Electric Potential at Regions near the Two Specific Thiols of Heavy Meromyosin Determined by the Fluorescence Quenching Technique

I. Effect of ATP

Abstract

Electric potentials at regions near the two specific thiol groups, SH₁ and SH₂, of the heavy meromyosin (HMM) molecule were studied by the fluorescence quenching technique. The effects of binding of ATP to HMM upon the electric potentials were also studied. N-(p(2-Benzimidazolyl)phenyl)maleimide (BIPM) was used as a thiol-directed fluorescent reagent. Prior to the labeling of SH₂ with BIPM, the SH₁ group was blocked with N-ethylmaleimide (NEM). Iodide ions (I^-), thallium ions (Tl⁺), and acrylamide were used as quenchers of fluorescence. The sign of the electric potential was collectively determined from the dependence of the Stern-Volmer constants upon the ionic strength of solutions.
1. The region near SH₁ was at a negative electric potential, whereas the electric potential at the region near SH₂ was almost zero.
2. On the addition of ATP, the fluorescence intensity of BIPM bound to SH₁ was unchanged, whereas that of BIPM bound to SH₂ was greatly decreased to about 50% of the original level. The fluorescence intensity recovered as the added ATP was split into ADP and orthophosphate, and became saturated. The saturated level of the fluorescence intensity was, however, smaller than the original one, due to binding of the produced ADP to HMM.
3. On the addition of ATP, the negative electric potential at the region near SH₁ was unchanged, whereas a negative electric potential with large gradient was newly introduced at the region near SH₂. The value of the newly introduced electric potential was calculated on the basis of various assumptions. These results are discussed in connection with the functions of myosin.