"It won't move..."
Kana stared at the electrophoresis gel. One band remained in place.
Milia explained. "Isoelectric point. The pH where protein charge becomes zero."
"Zero charge?"
"A state where positive and negative charges are balanced."
Rei added. "Proteins are made of amino acids. Amino acids have both acidic and basic groups."
"Both?"
"Carboxyl group is acidic. Amino group is basic."
Kana wrote in her notebook. "Charge changes with pH?"
"Yes," Milia continued. "Under acidic conditions, amino groups accept protons. Become positively charged."
"Under basic conditions?"
"Carboxyl groups lose protons. Become negatively charged."
Rei drew a diagram. "As you change pH, charge goes from plus to zero, zero to minus."
"That 'zero' is the isoelectric point?"
"Exactly. Denoted as pI."
Kana asked. "What happens at the isoelectric point?"
"Electrically neutral, so won't move in electric field," Milia answered.
"That's why it stops on the gel."
Rei continued. "Another important property. Solubility becomes minimum."
"Why?"
"With charge, water molecules are attracted. But at zero, intermolecular interactions strengthen."
"It precipitates?"
"Yes. Isoelectric precipitation. Used for protein purification."
Milia showed her tablet. "Each amino acid has a characteristic isoelectric point."
"All different?"
"Determined by side chain properties. Acidic amino acids have low pH, basic amino acids high pH."
Rei gave examples. "Glutamic acid pI is about 3.2. Lysine about 9.7."
Kana calculated. "What about whole protein isoelectric point?"
"Determined by constituent amino acid composition. Complex calculation, but predictable."
Milia continued. "Hemoglobin pI is about 6.8. Pepsin about 1."
"1? Very acidic."
"It's an enzyme working in the stomach. Adapted to acidic environment."
Rei emphasized. "Isoelectric point is like a protein's fingerprint."
"Can be used for identification?"
"Isoelectric focusing. Becomes one axis in two-dimensional electrophoresis."
Kana was confused. "Two-dimensional?"
"First axis is isoelectric point, second is molecular weight. Can separate thousands of proteins."
Milia showed an image. Countless dots scattered.
"Each one is a protein?"
"Yes. Basic technology for proteome analysis."
Rei brought up another topic. "pH is important in living organisms too."
"What do you mean?"
"Intracellular pH is about 7.4. But differs by organelle."
"Differs?"
"Lysosomes are pH 5. Many enzymes work in acidic environments."
Kana understood. "Function controlled by pH?"
"Yes. Near isoelectric point, enzyme activity can change."
Milia added. "When charge changes, substrate binding changes."
"That's why pH is important?"
"Much of life activity only functions in narrow pH range."
Rei said. "That's why buffer solutions are needed."
"To keep pH constant?"
"Yes. In living organisms, there's bicarbonate buffer system."
Kana looked at the gel again. "The stationary band, stopped at isoelectric point."
"The moment of zero charge," Milia said quietly.
"But that has special meaning."
Rei nodded. "The beauty of chemistry is in such singular points."
Kana murmured. "I feel I understand the secret of isoelectric point a little."
"You've just begun to understand," Milia laughed.
"But a good start," Rei acknowledged.
The three stared at the gel. The stationary band speaks volumes.