KARL F. GUTHE 



73 



complex, since the substrate concentration is high. This breakdown pro- 

 ceeds with a volume increase of 56 cc/mole, from the size of the observed 

 inhibition. The activity under pressure reflects the pH dependence of the 

 rate process (splitting per active site) , and it approaches a direct pro- 

 portionality to hydroxyl ion concentration, in agreement with Mommaert's 

 measurements of atmospheric rates in his best myosin preparations (17). 

 To find the pH dependence of reversible denaturation, the observed rates 



2 



I 



.5 



2 



.1 

 .051- 



.02 



"1 1 



30° 

 3.6 mM ATP 



5 mM Ca 



1 r 



INCREASE IN INACTIVE 

 FRACTION WITH pH 



.01, 



Fig. 1. Left: dependence of myosin ATP-ase activity on pH at two pressures (4). 

 The dashed line, which is approached by the data for 7,800 pounds per square inch, 

 corresponds to a direct proportionality between activity and hydroxyl ion concen- 

 tration. Right: corrected relative pressure increment inactivity as a function of pH 

 (4). Vp' is the observed activity corrected for direct pressure inhibition (see text). 

 Points are derived from the smooth ciu-ves of the preceding graph. Curves are 

 theoretical titration curves for a monobasic acid. The abscissa is proportional to the 

 fraction of reversibly denatured sites. The left-hand curve is calculated. 



under pressure (vp) are first increased by a constant factor to correct for 

 the direct inhibition of the rate process. The corrected pressure increment 

 (Vp'/v — 1) is then plotted against pH (fig. 1, right) . For convenience, only 

 the points obtained from the smooth curves on the left are shown. The 

 points follow the curve, which was calculated as a Henderson-Hasselbach 

 titration curve for dissociation of a single hydrogen ion. The good fit 

 justifies the assumptions, and it shows that reversible denaturation pro- 

 ceeds with the loss of a single hydrogen ion with a pK of 7.85 at a pressure 

 of 7800 pounds per square inch. From the size of the pressure activation, 

 the volume increase is 97 cc/mole, and the pK at atmospheric pressure 



