Stereospecific Reaction of Muscle Fiber Proteins with the 5' or 6' Isomer of (Iodoacetamido) Tetramethylrhodamine

The labeling of muscle fiber proteins with (iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (SI) but with different rates, and only the 5ʹ-IATR alters K⁺-EDTA-and Ca²⁺-activated ATPases. Absorption spectroscopic and ATPase studies of probe stoichiometry indicate that for 5ʹ-IATR there are two probes per myosin sulfhydryl 1 (SHI). Quantitative fluorograms of the SDS-PAGE gels confirm that there are one covalent and one noncovalent probe per SHI when SI is labeled with 5ʹ-IATR (5ʹ-IATR-Sl) and that there are one covalent and two noncovalent probes per SI when SI is labeled with 6ʹ-IATR (6ʹ-IATR-Sl). The 5ʹ-and 6ʹ-IATR probes have similar fluorescent lifetimes when bound to SI, but quenching studies with potassium iodide show that 5ʹ-IATR-Sl has a single class of strongly bound chromophores while 6ʹ-IATR-S 1 has two or more classes of chromophores. It is possible that 5ʹ-IATR labels SHI as a dimer. The polarization anisotropics of 5ʹ-and 6MATR-S1 indicate that 5ʹ-IATR is immobilized, while 6ʹ-IATR is moving independently, on the surface of SI. The emission spectrum from 5ʹ-IATR-S1 is unaffected by the addition of MgATP, while 6ʹ-IATR-Sl shows a spectral shift and total intensity change. When labeling muscle fibers, 5ʹ-IATR labels myosin SHI and differentiates between the fiber physiological states by indicating cross-bridge rotation in quantitative agreement with previous results [Burghardt et al. (1983) Proc. Natl. Acad. Sci. U.S.A.50, 7515]. 6ʹ-IATR reacts preferentially with actin in muscle fibers and does not differentiate between fiber physiological states as expected for an actin probe. The stereospecificity of the rhodamine isomers for SHI indicates features of the local protein structure. The experimental results are used with theoretical methods for determining molecular structure to suggest a qualitative scheme for the specific interaction of 5ʹ-IATR with its binding pocket on the surface of SI.