122 O. MEYERHOF, R. SHATAS, A. KAPLAN VOL. 12 (1953) 



However, if AF is large, small amounts of trimetaphosphate have to be determined in 

 the presence of relatively concentrated orthophosphate. No quantitatively reliable 

 analytical procedure to perform this task has yet been found. 



The second approach makes use of the thermodynamic equation of an isothermal 

 process : 



AF = AH— TAS 



AH, the change in enthalpy, can be calculated in principle from heats of combustion, 

 solution and dissociation or, if the temperature dependency of the equilibrium constant 

 is known, from van 't Hoff' s equation 



dlnK AH 



dT Rr- 



However, a direct calorimetric determination of the heat of reaction is the most reliable 

 method in evaluation of AH. Therefore, we have undertaken a series of measurements 

 of the heat evolved by the enzj-mic hj-droh^sis of trimetaphosphate. 



EXPERIMENTAL 

 The calorimeter 



The calorimetric measurements were carried out with an isothermal constant-flow calorimeter 

 similar to that first described by Ohlmeyer^^. By the use of electronic circuits, hisoriginal arrangement 

 has been adapted for a semi-automatic operation, with a simultaneous improvement in stability and 

 dependability * . 



Materials and preparations 



The trisodium trimetaphosphate used was a sample from Calgon Inc. (Pittsburgh, Pa.) of better 

 than 95 % purity. 



Trimetaphosphatase was prepared from baker's yeast as follows: a mixture of 6 g yeast, 0.5 ml 

 5 % XaHCOj, 2.5 ml o.i M glutathione (pH 7) and 16 ml water was vibrated for 75 min in a Raytheon 

 magnetostrictive oscillator at 9 kc/s. The preparation was kept cold by a circulating water system, 

 the temperature at the outlet being 8-10°. The vibrated mixture was centrifuged at 27,000 g for 5 

 min in a refrigerated centrifuge**. About 17.5 ml of supernatant resulted from each batch of vibrated 

 yeast. 



Solid ammonium sulfate was added to the supernatant to bring the concentration up to 30 % 

 and centrifuged at 27,000 g. The precipitate was discarded, with loss of some activity but much 

 inert protein. The concentration of ammonium sulfate was raised to 60%, the solution centrifuged at 

 27,000 g, the supernatant discarded, and the precipitate (resulting from 17.5 ml of the original 

 supernatant) dissolved in i ml o.i M sodium maleate at pH 7.0. Here the total volume was about 

 2.5 ml. This was dialyzed overnight against glass-distilled water buffered with o.oi M maleate pH 7 

 with three changes of fluid. The enzyme solution was stored in the frozen state. 



These partial purification were usually made with 35 ml of j-east supernatant yielding 4-5 ml 

 enzyme solution. The Qpoithe preparation was 400***. Glass distilled water was used in all solutions. 



Analytical methods 



The purity of trimetaphosphate was assayed by the method of Jones^®. Within the limits of 

 accuracy of this method, no contamination by other phosphates was found. The metachromatic 

 reaction^ was also used to identify hexametaphosphate. The trimetaphosphate was not hj'drolyscd 

 at all by crystalline pyrophosphataset. If not mentioned exphcitly, the assay was as follows: a 

 mixture containing 0.2 ml 0.1 M trimetaphosphate at pH 7.0, 0.4-0.5 ml 0.1 M buffer pH 7, 0.2-0.1 ml 

 enzyme and 0.2 ml of either the solution specified in the tables or o.oi M MgS04 was incubated at 

 33° C for ten minutes. The reaction was stopped by the addition of i ml 5% trichloracetic acid and 

 the supernatant analyzed for orthophosphate by the method of Fiske .\nd Subb.\ Row as modified 



by LOHMANN AND JeNDRASSIK". 



The nucleic acid/protein ration and the protein content during the purification of enzyme was 

 determined in a Beckmann spectrophotometer** by the method of Warburg and Christian^*. 



* Details will be published in The Journal of The Franklin Institute. 



** Loaned by The American Philosophical Society. 



*** Qp = /il H3PO4 formed per i mg dry substance in i h at 33° C. 



t We wish to thank Dr M. Kunitz for a generous sample of crystalline pyrophosphatase. 



References p. izy . 



