Development of myofibrillar ATPase assay system on pH stat

The ATPase assay system on pH stat apparatus was developed. For the ATPase activity measurement, hydrogen ion (H⁺) concentration delivered from inorganic phosphate (Pi) as a hydrolysis production of ATP was estimated by titrating with 20 mM NaOH solution instead of colorimetric measurement of Pi. Inclusion of 0.5 M KCl in the ATP stock solution and 2 mM Tris-maleate (pH 7.0) buffer in the reaction medium reduced the extent of pH change upon addition of ATP to initiate the ATPase reaction. The amount of H⁺ liberated from Pi was strongly affected by the set pH for the ATPase assay with a promotion at alkaline pH. Thus, it was required to estimate the coefficient to convert H⁺ to a Pi concentration at a specific pH. The specified coefficient at pH 7.0 was 1.248. ATPase assay on pH stat allowed us to follow the ATP hydrolysis by myofibrils continuously showing a curvature profile at low salt medium (≤0.2 M KCl) or a linear profile at high salt (≥0.3 M KCl).