-type subunits of creatine kinase. From studies of five different donor/acceptor combinations, the efficiency of energy transfer was found to occur over a range of 5-14%, indicating that the reactive thiols are well separated. Overlap integrals and quantum yields were evaluated, and estimates of the range of orientation factor were obtained to determine a range for the distance between the active sites of creatine kinase. When the ranges are overlapped, a limited distance of 48.6-60.4 A is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)
Grossman
S H
SH
Department of Chemistry, University of South Florida, Tampa 33620.
D000818:Animals; D001665:Binding Sites; D003402:Creatine Kinase / Q000032:analysis*; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007527:Isoenzymes; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D009218:Myosins / Q000032:analysis; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis
D005456:Fluorescent Dyes; D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D003402:Creatine Kinase; D009218:Myosins