CHEMICAL PKOPERTIES 13 



0.74 unit lower than at an ionic strength of zero. The free energy of forma- 

 tion of Mg-malonate is — 3.90 kcal/mole, while .1//° = 3.2 kcal/mole, 

 and J>S'o = 23.9 cal/degree (Evans and Monk, 1952; Chaberek and Martell, 

 1959, p. 139). The entropy term is large and probably results mainly from 

 displacement of water from the charged groups. 



The importance of this chelation in inhibition work lies in the reductions 

 in the concentrations of the free ions it may bring about, both the metal 

 ions and malonate. The decrease in metal ion concentration can easily alter 

 enzyme activity or cellular function and such changes are apt to be attrib- 

 uted to a direct action of malonate. Conversely, the malonate concentra- 

 tion may be reduced appreciably. Examples of mutual concentration re- 

 duction are given in Table 1-5. The concentrations of Mg++ chosen approxi- 

 mate those in Tyrode solution (0.11 niM), Krebs-Ringer phosphate medium 

 (1.18 mM), the usual media for mitochondria (5 mM), and sea water (53.6 

 TCiM), while the concentrations of Ca"'""'" correspond to Krebs-Ringer phos- 

 phate medium (2.54 mM) and sea water (10.24 mM). It may be noted that 

 very significant reductions in Mg^^ and Ca++ can occur with concentrations 

 of malonate commonly used; e.g., malonate at 10 mM will reduce the Mg++ 

 49% and the Ca++ 23% in Krebs-Ringer medium, and higher concentra- 

 tions may almost deplete these ions from the solution. When the concen- 

 trations of these cations are high, as in sea water, the effective malonate 

 concentration may be reduced markedly; e. g. malonate added to sea water 

 at a total concentration of 10 mM will result in a 1.5 mM solution of free 

 malonate ion. Such phenomena have usually been ignored or forgotten 

 despite their possibly large magnitudes. One way of determining the impor- 

 tance of cation reduction in malonate studies is to calculate the reduction 

 to be expected in the medium used and at the malonate concentration, and 

 then to test the effects of lowering the cation concentration to this extent 

 (Rice and Berman, 1961). Other metal cations may be reduced to a greater 

 extent than Mg++ and Ca++. Media initially 1 mM in Co++, Mn++, or Cu++ 

 will in the presence of 5 mM malonate contain these ions at concentrations 

 of 0.65 mM, 0.34 mM, and 0.0052 mM, respectively. If such metal ions are 

 normally bound to enzymes, the degree of removal from the enzyme will, 

 of course, depend on the relative affinities of the metal ion for the enzyme 

 and malonate. There is also the possibility that malonate may chelate with 

 metal ions combined with the enzyme, inactivating them for their catalytic 

 role. It should also be remembered that similar phenomena may occur with 

 succinate, and the inhibition kinetics may be distorted when malonate is 

 used due to the differential reduction in the concentrations of these anions. 



Detection and Determination of Malonate 



Methods have been developed for the separation and identification of 

 organic acids from animal and plant tissues. Earlier determinations involved 



