Protein kinetics define the metabolism of plasma proteins in normal physiology, pathophysiology, and drug-altered physiology. Kinetic parameters are usually determined by incorporating either radioactive or stable isotopes into the protein to be studied. Proteins labeled with radioactive isotopes are easy to detect but emit radiation energy that is dangerous to the health of the study subject and investigators. It also generates radioactive waste that is an environmental biohazard. While stable isotopes do not emit radiation, they require tedious protocols to isolate the labeled protein, isolate the labeled amino acid (aa), derivatize the aa and detect the labeled and unlabeled aa on expensive GC-mass spectrometry equipment. Furthermore, once the data is generated it requires special modeling techniques to generate the key kinetic parameters. We propose to develop a method that will exogenously label sialated proteins by enzymatically replacing their sialic acid residues with 9-O-acetyl sialic acid then isolating the labeled protein from plasma with the marine crab lectin which specifically binds to this acetylated sialic acid. The plasma concentration of both the labeled and unlabeled protein will then be detected by standard immunoassay methods, providing a very rapid and inexpensive method to determine plasma protein kinetics, without the use of isotopes.
