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98 Bioconjugate Chemistry

chemistry. Conjugates of antibodies with enzymes are reveal the binding of a small molecule to a macromolecule.
used for assays of all sorts of biological molecules by Electron paramagnetic resonance provides related infor-
enzyme-linked immunosorbent assay (ELISA). For ex- mation about changes in tumbling behavior, and can also
ample, a protein in a biological fluid can be analyzed by be used to study the distances between paramagnetic cen-
first trapping it using an immobilized antibody that binds ters. Electron microscopy can show the spatial relations
specifically and strongly to an epitope on that particu- between metal clusters attached to macromolecules as
lar protein. After washing away unbound substances, a probes. Recently, an approach has emerged that involves
second antibody that binds another epitope on the tar- cleaving the sugar-phosphate backbone of a nucleic acid
get protein is allowed to bind to the immobilized target. or the polypeptide backbone of a target protein, using a
This second antibody is conjugated to an enzyme; at this cutting protein that binds to the target. The cutting protein
point, the sample contains a number of immobilized en- is prepared by conjugation with a small chemical reagent,
zyme molecules, related to the number of target protein which acts as an artificial nuclease or protease. Using tools
molecules in the original biological fluid. The immobi- such as nucleic acid sequencing or Western blotting, the
lized enzyme molecules are then quantified by standard sites on the target macromolecule that are within reach of
techniques of enzymology, such as catalytic production the cutting reagent can be identified. Chemical reagents
of a light-absorbing product that can be measured spec- designed for use in all these biophysical studies are com-
trophotometrically. Western blotting is another applica- mercially available from specialty vendors.
tion of antibody conjugates, wherein electrophoretic sep-
aration of proteins is followed by blotting the separated
bands onto a special membrane. The bands containing a SEE ALSO THE FOLLOWING ARTICLES
specific antigen are then stained and visualized by pro-
cedures not unlike those used for ELISA, except that the BIOPOLYMERS • CARBOHYDRATES • ENZYME MECHA-
colored product is precipitated where the bands are. NISMS • NUCLEIC ACID SYNTHESIS • PROTEIN
SYNTHESIS
2. Biophysical Studies
BIBLIOGRAPHY
Bioconjugate chemistry plays a prominent role in studying
conformational changes in macromolecules or the bind-
Bioconjugate Chemistry, a journal published by the American Chemical
ing of one macromolecule to another. Techniques such as Society (http://pubs.acs.org/BC).
chemical cross-linking, fluorescence energy transfer, flu- Hermanson, G. T. (1996). “Bioconjugate Techniques,” Academic Press,
orescence polarization, electron paramagnetic resonance, San Diego, CA.
or various other spectroscopy or microscopy experiments, Lundblad, R. L. (1995). “Techniques in Protein Modification,” CRC
Press, Boca Raton, FL.
generally involve preparation of a bioconjugate tagged in
Means, G. E., and Feeney, R. E. (1971). “Chemical Modification of
some way by a reporter molecule. Fluorescence energy Proteins,” Holden-Day, San Francisco, CA.
transfer from an excited donor moiety to an acceptor moi- Aslam, M., and Dent, A. (1998). “Bioconjugation: Protein Coupling
ety depends on the distance between donor and acceptor; Techniques for the Biomedical Sciences,” Grove’s Dictionaries Inc.,
˚
it is useful for measuring distances in the 10 100 A range, New York.
“Pierce Catalog and Handbook” (1994, 1995). Life Science & Analytical
appropriate for macromolecular complexes. Fluorescence
Research Products, Pierce Chemical Company, Rockford, IL.
polarization is sensitive to difference in the rotational dif- Wong, S. S. (1991). “Chemistry of Protein Conjugation and Cross-
fusion (tumbling) of molecules in solution, and can readily Linking,” CRC Press, Inc., Boca Raton, FL.