Resonance energy transfer between the active sites of creatine kinase from rabbit brain
Abstract
Resonance energy transfer was measured between the active site domains of the brain isozyme of creatine kinase (CK-BB). The reactive thiol near the active sites, one on each subunit of the dimeric protein, was derivatized using 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (AED), 2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid (AANS) and 5-iodoacetamidofluorescein (AF). Suitable donor/acceptor protein conjugated hybrids were prepared by controlled kinetics producing CK-BB-AED/AF and CK-BB-AANS/AF. Transfer efficiencies, measured from the quenching of the donor lifetime and steady-state sensitized acceptor emission, ranged from 0.10 to 0.17. From determination of the donor/acceptor overlap integrals, donor quantum yields and attempts to delimit the orientation factor using steady-state and phase-resolved anisotropy measurements, it was found that a suitable estimate of the range between the active sites was between 45 and 57 A. This range is similar to that reported previously for the muscle isozyme of creatine kinase (Grossman, S.H. (1989) Biochemistry 28, 4894-4902) but is a significantly greater distance than detected for the hybrid, myocardial specific isozyme (Grossman, S.H. (1983) Biochemistry 22, 5369-5375).
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