In Vitro Studies May Overlook Important Interactions among Molecules

One approach to understanding a biological process is to study purified molecules in vitro (“in glass”—in the test tube), without interference from other molecules present in the intact cell—that is, in vivo (“in the living”). Although this approach has been remarkably revealing, we must keep in mind that the inside of a cell is quite different from the inside of a test tube. The “interfering” components eliminated by purification may be critical to the biological function or regulation of the molecule purified. For example, in vitro studies of pure enzymes are commonly done at very low enzyme concentrations in thoroughly stirred aqueous solutions. In the cell, an enzyme is dissolved or suspended in a gellike cytosol with thousands of other proteins, some of which bind to that enzyme and influence its activity.

Some enzymes are parts of multienzyme complexes in which reactants are channeled from one enzyme to another without ever entering the bulk solvent. Diffusion is hindered in the gel-like cytosol, and the cytosolic composition varies in different regions of the cell. In short,  a given molecule may function quite differently in the cell than in vitro. A central challenge of biochemistry is to understand the influences of cellular organization and macromolecular associations on the function of individual enzymes and other biomolecules—to understand function in vivo as well as in vitro.