VARIATION OF INTRACELLULAR INHIBITION WITH pH 727 



of a cell, the more HI will penetrate and the greater the concentration of 

 I~ or total inhibitor in the cells. Actual cells are neither unbuffered nor 

 completely buffered and so will respond in an intermediate fashion. It 

 would be very useful to know the buffer capacity of cells, particularly 

 those commonly used in inhibition studies, but no adequate quantitative 

 work has yet been done. The new^er intracellular methods for determining 

 pH combined with the use of a radioactively labeled or chemically deter- 

 minable weak acid or base could provide this information. One must know 

 how much H"^ has been released within the cell from the HI that penetrates 

 before accurate estimates of the buffer capacity can be made. 



Certainly the intracellular buffer capacity is not great because changes in 

 pH , occur rapidly and readily with alterations of the activity or the produc- 

 tion of injury. The pH, is particularly sensitive to changes in COg tension 

 in the medium. Caldwell (1958) has found that Carcinus muscle and Lo- 

 ligo axons show a drop in the pH^ of 1.1-1.3 units within 3 min following the 

 exposure to sea water saturated with CO.^ around a pH of 5. The rapidity 

 of penetration of COg accounts for this sudden fall in pH,. When the Car- 

 cinus muscle cells were exposed to 30 mM sodium hydrogen phthalate 

 (pHo = 3.4), the drop in pH, was much slower (0.8 units in 30 min. and 1.4 

 units in 60 min) and with phosphate at 30 mM and pH^ = 5.7 the changes 

 were even slower (0.3 units in 100 min). These two substances, however, 

 occur at these pH's mainly in the singly ionized state and there is very little 

 of the undissociated acid, so it is not surprising that penetration was slow. 

 The effects of monobasic acids at pH^'s near or below their piiL^/s would 

 probably occur with a rapidity comparable to the effects of COj. Although 

 from this work one cannot calculate the internal buffer capacity, it does 

 provide good evidence that the pH, does change appreciably when cells 

 are exposed to weak acids. 



It is difficult to calculate the buffer capacity of cells because there are 

 so many components capable of playing a significant role. Proteins have 

 rather weak buffering power from pH 4 to 10, but exert much more efficient 

 buffering outside this range. The inorganic and organic phosphates usually 

 buffer best between pH5.5 and 7.5 while the organic acids are effective be- 

 tween pH 3.6 and 6. There are many other substances that can play minor 

 roles. From the titration curves of a number of proteins and the mean pro- 

 tein content of cells (1.5 mM), one may estimate that (?pH/fZX, with respect 

 to protein, is around 80 between pH 4 and 10 and 110 between pH 3 and 4. 

 Assuming the total intracellular phosphate to be 20 mM with an average 

 pjfiTg of 6.7, d-pRjdX, with respect to phosphates, would vary from 10 to 

 20 in the pH range of 6 to 7.5. Thus the phosphates probably account for 

 the greater portion of the buffer capacity in the neighborhood of the normal 

 pH,. Below a pH^ of 6, dpH/dX would rise, the level to a great extent 

 being determined by the concentration of organic acids in the cell; since 

 this varies widely, all one can say is that dpH/dX probably ranges from 20 



