Trypsinization is a biochemical technique that uses the enzyme trypsin to break down protein molecules, a process known as proteolysis. This protein-cleaving action is leveraged in various scientific contexts, most notably to detach living cells from surfaces in a laboratory setting. By temporarily dissolving the proteins that anchor cells, trypsinization enables researchers to handle and transfer cell cultures for continued study.
What is Trypsin
Trypsin is a protease enzyme that hydrolyzes proteins into smaller components. It belongs to the family of serine proteases, characterized by a specific serine amino acid residue in the active site. It is naturally produced in the pancreas as an inactive precursor called trypsinogen. Trypsinogen is converted into the active form in the small intestine, which prevents the enzyme from digesting the pancreas itself.
Trypsin catalyzes the hydrolysis of peptide bonds in protein chains, an action that is highly specific. It is isolated, often from bovine or porcine sources, for use in laboratory applications. Its controlled protein-cleaving ability makes it a tool for manipulating proteins or separating cells.
The Mechanism of Trypsinization
Trypsinization relies on the enzyme’s high degree of substrate specificity. Trypsin selectively cleaves peptide bonds, which link amino acids in a protein chain. It targets the peptide bond on the carboxyl side of lysine and arginine. This selectivity is determined by a negatively charged aspartate residue in the active site, which attracts the positively charged side chains.
In the laboratory, this targeted action dissolves the proteins that secure cells to a culture dish. Adherent cells are attached to the surface by anchoring proteins, often part of the extracellular matrix. When trypsin is added, the enzyme breaks the peptide bonds within these adhesive proteins, dissolving the cellular “glue.” This causes the cells to detach, resulting in a suspension of individual cells.
Essential Use in Cell Culture
Trypsinization is a routine procedure for maintaining populations of adherent cells in cell culture. Cells proliferate until they cover the available surface area, a state known as confluence. Before the culture becomes too crowded, the cells must be detached and transferred to a new vessel, a process called passaging or subculturing.
Trypsin creates a single-cell suspension, which is necessary for counting cells, splitting the culture, or preparing cells for analysis. Researchers must control the trypsin concentration and incubation time to ensure cells detach without damage. If exposed too long, trypsin can cleave proteins on the cell surface, potentially altering function or viability. After detachment, trypsin activity is halted by adding a serum-containing growth medium that contains natural protease inhibitors.
Trypsin’s Natural Function in the Body
While its laboratory application is technical, trypsin’s original purpose is the digestion of dietary protein within the body. It is a central component of the digestive system, working in the small intestine to process food. Trypsin acts on large protein molecules consumed in the diet, which are too large to be absorbed directly into the bloodstream.
The enzyme hydrolyzes these protein chains into smaller peptide fragments. These fragments are then further broken down by other digestive enzymes into individual amino acids. Amino acids are the building blocks the body can absorb and utilize for processes such as tissue repair, hormone production, and growth.