H-1-NMR MEASUREMENT OF FRACTIONAL DISSOCIATION OF IMIDAZOLE IN INTACT ANIMALS

The alphastat hypothesis states that intracellular acid-base status is regulated to maintain constancy of the fractional dissociation of intracellular protein and enzyme imidazole-histidine (alpha-imidazole). A major drawback of this theory has been the lack of a means to directly measure alpha-imidazole in intact animals. We developed a method for directly measuring alpha-imidazole in intact unanesthetized animals using H-1-nuclear magnetic resonance spectroscopy (NMR). We measured carnosine a-imidazole of white skeletal muscle from intact unanesthetized newts at three body temperatures (10, 20, and 30 degrees C). alpha-Imidazole remained constant, similar to 0.56, with alterations in body temperature, whereas intracellular pH (pH(i)) changed significantly (-0.015 U/degrees C), affirming the validity of the imidazole alphastat hypothesis for this tissue. This method was also used to determine the pK values of the imidazole moiety of carnosine and the imidazole moiety alone over a temperature (T) range 4-40 degrees C. The pK values of carnosine differed from those of imidazole, but the Delta pK/Delta T was the same. pH(i) was also determined using P-31- NMR and found to be the same as that calculated from carnosine alpha-imidazole values. Therefore Pi and carnosine share a similar pH(i) environment. We describe a novel technique to directly measure alpha-imidazole in intact tissue.