CHEMICAL COMPOSITION OF THE MEDIA 429 



dition to the enzyme and substrate; when a requirement for a coenzyme 

 or cofactor is known, such may also be added. The pH may be chosen ar- 

 bitrarily or as that giving maximal enzyme activity, but is seldom selected 

 with any reference to the intracellular pH. It must be admitted that, al- 

 though much attention has been given to the designing of appropriate ex- 

 tracellular media, a minimal effort has been expended in the attempt to 

 test enzymes under reasonably normal conditions. The enzymologist has 

 been more interested in characterizing an enzyme in chemical or catalytic 

 terms than in determining its behavior under physiological conditions. In 

 attempting to extend inhibition studies on isolated enzymes to cellular 

 levels, it must be realized that most enzyme media used are grossly un- 

 physiological. 



A cursory examination of some five dozen studies on enzymes isolated 

 from mammalian tissues (in the 1959 issues of the Journal of Biological 

 Chemistry) showed that in 60% only a buffer was used, in 35% a cofactor 

 was added (such as Mg^^), and in the remaining 5% some stabilizer, such 

 as glutathione, cysteine, or EDTA, was present. No attempt was made 

 to create a medium ionically physiological. In some cases sodium buffers 

 were used rather than potassium buffers, despite the preponderance of 

 potassium over sodium in the cell. A similar impression may be obtained 

 by perusing the standard testing conditions for enzymes in "Methods in 

 Enzymology" (Colowick and Kaplan, 1955, Vols. I and II). In the papers 

 examined above, the pH chosen was anywhere from 5 to 10, with most 

 between 7.4 and 7.8; the pH was above 7 in 82% despite the fact that the 

 intracellular pH is generally somewhat less than 7. A variety of buffers was 

 used — potassium phosphate, sodium acetate, glycine, glycylglycine, so- 

 dium pyrophosphate, sodium citrate, tris(hydroxymethyl)aminomethane 

 (Tris), barbital, or combinations of these — and these were in concentra- 

 tions from 7 to 160 mil/. There is little rationale in the use of an odd as- 

 sortment of unnatural buffers and if experiments were made nearer the 

 physiological pH there would be no need for buffers effective outside this 

 range. The testing of enzyme activity in several buffers and choosing the 

 one giving the highest activity also is not conducive to physiological re- 

 sults. It seems reasonable to suggest that the lowest concentration of buf- 

 fer, commensurate with controlling the pH, should be selected; indeed, in 

 some reactions it is not necessary to have a buffer, at least over the interval 

 measured. Mg"^"^, when used, varied between 2.8 and 25 mM. Tempera- 

 tures ranged from 23° to 38o (in 10% of the reports it was not specified) 

 and in approximately half the cases it was physiological (370-38°). The 

 balance between K"^ and Na"^ ions was seldom considered. 



The over-all concentrations of the common physiological inorganic ions 

 in vertebrate cells are reasonably well known and selected values are given 

 in Table 9-1. It is evident that K"*" is usually at a much higher concentra- 



