What is the pH of a cell?
Hydrogen ions play a central role in the lives of cells. For example, as
illustrated by the wide variety of pK values for different macromolecules
and specifically for proteins, changes in hydrogen ion concentration are
intimately tied to the charge of side chains in proteins. This charge
state, in turn, affects the activity of enzymes as well as their folding and
even localization. Further, certain key molecular machines of the cell
such as the famed ATP synthases that churn out the ATPs that power
many cellular processes are driven by gradients in hydrogen ions across
the membranes that occupy them.
The abundance of these ions and, as a result, the charge state of many
compounds is encapsulated in the pH defined as

pH   log 10 [ H  ] [1M ])

where [] denotes the concentration or more formally the activity of the
charged hydrogen ions (H+ or more accurately the sum of hydronium,
H3O+, as well as the functionally important but often overlooked Zundel,
H5O2+, and Eigen, H7O3+, cations). We are careful to divide the hydrogen
ion concentration by the so-called “standard state” concentration,
namely 1M, in order to ensure that when taking the log we have a
unitless quantity. This step is often skipped in textbooks. The integer 7
is often etched in our memory from school as the pH of water, but there
is nothing special about the integral value of 7. Water has a neutral pH of
about 7, with the exact value varying with temperature, ionic strength
and pressure. What is the pH inside the cell? Just like with other
parameters describing the “state” of molecules and cells, the answer
depends on physiological conditions and which compartment within the
cell we are considering (i.e. which organelle). Despite these provisos,
crude generalizations about the pH can be a useful guide to our thinking.
An E. coli cell has a cytoplasmic pH of ≈7.2-7.8 (BNID 106518). This pH
value is a result of both active and passive mechanisms required for the
ability of E. coli to colonize the human gastrointestinal tract with niches
of pH ranging from 4.5 to 9 (BNID 106518). A passive mechanism for
maintaining this pH relies on the buffering capacity provided by the cell’s
metabolite pool, that is, the fact that a change in pH will result in a
release or absorption of hydronium ions which counteracts the original
change. As shown in the vignette on metabolite concentrations within
cells, the main ingredients of the metabolite pool are glutamate,
glutathione and free phosphate, all with concentrations in the mM range,
i.e. millions of copies per bacterial volume and a pKa in the neutral range
ensuring that they will tend to counteract changes in pH. Active
mechanisms for controlling the hydrogen ion concentration include the
use of transporters such as ATPases, that are driven by ATP hydrolysis to
pump protons against their concentration gradient. These transporters
are regulated such that the cell can actively involve them in order to
sculpt the intracellular pH.
As a second depiction of an organism’s characteristic pH range, budding
yeast is reported to have a cytoplasmic pH of ≈7 when growing on
glucose (Orij 2009, but also see Valli, 2005 for lower values). As shown in
Figure 1A, these measurements were carried out using more fluorescent
protein tricks, this time with a pH-sensitive fluorescent protein. By
examining the ratio of the light intensity emitted by these proteins at two
distinct wavelengths, it is possible to calibrate the pH as shown in Figure
1B. Yeast flourish when the external pH is mildly acidic as their
transport into the cell is often based on co-transport with an incoming
proton and is thus more favorable if the external pH is lower than its
value within the cell (Orij 2011). Pumping excess protons to the vacuole
is an efficient way of maintaining a high cytoplasmic pH, indeed the
vacuole has a more acidic pH of ≈5.5-6.5, while these same fluorescence
measurements reveal that the yeast mitochondria are characterized by a
pH of 7.5. Interestingly, as shown in Figure 1C, the internal pH of these
cells is kept almost constant under very different pH of the surrounding
medium. Using such pH sensitive probes in Hela cells revealed the
cytoplasm and nucleus had a pH of ≈7.3, mitochondria ≈8.0, ER ≈7.45
and Golgi ≈6.6 (BNID 105939, 105940, 105942, 105943).
Even though hydrogen ions are ubiquitous in the exercises sections of
textbooks, their actual abundance inside cells is extremely small. To see
this, consider how many ions are in a bacterium or mitochondrion of
volume 1 m3 at pH 7. Using the rule of thumb that 1 nM corresponds to
Figure 1: Measuring the pH of cells in vivo using pH sensitive fluorescent protein.
Adapted from Rick Orij, Microbiology, 155, 268–278, (2009).
≈1 molecule per bacterial cell volume, and recognizing that pH7
corresponds to a concentration of 10-7 M (or 100 nM), this means that
there are about 100 hydrogen ions per bacterial cell at the typical pHs
found in such cells as worked out in more detail in the calculation shown
in Figure 2. This should be contrasted with the fact that there are in
excess of a million proteins in that same cellular volume, each one
containing several ionizable groups each of which has a pKa close to 7
and thus the tendency to gain or release a hydrogen ion.
How can so many reactions involving hydronium ions work with so few
ions in the cell ? On average, how long does an active site need to wait to
find a charge required for a reaction? It is important to note two key
facts: (i) cells have a strong buffering capacity as a result of metabolites
and amino acid side chains and (ii) the hopping time of charges between
different water molecules is very short in comparison with the reaction
times of interest.
We begin by discussing buffering. If the 100 hydrogen ions we have
estimated are present in each cell were all used up to alter the charge
state of macromolecules, the pH still does not change significantly as
there are literally millions of groups in proteins and metabolites such as
ATP that will compensate by releasing ions as soon as the pH begins to
change. Hence, these 100 ions are quickly replenished whenever they
are consumed in reactions. This implies that there are orders of
magnitude more ion utilizing reactions that can take place. This
capability is quantified by the cell’s buffering capacity. But how does a
reaction “find” the hydronium ion to react with if they are so scarce? The
lifetime of a hydronium ion is extremely brief, about 1 picosecond (10 -12,
BNID 106548). Lifetime in this context refers to the “hopping” timescale
when the charge will move to another adjacent water molecule (also
called the Grotthuss mechanism). The overall effective diffusion rate is
very high ≈7000 m2/s (BNID 106702). The lifetime and diffusion values
can be interpreted to mean that for every ion present in the cell, on
average, 1012 water molecules become charged very briefly every
second. In an E. coli cell there are about 1011 water molecules. Thus
every single ion “visits” every molecule 10 times a second and for 100
ions per cell every molecule will
be converted to an ion 103 times
per second, even if very briefly in
each such case. As a result, an
enzyme or reaction that requires
such an ion will find plenty of
them in the surrounding water
Figure
2:
back
of
the
envelope
calculation of the number of hydrogen
ions in a cell volume.
assuming the kinetics is fast enough to allow utilization before the ions
neutralize to be formed somewhere else in the cell.
The scale of the challenge of keeping a relatively constant pH can be
appreciated by thinking of the first steps of sugar utilization. As glucose
is catabolized in the process of glycolysis, internal electron
rearrangements known as substrate-level phosphorylation, convert noncharged groups into a carboxyl acid group which releases a hydrogen
ion. Consuming over 106 such glucose molecules per second the pH of
the cell would rapidly change if not for the buffering effects and active
regulation just discussed. This is but one example that illustrates the
powerful and tightly-regulated chemistry of hydrogen ions in the
molecular inventory of a cell.