Intracellular pH (pH i) measures how acidic or basic the fluid inside a cell is. This pH level is important for processes like moving materials across cell membranes and other activities inside the cell. If the pH i is not correct, cells may not work properly. To keep cells healthy, pH i is carefully controlled to support normal cell function, growth, and processes. The ways pH i is controlled usually involve transporters in the cell's outer layer. These transporters are divided into two groups: those that depend on bicarbonate (HCO₃⁻) and those that do not. Normally, the pH inside most cells is between 7.0 and 7.4, but this can vary by tissue type. For example, muscle cells in mammals often have a pH i of 6.8 to 7.1. Different parts of the cell, called organelles, also have varying pH levels, which can range from about 4.5 to 8.0. There are several methods to measure pH i.

Homeostasis

The pH inside a cell is usually lower than the pH outside the cell because there is less bicarbonate (HCO₃⁻) inside. When the level of carbon dioxide (CO₂) outside the cell, such as in the blood, rises above 45 mmHg, it forms carbonic acid. This acid breaks apart into smaller parts, which lowers the pH inside the cell.

Cells contain fluids that can act as buffers, helping to keep the pH inside the cell stable within a certain range. When the pH inside the cell becomes more acidic or more basic, the cell uses sensors in its membrane to adjust. These sensors allow hydrogen ions (H⁺) to move in or out of the cell, linking the pH inside the cell to the pH outside the cell.

Important buffer systems inside the cell include those involving proteins and phosphates. Proteins have parts that can act as acids or bases, allowing them to either give off or take in protons to help keep the pH inside the cell steady. Phosphate buffers also help by using weak acids (like H₂PO₄⁻) and their matching weak bases (like HPO₄²⁻) to either donate or accept protons, which helps maintain a stable pH inside the cell.

In organelles

The pH inside a specific organelle is adjusted to match its role in the cell.

For example, lysosomes have a pH of about 4.5, which is very acidic. Special microscope techniques have shown that phagosomes also have a low internal pH. Both lysosomes and phagosomes are organelles that break down other substances. To do this, they need a very acidic environment to carry out their function effectively.

In contrast, the mitochondrial matrix has a pH level of about 8.0. This is about 0.9 units higher than the pH inside the intermembrane space. Since the process of oxidative phosphorylation happens inside the mitochondria, the difference in pH is needed to create a gradient across the membrane. This gradient helps the mitochondria produce large amounts of ATP.

Measurement

There are several common ways to measure intracellular pH (pH i ). These include using a microelectrode, a pH-sensitive dye, or nuclear magnetic resonance (NMR) techniques. To measure pH inside organelles, scientists may use a method involving pH-sensitive green fluorescent proteins (GFPs).

Each method has its own strengths and weaknesses. Using dyes is often the easiest and fairly accurate method. However, NMR is less precise compared to other methods. Using a microelectrode can be difficult when working with very small cells or when the cell membrane must remain intact. GFPs are unique because they allow scientists to measure pH inside organelles without harming the cell, but this method is not the most precise for measuring pH.

The microelectrode method involves placing a tiny electrode into the cytosol of a cell by creating a small hole in the cell's plasma membrane. The microelectrode contains fluid with a high concentration of hydrogen ions (H+). This creates a voltage difference between the inside and outside of the electrode due to the pH difference. Scientists use this voltage and the known pH of the fluid inside the electrode to calculate the intracellular pH of the cell.

Another method uses pH-sensitive dyes that change their fluorescence depending on the pH level. This technique, called fluorescence spectroscopy, involves adding the dye to the cytosol of a cell. Scientists then use light to excite the dye and measure the wavelength of light it emits as it returns to its normal energy state. This helps identify the type of dye and determine the cell's intracellular pH.

Nuclear Magnetic Resonance (NMR) spectroscopy is another method for measuring pH i . NMR works by placing a cell in a strong magnetic field. Scientists analyze the ratio of protonated to deprotonated phosphate compounds inside the cell to determine its internal pH. NMR can also detect intracellular sodium levels, which can provide additional information about pH i .

Studies using NMR have shown that lymphocytes maintain a constant internal pH of 7.17±0.06. However, like all cells, their internal pH changes in the same direction as the extracellular pH.

To measure pH inside organelles, scientists often use pH-sensitive GFPs. This method is noninvasive and effective. Scientists use cDNA and specific primers to produce GFPs in the cytosol. These proteins can target specific areas within the cell, such as the mitochondria, Golgi apparatus, cytoplasm, and endoplasmic reticulum. If highly pH-sensitive GFP mutants are used, the amount of fluorescence produced can indicate the pH of the surrounding environment.