The differences in proteins, especially in enzymes, account for differences among cells. It is now appropriate to ask what makes one protein different from another. We know that the structure of a protein depends upon several factors, such as the molecular weight. But the main differences among proteins depend upon the sequence, or order, of the amino acids that are linked together in the protein molecules. Amino Acids and Protein Structure Amino acids are the fundamental structural units of proteins. There are 20 amino acids found frequently in mammalian proteins, and these molecules may be linked to one another to form a chain called a polypeptide chain. The structure of a protein then depends on: (1) the quantity of each amino acid present; (2) the sequence of amino acids in the polypeptide chain; (3) the length of the polypeptide chain, that is, the molecular weight; and (4) the folding and the side (nonlinear) arrangement of the polypeptide chain molecules, that is, the secondary and tertiary structures. How can we investigate protein synthesis by using radioactive isotopes? Since proteins are made up of amino acids, the logical conclusion, after what we have learned about DNA synthesis and RNA synthesis, is that the best way would be to mark an amino acid and follow its incorporation into a molecule of protein. We could label a mixture of several amino acids, but, for the sake of clarity, we will describe the incorporation of a single labeled amino acid. Labeling an Amino Acid with a Radioactive Isotope Suppose we have the amino acid leucine labeled with ¹⁴C and we inject a solution containing it into an experimental animal. Since leucine is incorporated into proteins, if we isolate the proteins and determine both the amount of proteins and the amount of radioactivity, we can measure fairly accurately the rate of protein synthesis. Autoradiography, by the way, is of little help in studying most protein synthesis because all cells are always synthesizing proteins and so are all labeled after a single exposure to a radioactive amino acid. With RNA precursors autoradiography at least told us where RNA was being made, but with amino acids we do not even get this information because proteins are synthesized both in the nucleus and in the cytoplasm. Under these circumstances radiochemical methods are better for studying protein synthesis. Proteins are isolated from the residue left after a nucleic-acid extraction process similar to that described previously, and the amount of protein is determined by a simple colorimetric analysis based on comparison of the color of the solution with a standard color. The amount of radioactivity (remember that we are now using a precursor labeled with ¹⁴C) can be determined with a gas-flow counter, which is probably more widely used at present than any other instrument for counting beta emitters, chiefly because of its reliability and low cost.