2'(or 3')-O-(2, 4, 6-Trinitrophenyl) adenosine 5'-Triphosphate as a Probe for the Binding Site of Heavy Meromyosin ATPase

Abstract

1. From NMR, IR and visible absorption studies of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine (TNP-Ad), and l-(2'-hydroxyethoxy)-2, 4, 6-trinitrobenzene (TNP-EG), it was concluded that there is an intramolecular interaction between the base and 2, 4, 6-trinitrophenyl (TNP) moieties in the TNP-ATP molecule.
2. A broad new absorption band was observed in the 530-630nm region when excess indole was added to reaction mixtures containing TNP-ATP dissolved in 50% methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, TNP-ATP and TNP-Ad underwent spectral shifts in the 400-550nm region. The formation of a 1:1 complex apparently occurred between TNP-ATP and aromatic amino acid derivatives, and the complex with N-acetyltryptophan was stable in 50% methanol. The difference spectrum of TNP-EG vs. TNP-ATP closely resembled that induced by the addition of N-acetyltryptophan to the TNP-ATP solution.
3. The binding of 2'(or 3')-0-(2, 4, 6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) to heavy meromyosin (HMM) was studied by the rapid gel equilibrium method using Sephadex G-25. A dissociation constant of 1.4 μM and a maximum binding number of 1.8 were obtained in 0.15_M KC1, 10m_M MgCl₂, and 50m_M Tris-HCl (pH 8.0) at 25°. TNP-ADP bound to the enzyme caused a characteristic spectral shift in the visible region. This spectral shift was explained in terms of an interaction between tryptophanyl residues and the adenine base of TNP-ADP bound to the enzyme. TNP-ADP quenched the tryptophanyl fluorescence, but TNP-EG and TNP-Ad did not. In the presence of 6_M guanidine hydrochloride, TNP-ADP scarcely quenched the tryptophanyl fluorescence, its effect being comparable to that of TNP-Ad.