Resonance energy transfer between the active sites of myocardial-type creatine kinase (isozyme MB)

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Abstract
The single reactive sulfhydryl group, located in the active site of each subunit of dimeric creatine kinase from rabbit muscle (isozyme MM), was selectively labeled with 3-(4-maleimidylphenyl)-7-(diethylamino)-4-methylcoumarin (CPM). Isozyme BB, purified to homogeneity from rabbit brain, was conjugated with the sulfhydryl-specific reagent 5''-(iodoacetamido)fluorescein (5''-IAF). Spectral analyses demonstrated that 1.8 mol of CPM and 1.9 mol of 5''-IAF had reacted/mol of protein. Labeled isozymes were combined, denatured in 8 M urea, and renatured by dialysis, producing the parent labeled homodimers and forming the heterolabeled hybrid dimer, creatine kinase MB. Similar hybridizations were performed to prepare singly labeled hybrids, starting with labeled and unlabeled homodimers. The hybrid isozymes were isolated by ion-exchange chromatography, and spectral analyses of singly labeled heterodimers revealed overlap between the absorption spectrum of MB labeled with acetamidofluorescein on the B subunit and the corrected fluorescence emission spectrum of MB labeled with CPM on the M subunit. Analyses included evaluation of the quantum yield of the CPM-labeled hybrid, estimation of the range of the orientation factor K2 from fluorescence polarization and anisotropy studies, and determination of J, the spectral overlap integral for the fluorescence donor (CPM-labeled MB) and acceptor (acetamidofluorescein-labeled MB). Results of these experiments permitted an estimation of R0, the distance between the donor and the acceptor at which energy transfer is 50% efficient. Comparison of the relative fluorescence of the donor in the presence (heterolabeled hybrid) and absence (hybrid conjugated with CPM on the M subunit) of the acceptor or determination of the normalized sensitization of the acceptor fluorescence led to an evaluation of the transfer efficiency and the actual transfer distance of between 27 and 52 .ANG.. The kinetics of quenching of CPM fluorescence, during hybridization with CPM-labeled MM and acetamidofluorescein-labeled BB, compared to hybridization with unlabeled BB, suggest that substantial refolding of denatured subunits precedes reassociation.