VOL. 12 (1953) 



HE.\T OF HYDROLYSIS OF TRIMETAPHOSPHATE 



125 



decreased to 60 % of the value expected for a reaction of the first order: however, this 

 discrepancy was explained quantitatively by the inactivation of the enzyme. 



60 min 



Fig. I. Kinetics of enzymic hydrolysis of trimeta phosphate in semilogarithmic coordinates. Ordinate, 

 substrate concentration, abscissa, duration of hydrolysis. 



Calorimetric measurements 



By an appropriate excess of buffer the constancy of hydrogen ion concentration of 

 the assay mixture was maintained while trimetaphosphate was hydrolysed to ortho- 

 phosphate. If the acidity of reactants and reaction products is different, the measured 

 heat effect includes heat evolved by the neutralization of excess acidity by the buffer, 

 which can be computed easily from the constants and enthalpies of dissociation". 



However, thus far no reliable data were available concerning the trimetaphosphoric 

 acid. On the other hand, by use of a buffer with a low enthalpy of dissociation, the 

 numerical value of measured heat effect will differ only by a small corrective term from 

 the change in enthalpy of that reaction. Therefore, we choose maleate (piv = 6.6, 

 AH112 = 380 cal/mole)^^ as a buffer. 



In the purification of the enzyme employed in the heat measurements, we were 

 interested primarily in achieving a low ratio of nucleic acids to protein. The necessity 

 of a low nucleic acid content is based on the fact that their heat of neutralization is 

 considerable. Thus, nucleic acids if present in the reaction mixture in an appreciable 

 concentration may participate in the neutralization of excess acidity and contribute to 

 the heat effect of reaction in an uncontrollable manner. 



TABLE VI 



HEAT OF HYDROLYSIS OF TRIMETAPHOSPHATE 



Temperature, 33°C; reaction mixture, o.oi M (NaP03)3, 0.02 M MgSO^, 0.03 M sodium maleate buffer, 



pH 6.95 



Run No. Total Volume 

 ml 



Enzyme 

 ml 



PR 

 mW 



Qh-Q'h 



Duration of 



measurement 



sec 



Heat evolved 

 meal 



Turnover 

 fimoles 



Heat of 



Hydrolysis 



cal/mole 



