Effects of pH and molecular charge on dipolar coupling interactions of solutes in skeletal muscle observed by DQF, [formula omitted] NMR spectroscopy

Abstract

In this study we tested the effect of molecular charge and chirality as well as tissue pH on dipolar coupling interaction in skeletal muscle. These results were demonstrated by double quantum filtered, DQF, 1 H NMR spectra acquired on permeable skeletal muscle samples dialyzed against buffered solutions containing three classes of solutes—electrolytes (lactate and Tris), zwitterions (alanine and glycine), and non-electrolytes (dioxane and ethanol)—as a function of pH ranging from 5.0 to 8.5. The results show that charge density on the protein filaments strongly influences dipolar coupling of solutes in muscle whereas charge on the solutes themselves has only a small effect. The frequency splitting of the dipolar coupled peaks for all the molecules tested was strongly affected by muscle pH. Higher pH increased negative charge density on the filaments and resulted in weaker dipolar coupling for anions and zwitterions but stronger coupling for the cation TRIS. Molecular charge per se or chirality did not affect the frequency splitting of the dipolar coupled peaks. The molecules, lactate, ethanol, and alanine, have scalar coupled spins and consequently a double quantum signal in solution. However, spectra acquired from these molecules in muscle showed an additional frequency splitting due to additional dipolar coupling interactions. Due to lack of scalar coupling, spectra from Tris, glycine, and dioxane showed no double quantum signal in solution but did when in muscle. All these observations can be explained by the fact that the net charge on protein filaments dominates the mechanism of dipolar coupling interactions in the highly anisotropic structures in muscle.